Nova Science Publishers, Inc.

Transkript

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TUMOR SUPPRESSOR GENES
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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS
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FUNCTIONS, REGULATION
AND HEALTH EFFECTS
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CANCER ETIOLOGY,
DIAGNOSIS AND TREATMENTS
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Additional e-books in this series can be found on Nova‘s website
under the e-book tab.
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Additional books in this series can be found on Nova‘s website
under the Series tab.
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GENETICS - RESEARCH AND ISSUES
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Additional books in this series can be found on Nova‘s website
under the Series tab.
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Additional e-books in this series can be found on Nova‘s website
under the e-book tab.
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TUMOR SUPPRESSOR GENES
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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS
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FUNCTIONS, REGULATION
AND HEALTH EFFECTS
MEHMET GUNDUZ
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EDITORS
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ESRA GUNDUZ
New York
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Copyright © 2013 by Nova Science Publishers, Inc.
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For permission to use material from this book please contact us:
Telephone 631-231-7269; Fax 631-231-8175
Web Site: http://www.novapublishers.com
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All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
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NOTICE TO THE READER
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contained in this publication.
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This publication is designed to provide accurate and authoritative information with regard to the
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AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
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Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data
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ISBN: 978-1-62808-665-2
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Library of Congress Control Number: 2013945710
Published by Nova Science Publishers, Inc. † New York
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Multifunction New Tumor Suppressor Gene Family from
Cancer to Metastasis: A Disintegrin and Metalloproteinases
with Thrombospondin Motifs (ADAMTS)
Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik,
Yudum Yaral, Birsen Dogan, Zisan Akcaaga,
Zahide Nur Unal and Mehmet Gunduz
45
Application of Cancer Gene Therapy Using Tumor
Suppressor Gene p53
Hiroshi Tazawa, Shunsuke Kagawa and Toshiyoshi Fujiwara
81
p63 and p73: Members of the p53 Tumor Suppressor Family
Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu,
Esra Gunduz and Mehmet Gunduz
Chapter 7
The Emerging Roles of Forkhead Box (FOX) Family
Proteins in Tumor Suppression
Pang-Kuo Lo
129
Molecular Basis of BRCA1 and BRCA2 and Clinical
Approaches to BRCA1/2 Mutation Carriers
Kemal Keseroglu, Fatih Aydogan and Mustafa Ozen
183
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Chapter 6
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Chapter 5
1
Von Hippel–Lindau (VHL) Gene and Protein (pVHL):
A Member of the Tumor Suppressor Gene Family
Ferah Armutcu, Kadir Demircan and Murat Oznur
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Chapter 4
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Chapter 3
The Inhibitor of Growth (ING) Gene Family: Potential Use
in Cancer Diagnostics and Therapy
Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul,
Senol Dane and Esra Gunduz
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Chapter 2
Physico-Chemical Properties of the Putative Tumor
Suppressor Protein, 101F6
Alajos Bérczi and Motonari Tsubaki
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Chapter 1
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Contents
Chapter 8
105
vi
Contents
DKK3, a Mysterious Tumor Suppressor Gene that Possesses
Multiple Functions in Tumor Progression
Naoki Katase, Tsutomu Nohno and Mehmet Gunduz
207
The Functions and Roles of the Unique Tumor
Suppressor Gene PTEN
Omer Faruk Karatas, Esra Guzel and Mustafa Ozen
233
Functions of the Tumor Suppressor Gene APC
Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa
and Yoshio Naomoto
253
Chapter 13
Structure and Function of the Tumor Suppressor Gene p16
Zeynep Tarcan, Catherine Moroski Erkul,
Bunyamin Isik, Esra Gunduz and Mehmet Gunduz
263
Chapter 14
The Functions and Roles of RB1 in Cancer
Erkan Koparir, Asuman Koparir and Mustafa Ozen
Chapter 15
Endoplasmic Reticulum Protein 29 (ERp29) and Cancer:
Molecular Functions, Mechanisms and Clinical Implication
Daohai Zhang
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Divergent Roles for Tumor Suppressor Genes in Cancer
Marina Trombetta-Lima, Thiago Jacomasso,
Sheila Maria Brochado Winnischofer and Mari Cleide Sogayar
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Index
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Chapter 12
Chapter 16
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Chapter 11
195
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Chapter 10
On The Verge of Being a Tumor Suppressor Gene
or an Axonal Guidance Molecule: DCC
Omer Faruk Karatas, Betul Yuceturk and Mustafa Ozen
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Chapter 9
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301
333
355
ISBN: 978-1-62808-665-2
© 2013 Nova Science Publishers, Inc.
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In: Tumor Suppressor Genes
Editors: Mehmet Gunduz and Esra Gunduz
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Chapter 1
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Physico-Chemical Properties of the
Putative Tumor Suppressor Protein,
101F6
Alajos Bérczi1 and Motonari Tsubaki2
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Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences,
Szeged, Hungary
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Department of Chemistry, Kobe University Graduate School of Science, Kobe, Japan
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Abstract
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Based on structural similarities, the putative human tumor suppressor protein, 101F6,
and its mouse ortholog, TSP10, were identified as members of the cytochrome b561
(Cyt-b561) protein family about 10 years ago. Both proteins have been successfully
expressed in different yeast expression systems and their basic physico-chemical
properties have been established. MALDI-TOF mass analysis indicated that no posttranslational modification of the proteins in yeast cells occurred. These integral
membrane proteins have six trans-membrane domains (TMD) and two b-type hemes with
different midpoint redox potentials; one heme on each side of the membrane. The two
hemes are coordinated by two pairs of His residues that are localized on the four central
trans-membrane domains. The recombinant tumor suppressor (TSCytb) proteins could be
reduced by ascorbate (ASC) and by dihydrolipoic acid (DHLA) at about the same level
but maximal reduction of TSCytb could be reached by dithionite. The 50% reduction of
the high-potential and low-potential hemes by ASC could be obtained at about 0.25 mM
and 10 mM, respectively. The reaction rates of the electron donation from the ASCreduced human TSCytb to the pulse-generated monodehydroascorbate radical was found
to be about 2~5-times faster than in case of other Cyt-b561 proteins, suggesting that
human TSCytb is very effective for scavenging monodehydroascorbate radicals in cells.
Low-temperature EPR spectroscopy has revealed the presence of a highly anisotropic
low-spin (HALS) heme and a rhombic low-spin heme with gz values of 3.61 and 2.96,
respectively, for the mouse TSCytb. However, only two overlapping HALS heme signals
with gz values around 3.7 appeared for human TSCytb. Resonance Raman spectroscopy
could not detect difference between the in-plane and vinyl vibrational modes of the two
2
1. Introduction
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hemes as well as any out-of-plane vibrational modes. These results indicate that (1) the
local electrostatic environment within the protein at the two hemes are slightly different
from that in other Cyt-b561 proteins, (2) the orientation of the imodazole plane of the two
heme-coordinating His residues are almost perpendicular at one HALS heme but the
other heme might be easily converted from a HALS-type to a rhombic-type heme (where
the two imidazole planes are parallel to each other), and (3) both hemes are in relaxed
state (no constraint from the protein body) where the central iron lies within the
porphyrin plane. Comparison of these results with those obtained for other Cyt-b561
proteins might help in understanding the precise biological function and mechanism of
the human 101F6 protein.
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Frequent genetic alterations, like allelic loss or homozygous deletions, in the short arm of
the human chromosome 3 are among the earliest molecular changes occurring during the
tumor developments in lung, breast, kidney, and other organs (Hung et al. 1995, Kok et al.
1997, Wistuba et al. 2000, Zabarovsky et al. 2002). Investigation on this chromosomal region
has identified many tumor suppressor gene candidates (TSGs) in the gene-rich 3p21.3
subregion (Sekido et al. 1998; Sundaresan et al. 1998). Identification of nested 3p21.3
homozygous deletions in small cell lung cancers and a breast cancer line directed positional
cloning efforts to a 630-kb region, which was subsequently narrowed to a 120-kb subregion
by a breast cancer homozygous deletion (Sekido et al. 1998, Lerman and Minna 2000). The
120-kb region contains 8 putative TSGs, one of which is the 101F6 gene. 101F6 mRNA is
widely expressed in tissues and the mouse mRNA was especially abundant in liver, kidney,
and lung (Mizutani et al. 2007) while the human protein was most abundant in liver, placenta,
and lung (Lerman and Minna 2000).
Elevated expression of 101F6 in tumor cells significantly inhibited cell growth; and
intratumoral injection of recombinant adenovirus-101F6 gene vectors as well as systemic
administration of protamine-complexed adenovirus-101F6 gene vectors significantly
suppressed tumor xenograft growth (Ji et al. 2002). Ohtani et al. (2007) recently found that
nanoparticle-mediated 101F6 gene transfer and a sub-pharmacological concentration of ASC
synergistically and selectively inhibited tumor cell growth by caspase-independent apoptosis
or autophagy both in vitro and in vivo. The C-terminal myc-tagged mouse 101F6 protein was
expressed in Chinese hamster ovary (CHO) cells, and immunofluorescence microscopy was
used to localize the recombinant proteins; they were found in small vesicles, including
endosomes and endoplasmic reticulum of the perinuclear region (Mizutani et al. 2007). It was
also shown that CHO cells expressing the myc-tagged mouse 101F6 protein showed higher
ferric ion and azo-dye reduction level than the control CHO cells. Mizutani et al. (2007)
concluded that mouse 101F6 proteins played roles in the ferri-reduction via a yet unresolved
mechanism. These results clearly show the tumor suppressor activity of 101F6 protein.
At the level of genomic and predicted protein sequences, a human tumor supressor
protein (the 101F6 gene product) has been identified as a putative member of the cytochrome
b561 (Cyt-b561) protein family (Lerman and Minna 2000, Ponting 2001, Tsubaki et al.
2005). The mouse homologue was also discovered, sequenced, and shown to be 85% and
95% identical with the human sequences on the cDNA and protein sequence level,
respectively (Lerman and Minna 2000). Both the human and the mouse 101F6 genes were
Physico-Chemical Properties of the Putative Tumor Suppressor Protein, 101F6
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found to encode a protein consisting of 222 amino acids (Figure 1). Both proteins have 6
trans-membrane α-helices, the central four of which make up the ‗cytochrome b561-core‘, the
N- and C-termini in the cytoplasm, and 4 well-conserved histidine residues for binding two
heme-b prosthetic groups, one on each side of the membrane.
The adrenal cytochrome b561, as a representative and the most studied protein of this
family, is a highly hydrophobic protein with a molecular mass of 28 kDa and its expression is
localized in the secretory vesicle membranes of adrenal chromaffin cells. This protein is
involved in a trans-membrane electron transfer reaction from cytosolic ascorbate (ASC) to
intravesicular monodehydroascorbate (MDA) radical that replenishes reducing equivalents to
maintain physiological levels of ASC inside the vesicles (Kobayashi et al. 1998, Seike et al.
2003). For the efficient electron transfer, the adrenal gland cytochrome b561 contains a
putative ASC-binding motif on the cytosolic side and a putative MDA-radical binding motif
on the intravesicular side (Okuyama et al. 1998), respectively (Figure 1). Comparative
analysis on the amino acid sequences of seven subfamilies of the cytochrome b561 protein
family showed that 101F6 protein does not contain the putative MDA-radical binding motif
and the ASC binding motif was significantly modified (―modified motif 1‖; Tsubaki et al.
2005).
These results suggested that redox active biofactor(s) other than ASC or MDA radical
might be responsible for the redox activity of the 101F6 protein. It is very intriguing to
consider that the 101F6 protein has a role for trans-membrane redox signaling via unknown
redox-linked activity. Clarification of biophysical and biochemical properties of the 101F6
protein is highly essential for understanding the role of this trans-membrane protein as a
candidate for the tumor suppressor activity in cancer.
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Figure 1. Multiple alignment of bovine (Bt) CGCytb, mouse (Mm) TSCytb, and human (Hs) TSCytb by
using Clustal W (v. 2.0.1) software (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Predicted transmembrane segments are bold faced, underlined, and obtained by using HMMTOP (http://www.enzim.
hu/hmmtop/). The highly conserved His residues binding the two hemes are labelled in pairs with stars
(* and +). Gray triangles (▲) show the 10 places where the mouse and human TSCytb are different.
The putative ASC-binding site (box with full line) and monodehydroascorbate binding site (box with
dashed line) are shown on the bovine protein. While the former one is present, in modified form, in the
TSCytb proteins, the latter one is absent in them.
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2. Expression Systems
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Except for the cytochrome b561 protein localized in the adrenal gland chromaffin granule
membrane, all members of the Cyt-b561 protein family are present in a very small amount in
cells and tissues. For the biochemical and biophysical characterization of these proteins in
detail, high yield expression system to produce the recombinant protein in study might be
required. In the case of 101F6 protein, potent expression systems for the mouse and the
human protein have recently been established (Bérczi and Asard 2008, Recuenco et al. 2009).
While the His6-tagged mouse 101F6 protein was expressed in Saccharomyces cerevisiae
(Bérczi and Asard 2008), the His8-tagged human ortologue was expressed in Pichia pastoris
(Recuenco et al. 2009). The His8-tagged human 101F6 protein contains a thrombin-specific
cleavage sequence (LVPRGS) between the wilde-type 101F6 protein moiety and the His8-tag
making thus possible the removal of the tag after the His-tag affinity purification step. The
name of TSCytb will be used for the recombinant 101F6 proteins. In order to distinguish the
native chromaffin granule cytochrome b561 from its recombinant form, the recombinant
protein will be called as CGCytb (see also i.e. Bérczi and Asard 2008).
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3. Biochemical Properties
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In the case of a putative electron transporting protein, the value of midpoint potential of
redox chromophores, the nature of electron donors and acceptors, and the oxidation-reduction
mechanism(s) should be considered as the most characteristic biochemical properties from the
point of view of their biological functions.
3.1. Midpoint Redox Potentials
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Presence of two heme b prosthetic groups with different midpoint redox potential (E0')
values is the most characteristic aspect of the Cyt-b561 proteins. Using a broad range of redox
mediators and sodium dithionite, the redox titrations under anaerobic conditions resulted in a
set of experimental values that could be best approximated by a Nernst-equation containing
two independent one-electron redox centers. Although the primary structure of the mouse and
human TSCytb proteins differ only by 10 amino acids, the obtained E0' values showed
somewhat significant alterations. While the two E0' values were +140 mV and +40 mV for the
mouse TSCytb (Bérczi et al. 2010), they were +109 mV and +26 mV for the human protein
(Figure 2) (Recuenco et al. 2013). Although the purification protocols for the two His-tagged
proteins differred at some points and the TSCytb proteins were obtained in two different
detergent micelles (sucrose monolaurate for the mouse protein, octyl-β-D-glucoside for the
human protein), redox titrations were performed under similar conditions (50 mM phosphate
buffer, pH 7, 10% glycerol, anaerobic condition, redox mediators, dithionite titration). There
were 90~100 mV differences between the two E0' values for both proteins, which is, however,
in agreement with the results obtained for other members of the Cyt-b561 protein family
(Flatmark and Terland 1971, Takeuchi et al. 2001, Nakanishi et al. 2009a, 2009b, Bérczi et al.
2005, 2007, Liu et al. 2007).
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Figure 2. Determination of the midpoint redox potentials of the purified human TSCytb. Experimental
results were analyzed on the basis of the Nernst equation. If two independent one-electron redox
components were assumed to exist, the approximation resulted in the midpoint potentials of +109 mV
and +26 mV (solid line) for the high-potential and the low-potential hemes, respectively. If only a
single one-electron redox center was assumed to exist, the approximation resulted in a midpoint
potential of +63 mV and an improper fit (dotted line).
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3.2. Reduction by Ascorbate and Dihydrolipoic Acid
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Ascorbate-reducibility is the most well-known characteristic of Cyt-b561 proteins.
Purified TSCytb proteins are in their fully oxidized state by the end of purification protocols,
if no ASC is included in buffers. Gradual increase of the concentration of ASC in the medium
up to 100 mM reduced the mouse TSCytb and resulted in an ASC-dependent reduction
profile (Figure 3) that could not be explained (and mathematically described) by assuming a
single ―apparent affinity‖ (or ―binding‖) constant but a mathematical equation with two such
constants resulted in a satisfactory approximation (Bérczi and Asard 2008). This result is in
full agreement with those obtained for other Cyt-b561 proteins (i.e. Bérczi et al. 2005, Bérczi
and Asard 2006, Kamensky et al. 2007), although TSCytb does not have the so-called ―ASCbinding motif‖ (Tsubaki et al. 2005).
It should be noted that effective binding of ASC has never been proved with any of the
members of the Cyt-b561 protein family. Mentioning ―apparent affinity constants‖ (or
―binding constants") in the mathematical descriptions are misleading. These parameters are
namely the ―midpoint ASC concentrations‖ that characterize the redox transition of the hemeb centers in Cyt-b561 proteins while ASC concentration in the medium increases. For the
mouse TSCytb, 0.25 mM and 7 mM are the two midpoint ASC concentrations (Bérczi et al.
2013). These values are about 5-10 times higher than those obtained for the mouse CGCytb
(Bérczi et al. 2006).
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Figure 3. ASC-dependent reduction of purified mouse TSCytb in detergent micelles at pH 7. The
experimental points cannot be approximated by assuming only one interaction site between ASC and
TSCytb (dashed line) but rather good fit can be obtained by assuming two interaction sites (continuous
line). The two interactions are characterized by the cH and cL parameters (ASC concentrations), where H
and L refer to the high-potential and the low-potential heme-b centers, respectively.
Figure 4. Reduced-minus-oxidized difference spectra of purified mouse TSCytb in detergent micelles at
pH 7 by different reducing agents. Dithionite (DTH), ASC, and DHLA concentrations were 1 mM, the
concentration of the other reducing agents were 10 mM. DTT refers to dithiothreitol.
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3.3. Reduction by Dithionite and Auto-Oxidation
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Detailed spectrum analysis of the α-band (absorption profile between 545 nm and 575
nm) in the spectra of the dithinite-reduced and the ASC-reduced Cyt-b561 proteins revealed
that the heme-b that is characterized by the higher E0‘ value was reduced first; thus the hemeb center with the higher E0‘ value can be characterized by the lower midpoint ASC
concentration (Bérczi et al. 2013). This heme is called the high-potential (HP) heme, while
the other one is called the low-potential (LP) heme.
The mouse TSCytb can be reduced not only by ASC and dithionite but also some dithiol
reagents (Bérczi et al. 2013). Reduced pyridine nucleotides (NAD(P)H) or glutathione (GSH)
could not reduce this protein more than 2%, as compared to the reduction by dithionite, even
above 10 mM concentration of these reducing agents (Figure 4). Dihydrolipoic acid (DHLA)
and dithiothreitol however reduced the mouse TSCytb rather efficiently. It was shown earlier
that DHLA could reduce the mouse CGCytb more efficiently than ASC did
(Lakshminarasimhan et al. 2006).
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There are two indications that TSCytb is an auto-oxidizable protein. First, if no ASC is
present in the buffer, or if the ASC-containing buffer is replaced by an ASC-free buffer by a
fast desalting chromatography step at the end of the purification protocol, the highly purified
TSCytb is always in its fully oxidized form.
Secondly, when the protein was reduced by addition of dithionite, the reduced state of
TSCytb was lost after a while and the fully oxidized state appeared (Bérczi et al. 2010). The
higher the concentration of dithionite was, the longer the protein stayed in its reduced state.
This phenomenon was not observed, if reduction of TSCytb by dithionite was carried out
under anaerobic conditions (under continuous streaming of humidified nitrogen or argon gas
in the cuvette).
Since administration of dithionite not only reduces TSCytb but also depletes dioxygen in
the medium, the only difference between the experiments under aerobic and anaerobic
conditions was the possibility of re-airation of the medium under aerobic conditions. Under
conditions used in the experiments, dithionite was always present in great excess to TSCytb;
in such cases and in concentrations applied, it is assumed that SO2¯ serves as reducing agent
(Mayhew 1978). How re-airation resulted in re-oxidation of the reduced TSCytb was left for
further studies.
This result even does not allow speculating whether hydrogen peroxide generation could
occur; a chemical agent that plays a central role in killing cancerous cells (Chen et al. 2005,
Du et al. 2010).
4. Biophysical Properties
Biophysical properties of TSCytb include a wide range of parameters that can be
obtained by using different spectroscopic methods and which characterize either the whole or
just a part of the TSCytb molecule. These properties can be either kinetic (dynamic) or
steady-state parameters and mostly refer to some structural feature of the protein under study.
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4.1. UV-Vis Spectroscopy
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The fully oxidized TSCytb has a sharp absorption band, the so-called Soret band, with
absorption maximum at 417 nm and a wide, very shallow absorption band between 500 nm
and 600 nm with two hardly resolvable absorption maxima, in the visible wavelength region
(Figure 5). Addition of ASC causes a characteristic change and the reduced spectrum of
TSCytb has three characteristic bands. The Soret band maximum shifts to 427 nm, and the
absorbance increases. Parallel with this change, two new bands with absorption maximum at
529 nm (β-band) and 561 nm (α-band) show up. Both bands are asymmetric and have more
than one component. Detailed spectrum analyses of the α-band of the reduced spectrum of the
ASC-reduced TSCytb revealed the presence of two, distinct, split α-bands (Bérczi et al. 2010,
2013). These results point to the fact that the two b-type hemes are located in a very
anisotropic electrostatic field. The anisotropy might origin from the presence of charged
amino acid side chains in the vicinity of the hemes. Different side chains result in different
local electrostatic fields which can explain the minor differences between the shapes of the
split α-bands (Bérczi et al. 2013).
Similar results were obtained when mouse TSCytb was reduced by dihydrolipoic acid.
Concentration-dependent reduction of TSCytb by reducing agents and the detailed spectrum
analysis of the asymmetric α-band of the reduced spectra provide us a rather sensitive
experimental tool to detect the changes that affect the electrostatic field in the vicinity of
heme pockets.
Figure 5. UV-visible absorption spectra of purified human TSCytb in oxidized (solid line), ascorbatereduced (dotted line), and dithionite-reduced (broken line) states. Inset shows a result of pyridine
hemochrome assay of the purified sample, indicating the presence of heme B with 1.59 (±0.06) mole of
heme B/mole protein.
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4.1.1. Stopped-Flow Analysis on the Reduction by Ascorbate
The fast reduction process of oxidized human TSCytb with ASC was analyzed by a
stopped-flow method and was found to be independent of pH (Recuenco et al. unpublished).
It is in contrast to those observed for chromaffin granule and Zea mays Cyt-b561 proteins in
which both cytochromes exhibited very slow rates at pH 5.0 but faster at pH 6.0 and 7.0,
although the electron transfer rates were not significantly slower than those previously
reported (Takigami et al. 2003, Nakanishi et al. 2009a). This might be reasonable, if we
consider the significant difference in the sequence of the putative ―ASC-binding site‖. The
well-conserved Lys, Arg, and Tyr residues among other members of the Cyt-b561 family
(Tsubaki et al. 2005), which are considered to be important for the interaction with ASC
(Nakanishi et al. 2009a, Rahman et al. 2012), were replaced with Ala, Ser, and Phe residues,
respectively, inferring that ASC might not be a physiological electron donor of human
TSCytb or ASC might utilize a different electron transfer mechanism.
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4.1.2. Effects of the Modification with DEPC
DEPC is well known as a chemical modification reagent with high selectivity toward a
de-protonated nitrogen atom of the imidazole ring of His residues. It was shown previously
that DEPC-treatment of bovine adrenal CGCytb in chromaffin granule membranes or in
detergent-solubilized and purified state caused a significant inhibition of the electron transfer
from ASC (Njus and Kelly 1993, Kipp et al. 2001, Njus et al. 2001, Tsubaki et al. 2000) both
in the final reduction level (30~35%) and in the initial reaction rate (~1/400). The inhibition
was caused mainly by specific N-carbethoxylation of the heme axial His residue, with the
bond between the heme and the axial His residue remaining intact (Takeuchi et al. 2001,
Takigami et al. 2003). This observation was central to the proposal of ―histidine cycle
mechanism for the concerted proton/electron transfer‖ from ASC to the heme iron of adrenal
CGCytb (Nakanishi et al. 2007, da Silva et al. 2012). Such specific inhibition on the electron
transfer from ASC by the DEPC treatment was observed for other Cyt-b561 proteins (Preger
et al. 2005, Nakanishi et al. 2009b, Cenacchi et al. 2012). Very interestingly, such inhibition
on the electron transfer from ASC was not observed for human TSCytb at all (Recuenco et al.
2013), despite the fact that more than half of total His residues were modified. This
observation suggested that human TSCytb might use a different physiological electron donor
other than ASC or might use a different electron transfer mechanism, as suggested in the
previous section.
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4.1.3. Pulse Radiolysis Analysis on the Reactions with
Monodehydroascorbate Radical and Ascorbate
Pulse radiolysis experiments were performed with an electronic linear accelerator as
described by Kobayashi et al. (1998). The human TSCytb sample solution with 10 mM ASC
was radiated with a pulse beam to generate monodehydroascorbate (MDA) radical. Oxidation
of the reduced heme of human TSCytb with pulse-generated MDA radical and following rereduction of oxidized heme with ASC were monitored by absorbance changes at 430 nm and
405 nm. The second order rate constant for the electron donation from the ASC-reduced
TSCytb to the pulse-generated MDA radical was found to be 5.0 x 107 M-1s-1 (Recuenco et al.
unpublished), about two-fold faster than that of bovine CGCytb (Kobayashi et al. 1998) and
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about five times faster than that of Zea mays Cyt-b561 (Nakanishi et al. 2009a), suggesting
that human TSCytb is very effective for scavenging MDA radicals in cells.
In a later time-domain of the pulse-generated reactions, re-reduction of oxidized heme
with ASC occurred, as observed earlier for other Cyt-b561 proteins. The second order rate
constants for the reaction of the MDA radical-oxidized form of TSCytb with ASC showed
significant pH-dependency (Recuenco et al. unpublished), which was very similar to the
corresponding re-reduction process of adrenal CGCytb (Kobayashi et al. 1998). Further, the
second order rate constant value at pH 6.5 was probably about 3~4 times faster than those of
corresponding values for the reaction of oxidized TSCytb with ASC obtained by stoppedflow technique (Recuenco et al. unpublished), which did not show any significant pHdependency. These discrepancies are apparently due to the difference in the starting point of
the electron transfer reaction. For the re-reduction phase of the MDA radical-oxidized form of
TSCytb, an ASC molecule might be already bound at a certain site of TSCytb, being ready for
a rapid electron transfer to the oxidized heme on the intravesicular side. On the other hand, in
the stopped-flow experiments, the heme reduction processes might be governed by several
factors; approaching of ASC to the catalytic site of TSCytb protein, ―binding of ASC‖ at the
catalytic site, and electron transfer from ASC to the oxidized heme by an unknown
mechanism.
4.2. EPR Spectroscopy of the Heme-b Centers
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EPR spectroscopy is a very sensitive technique for studying the properties of molecules
having unpaired electrons, particularly for the metal centers including heme iron. It provides
information on the coordination structure and electronic state of the metal centers by
analyzing the energy needed for the transition of unpaired electrons between the two spin
states. The information in an EPR spectrum is obtained as gmax or gz value. The larger the g
value is, the further the unpaired electron is from the free electron state (ge=2.0023). Both
hemes in TSCytb have unpaired electrons when the molecule is in its fully oxidized state. In
the fully reduced state, however, there are no unpaired electrons in TSCytb. The transition
between the two redox states can be followed not only by UV-Vis spectroscopy (as discussed
above) but also by low-temperature EPR spectroscopy.
The two b-type hemes in Cyt-b561 proteins have two distinct EPR signals. The two btype hemes in mouse TSCytb are characterized by gz=2.96 and gmax=3.61. While the latter
value is in good agreement with results obtained for other Cyt-b561 proteins, the former value
is significantly lower than the values between gz=3.13-3.16 obtained earlier for other Cytb561 proteins (Tsubaki et al. 1997, Takeuchi et al. 2004, Bérczi et al. 2005,2007, Liu et al.
2005,2007, Nakanishi et al. 2009a). EPR spectra of human TSCytb in oxidized state at 5 K
showed only a highly anisotropic low-spin (HALS) signal at gz=3.75. However, at 15 and 20
K, another HALS-type signal appeared at gz=3.65 being overlapped with that of gz=3.75.
These two HALS-type signals showed distinct power dependency (Recuenco et al. 2013). The
rhombic EPR signal at gz=3.16 previously seen in other Cyt-b561 proteins was not observed,
neither in the detergent-solubilized purified state nor in the microsomal membrane state. This
observation suggested that one of the heme irons would have distinctly different heme
environment from those of usual Cyt-b561 members and might be easily converted from a
HALS-type to a rhombic-type heme (or vice versa).
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4.3. Resonance Raman Spectroscopy of the Heme-b Centers
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Resonance Raman (RR) spectroscopy is a technique that provide information for the
motional freedom of characteristic chemical groups and/or bonds. Heme-b centers in the
mouse TSCytb (Bérczi et al. 2010) have already been subject for RR spectroscopy.
Resonance excitation occurred in the Soret band at 413.1 nm, close to the absorption
maximum of TSCytb in the Soret band (see above). The most prominent bands characteristic
for the different skeletal vibrational modes of the hemes, both for the oxidized and the
reduced forms, are located at almost the same frequency (wavenumber) values as those found
for the protein-matrix-free heme-b model complex (imidazole)2-protoporphyrine IX
{[(Im)2Fe3+PP] + and [(Im)2Fe2+PP] +, for the oxidized and reduced compounds, respectively;
Choi et al. (1982), Choi and Spiro (1983)}. These results are consistent with the presence of
fully-oxidized and/or fully-reduced, hexa-coordinated, low-spin heme-b centers. High
similarity holds also for the strongest bands characterizing the stretching and in-plane motions
of peripheral groups on the porphyrin ring. The presence of these RR bands attributable to
motions of the vinyl groups verifies the presence of b-type hemes in TSCytb. There was no
indication that would suggest a structural difference between the two heme-b centers in
TSCytb. The RR results support the view that both heme-b centers are in a rather relaxed
state; e.g. no constraint from the surrounding amino acid side chains can be observed that
would result in deformation of the porphyrin ring of heme-b centers. These results are
essentially very similar to that reported for the resonance Raman measurements for bovine
adrenal chromaffin granule cytochrome b561 (Takeuchi et al. 2004).
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5. Structure-Function Relationship
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It was generally assumed for decades that the function of a protein was closely linked to
its three-dimensional structure (Fetrow and Skolnick 1998). After identifying some
intrinsically unstructured proteins (IUPs) with well-defined biological function(s), however, it
became necessary to change or at least modify the old paradigm (Wright and Dyson 1999).
The Cyt-b561 protein family was identified on the basis of sequence similarity to the amino
acid sequence of bovine chromaffin granule cytochrome b561 (Asard et al. 2001, Verelst and
Asard 2003, Tsubaki et al. 2005). The biological function of this protein is transferring
electrons across the adrenal gland chromaffin granule membrane from the cytosolic ASC to
the intravesicular semidehydroascorbate (SDA) (Kent and Fleming 1987, Seike et al. 2003).
According to the structure-function paradigm, TSCytb should have biological function(s) and
physico-chemical properties similar to those of the chromaffin granule cytochrome b561.
However, the classical sequence-to-structure-to-function paradigm that was originally worked
out for globular and soluble proteins can hardly be applied for TSCytb. As it has been pointed
out, very basic structural (6 trans-membrane helices, 2 b-type hemes, highly conserved His
residues) and functional (ASC reducibility) properties of TSCytb are common in the Cytb561 protein family, but the biological function of different members of the Cyt-b561 protein
family show some variation (McKie et al. 2001, Griesen et al. 2004, Asard et al. 2013). The
biological function of TSCytb seems to be coupled to tumor suppression; some experiments
suggest that ASC might also be involved. ASC has been used in cancer treatment for many
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years in complementary and alternative medicine practices without having a clear action
mechanism (Cameron and Campbell 1974, Cameron and Pauling 1978, Chen et al. 2005,
Padayatty et al. 2010). Its action mechanism(s) seem(s) to involve hydrogen peroxide
generation (Chen et al. 2005, Du et al. 2010), inhibition of cell cycle progression (Belin et al.
2009, Frömberg et al. 2011), gene expression regulation (Belin et al. 2010), ATP depletion
(Chen et al. 2012), but somehow all end up at autophagy of cancer cells. How the biophysical
and biochemical properties of the tumor suppressor 101F6 protein prevail in this function will
be subject for further studies. Progress of X-ray crystallographic studies on TSCytb would be
extremely helpful for understanding the biological activity of this protein.
Acknowledgments
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One of the authors (A.B.) expresses his thanks to the Hungary-Romania Cross Border
Cooperation Program of the EU (HURO/0901/219) for the financial support. M.T. expresses
his thanks to Grant-in-Aid for Scientific Research (C) (22570142) from Japan Society for the
Promotion of Science for the financial support.
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Reviewed by emeritus professor Takashi Iyanagi (Department of Life Science, University
of Hyogo, Japan).
ISBN: 978-1-62808-665-2
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Chapter 2
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The Inhibitor of Growth (ING) Gene
Family: Potential Use in Cancer
Diagnostics and Therapy
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Mehmet Gunduz1,, Eyyup Uctepe1, Catherine Moroski Erkul1,
Senol Dane2 and Esra Gunduz1
Department of 1Medical Genetics, 2Medical Physiology,
Faculty of Medicine, Turgut Ozal University, Turkey
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Abstract
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Attempts to explain precisely how ING gene functions have been modified in tumors
have produced a number of theories; among these are loss of heterozygosity (LOH),
promoter CpG hypermethylation and protein mislocalization.
While ING transcript levels are often down-regulated in tumor cells, mutations in
these genes are very rare. However, it is known that inactivation of ING family genes at
genetic and epigenetic levels plays a major role in the tumorigenesis of many different
tumor types.
As a family of putative tumor suppressor genes, ING may prove to be of value as a
target for diagnosis and molecular therapy in a variety of cancers. Recent studies have
demonstrated that ING genes may play a role in regulating the response of cancer cells to
chemotherapeutic agents. Despite this compelling evidence, there are few in vitro studies
currently in the literature that investigate the potential use of ING family genes in gene
therapy.
Advancements in knowledge of ING family gene function(s) as well as their
interaction with p53 and other as yet unidentified molecules will shed light on their role
in the development of human cancers, and aid in determining their potential for use in
cancer diagnostics and therapy.

Corresponding author: Mehmet Gunduz, MD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut
Ozal University, Turkey. Anadolu Bulvari 16A Gimat Ankara, Turkey. Tel:+90-312-3977400/7221. Fax:+90312-221 3670. E-mail: [email protected].
18
Mehmet Gunduz, Eyyup Uctepe, Catherine Moroski Erkul et al.
Keywords: Tumor suppressor gene, ING1, ING family, cancer, gene therapy
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Introduction
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The first member of the Inhibitor of Growth (ING) family was discovered by Riabowol‘s
group in 1996 via subtractive hybridization [1, 2]. Designated ING1, this gene has been
mapped to chromosome 13 at locus 13q33-34 and encodes a 33-kDa protein (p33ING1b).
ING genes have been reported in many species including human, mouse, rat, cow, zebra fish,
venous, yeast, etc., highlighting their importance in central biological processes [3].
Our group characterized the genomic structure of human ING 1 [4]. Subsequently it is
determined that it has four exons and three introns. The ING1 gene can be alternatively
spliced to generate p47ING1a, p33ING1b, p24ING1c and p27ING1. The loss of ING 1 gene
expression has been demonstrated in numerous cancer types. Furthermore, ING 1 knockout
mice were found to be cancer prone [5]. Subsequently, four additional human ING genes
(ING2-5) were identified. By homology studies, ING2 (also known as ING1L) was cloned
[6]. It was mapped to chromosome 4 at locus 4q35.1 and encodes a single variant. In 2003,
ING3was identified through a computational domain search. p47ING3 encodes a 46.8-kDa
protein and contains a C-terminal plant homeodomain (PHD) finger motif [7]. It has been
mapped to chromosome 7 at the locus 7q31 and as a result of distinct splicing, encodes two
variants.
The other members of the ING family, ING4 and ING5 were also identified in 2003
through using a computational sequence homology search [8]. The ING4 gene was mapped to
chromosome 12 at locus 12q13.3 and encodes 8 variants as a result of alternative splicing.
The ING5 gene was mapped to chromosome 2 at locus 2q37.3 and encodes a single variant.
Also, an ING-like pseudogene, designated INGX, was identified and mapped to the X
chromosome at locus Xq12.9 [9].
As the nomenclature used for the multiple isoforms of the different ING family genes
aroused confusion in the literature, a new nomenclature was reported to standardize the
naming of the various ING genes and their transcripts [10].
This review summarizes the most recent research on ING family genes in relation to their
tumor suppressor function and potential role in cancer therapy.
a
All ING proteins are characterized by a highly conserved C-terminal domain that is also
commonly found in various chromatin remodeling proteins [11]. They have a high degree of
homology with one another, but the N-terminal region of each ING protein is unique and
determines each ING member‘s specific role(s) [4, 12] (Figure 1). There is at least one
nuclear localization sequence (NLS) and plant homeo domain (PHD) finger found in the Cterminal domain.
Additionally, they contain a nuclear conserved region (NCR), which can also direct the
ING proteins to the nucleus. This domain might specifically participate in binding to histone
acetylase (HAT) and histone deacetylase (HDAC) complexes. There are three nucleolar
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ING Family Structure
The Inhibitor of Growth (ING) Gene Family
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targeting signals (NTS) in ING 1 and ING 2. Two of them target ING 1 to nucleoli in
response to different stress.[9, 13] ING members also have other motifs such as a 14-3-3
binding motif, polybasic region (PBR), PCNA-interacting protein (PIP) bromodomain (PBD)
and leucine zipper like (LZL) motifs. [14] The PIP motif interacts with PCNA in a DNA
damage inducible manner.
Decreased expression of ING1b causes decline in PCNA monoubiquitination and
sensitizes cells in response to UV during S phase [15]. Also it was shown with nuclear
magnetic resonance study that ING1b PIP interacts PCNA with a very low affinity suggesting
ING1b PIP motif have not a second aromatic residue generally role in the canonical PIP motif
[16]. The14-3-3 family, which recognizes 14-3-3 binding motifs, and targets proteins to
different subcellular localizations.
The N-terminus of p33ING1b includes both a PIP and PB motifs, which control proteinprotein interactions during chromatin remodeling. Phylogeny studies have shown that ING1
and ING2 proteins and ING4 and ING5 proteins have an overall high homology with one
another and, therefore, could have closely related or redundant functions. Several studies have
shown that ING proteins play a significant role in multiple critical cellular processes such as
growth regulation, senescence, apoptosis, DNA repair, cell migration, etc [17-19].
p33ING1b was originally described as interacting physically with the tumor suppressor
protein p53 and as being necessary for p53 transcriptional activity. Thus, intact p33ING1b is
necessary for the efficient negative regulation of cell proliferation by p53 [20]. Further studies
have shown that all the ING proteins may have functions in [21] cell cycle arrest, apoptosis
and senescence [8, 20, 22-26]. However, a note of caution is necessary as studies on ING1
knockout mice and knockout cells derived from these mice suggest that in physiological
conditions, the function of ING1 proteins may be mostly independent of p53 signaling
pathways [5, 27].
Figure 1. ING proteins and their basic functions.
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The ING Family and Their Biological Functions
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ING family proteins regulate a variety of critical cellular and signaling processes such as
growth regulation, apoptosis, DNA repair, DNA demethylation [28], angiogenesis, cell
migration, tumorigenesis, cellular senescence, negative regulation of cell proliferation [1, 29,
30], chromatin remodeling [20, 31], hormone responses [32, 33] and regulation of tumor
growth via NF-kB (34) and hypoxia inducible factor pathways [35, 36]. They can form
complexes with HATs and HDACs [17]. In addition, suppression of ING proteins has been
shown to increase cell migration and to inhibit contact inhibition. Some studies have shown
that most of the ING proteins are important for proper p53 function [17, 21]. Although more
recent mouse model experiments indicate otherwise [27] p33ING1b has been shown to
physically interact with p53 and to have an essential role as a cofactor for p53-mediated cell
regulation and apoptosis [17, 18, 21]. Studies have also indicated that ING proteins are
involved in cell cycle checkpoints and cell cycle progression [17, 21].
ING1 expression is significantly repressed in 44% of human primary breast cancers and
100% of established breast cancer cell lines [18]. Decreased ING1 expression has been found
in many other forms of solid and blood tumors [37-41]. Similarly, the expression of ING2,
ING3 and ING4 is reduced in human melanomas [42-44]. All ING family proteins have been
shown to cooperate with p53 to induce apoptosis and cellular senescence [45-49] and, as a
result, the notion that the ING family proteins act as class II tumor suppressors has emerged.
It was recently shown that ING1 binds to the DGCR8 promoter, a protein involved in the
early steps of microRNA biogenesis, and controls its transcription through chromatin
regulation [50]. Eapen et al. characterize a mechanism by which ING2 contributes to muscle
differentiation.
In structure-function analyses, they found that the leucine zipper motif of ING2 drives
ING2-dependent muscle differentiation. By contrast, the PHD domain, which recognizes the
histone H3K4me3 epigenetic mark, blocks the ability of ING2 to induce muscle
differentiation [51]. Helbing et al. demonstrated that ING proteins modulate T helper
dependent immune responses and revealed a novel role for ING proteins in hormone
signaling which can influence disease states. They suggest that the induction of ING proteins
may facilitate TH receptor function during transformation in a tissue-specific manner [52].
Researchers have identified loss of heterozygosity (LOH), reduced mRNA expression,
loss of nuclear protein expression and mutation of ING genes in different tumors and tumor
cell lines (Table 1). Inhibitor of growth-4 has been shown to have a role in innate immunity.
It promotes IkappaB promoter activation thereby suppressing NF-kappaB signaling and
innate immunity.[53] Zhang et al. showed that in murine embryonic fibroblasts (MEFs)
derived from (interaction partners of Inhibitor of cyclin A1) Inca1 (+/+) and Inca1 (-/-) mice,
overexpression of ING5 suppressed cell proliferation only in the presence of INCA1, while
ING5 had no effect in Inca1 (-/-) MEFs. ING5 overexpression triggered a delay in S-phase
progression, which required INCA1. In aggregate, ING5 overexpression accelerated Fasinduced apoptosis in Inca1 (+/+) MEFs, while Inca1 (-/-) MEFs were protected from Fas
antibody-induced apoptosis [54].
ING2 was recently established as a novel regulator of spermatogenesis, functioning
through both p53- and chromatin-mediated mechanisms, suggesting that anHDAC1/ING2/
H3K4me3-regulated, stage-specific coordination of chromatin modifications is very
21
ING Family Genes and Human Tumors
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important for normal spermatogenesis, and provides an animal model to study idiopathic and
iatrogenic male infertility.
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Gene rearrangements in ING1 have been identified in a neuroblastoma cell line and
decreased expression is common in primary tumors and cell lines [1, 12]. Our group
characterized the genomic structure of the human ING 1 gene and subsequently identified its
tumor suppressor role for the first time by demonstrating its chromosomal deletion at 13q34
and tumor-specific mutations in head and neck squamous cell carcinoma (HNSCC) [4]. There
are many studies investigating the mRNA expression status of ING family genes in different
human cancers. Toyama et al. examined mRNA expression in breast cancer patients and
found 2 to 10 fold decreases in 44% of the tumors tested [18]. In addition, most of the breast
cancers exhibiting decreased ING 1 expression had metastasized to regional lymph nodes. In
comparison, only a subset of these cancers, which had increased ING1 expression, as
compared to adjacent normal tissues, were metastatic [18]. Other studies have also found
down-regulation of ING 1 mRNA in different cancer types such as lymphoid malignancies,
lung cancer, brain tumors and esophagogastric carcinomas, however no comprehensive
clinical correlation has been carried out [12, 17, 55] Rare missense mutations in ING 1 have
been identified in esophageal carcinomas [39] and colon cancer cell lines [56] while no
mutations have been found in studies of leukemias [40, 57], oral cancers [58] and lymphoid
malignancies [37]. Although mutations are not common, reduced expression of ING 1 mRNA
has been detected in breast cancers [13, 45], gastric cancers [56] and lymphoid malignancies
[37].
Some theories have been postulated to explain how changes in ING gene functions occur
in tumors, such as loss of heterozygosity (LOH), promoter CpG hypermetylation and protein
mislocalization [12, 17]. Using methylation-specific PCR, the p33lNG1b promoter was
determined to be methylated and silenced in approximately a quarter of all cases of primary
ovarian tumors [59]. Recent studies failed to find differences in ING expression in myeloid
leukemia or melanoma [40, 60].
Reduced ING2 expression has been detected in lung cancer, melanoma, colon cancer and
hepatocellular carcinoma (HCC) and was associated with tumor progression and a reduction
in overall survival [61]. Our group has also identified frequent deletions of the ING2 locus at
4q35.1, which are associated with advanced tumor stage in HNSCC [62]. ING2 may have a
role in melanoma initiation, since decline in nuclear ING2 has been demonstrated in the radial
and vertical growth phases in metastatic melanoma as compared with dysplastic nevi. [61].
These epidemiological studies suggest that ING2 loss or reduction may be essential for tumor
initiation and progression [1].
The third member of the ING family was recently identified (ING3) by our group [102].
ING3 allelic loss and reduced expression was identified in a limited number of HNSCC cases.
In our study a missense mutation at codon 20 of ING3 was identified but was very rare,
suggesting that there may be other inactivation mechanisms at play, perhaps via
transcriptional mechanisms like promoter methylation. Whatever the precise mechanism,
ING3 down-regulation is a significant step in the process of carcinogenesis [102].
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Table 1. ING Status and Aberrations in Human Malignancies
References
[41, 63]
[64]
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Mutation/Aberration Status
Loss of nuclear expression
Decreased mRNA expression
Silent and missense mutation
Astrocytoma
Basal cell carcinoma
Missense mutation
Bladder cancer
Brain tumors
Missense mutation, overexpression
Breast carcinoma
Decreased protein and nuclear expression/Decreased
mRNA expression, mutation
Colorectal carcinoma
Esophageal carcinoma
LOH, mutation
Gastric carcinoma
Hepatocellular carcinoma Decreased mRNA expression/Missense mutation ,LOH
HNSCC
LOH, mutation
Laryngeal squamous cell Mutation
carcinoma
Melanoma
Loss of nuclear expression/Missense mutation
Meningioma
Missense mutation
Neuroblastoma
NSCLC
Nonsense and missense mutation, Decreased mRNA
expression, LOH
Osteosarcoma
Nonsense and missense mutation
Ovarian carcinoma
Pancreatic carcinoma
LOH, mutation
Papillary thyroid
carcinoma
Seminoma
Skin basal cell carcinoma Missense mutation, overexpression
Lung cancer
No mutation/Decreased mRNA expression
Melanoma
Decreased nuclear expression
HNSCC
LOH
Breast carcinoma
LOH
HNSCC
Decreased or no mRNA expression/Missense mutation
[65]
[66]
[67]
[1, 67, 68]
[18, 41,
63]
[69, 70]
[39]
[56]
[4, 71, 72]
[12]
[73]
,I
Type of Tumor
ALL
Adenocarcinoma (EGF)
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ING1
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ING4
ING5
Melanoma
Ameloblastoma
Renal cell carcinoma
Brain Tumors
Breast carcinoma
HNSCC
Melanoma
Ovarian cancer
Lung cancer
Gastric adenocarcinoma
Colorectal Cancer
Myeloma
Hepatocellular carcinoma
HNSCC, Oral cancer
Decreased nuclear expression
LOH
LOH
Mutation, Chromosomal deletion
LOH, decreased mRNA expression
LOH, decreased mRNA expression
[19, 74]
[75]
[1, 32, 67]
[76, 77]
[78]
[59]
[79]
[63]
[63]
[66]
[80]
[66]
[62]
[81]
[3, 82, 83]
[83, 84]
[85]
[86]
[34, 87]
[88-90]
[6, 91]
[92, 93]
[94]
[95]
[96]
[97]
[98]
[99]
[100, 101]
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In another study, we found that down-regulation of ING3 occurred more frequently in
late-stage tumors as compared with early-stage patients and patients with reduced ING3
mRNA expression showed worse survival rates as compared to the patients with normal-high
ING3 expression [82].
ING4 mRNA levels have been found to decreased in glioblastoma and related with
advanced tumor progression [34]. Increased expression of IL-8 and osteopontin (OPN) has
been associated with reduced ING4 in myeloma [98]. In these reports, higher tumor grade and
increased tumor angiogenesis was accompanied by decreased ING4 expression. Also,
higherinterleukin-5 and osteopontin expression was identified in myeloma [98]. Expression of
ING4 was lower in malignant melanoma as compared to dysplastic nevi and was determined
to be an independent factor contributing to poor prognosis in these patients [92]. Another
study revealed that ING4 prevented the loss of contact inhibition and unrestrained growth.
Finally, some mutations and deletions were found in cell lines originating from human
cancers including breast and lung cancer [88].
We previously reported reduced ING4 expression and LOH in HNSCC [103]. Analysis
of LOH was performed at the12p12-13 region in 50 HNSCC tumors and allelic loss was
estimated at 66% in the informative cases. There were noING4mutationsdetected in these
patients. It was recently reported that ING4 may be an important regulator of chromatin
acetylation [104]. It was shown that adenovirus-mediated ING4 expression can inhibit tumor
invasion and angiogenesis, thereby causing suppression of lung carcinoma cell growth [104].
It was demonstrated that ING4 physically interacts with a HAT, p300, and p53, which
causesp53 acetylation. ING4 accelerated the transit of cells through the G1/S phase of the cell
cycle, p21 promoter activity and protein expression and apoptotic ratio in cells with wild type
p53, however the effect of ING4is lower in cells with mutant p53 [3, 21, 105, 106]. However,
no direct relationship with p53 mutation status and HNSCC patients has been determined in
our study [103]. Nagahama et al. demonstrated up-regulation of ING4 in a human gastric
carcinoma cell line (MKN-l) by triggering mitochondria-mediated apoptosis via the
activation of p53 [107].
Our group has also determined that the ING5 chromosome locus is implicated in a human
cancer. In our study, using 16microsatellite markers on the long arm of chromosome 2q2137.3, we found a high ratio of LOH in oral cancer [100]. Based on these preliminary findings,
ING 5 appears to be a strong candidate tumor suppressor; however, it should be noted that
several other candidate TSGs such as ILKAP, HDAC4, PPPI R7, DTYMK, STK25, and
BOK are also located in this area, where frequent deletions have been found [100]. Still, our
ongoing studies have shown mutation and reduced expression of ING5 mRNA in oral cancer
tumors, supporting its tumor suppressive role in cancer. A summary of alterations of ING
genes in human tumors is shown in Table 1.
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ING Family and miRNA
In certain cancers, including lymphoid malignancies, lung cancer, gastric cancer, brain
tumor, ING1b and ING2a expression loss could be involved in the tumor initiation and/or
progression. It is demonstrated that since miR-622 causes to decrease the ING1 mRNA
expression by targeting the ING1 3′UTR in gastric cancer suggesting decreased ING1b or
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ING2a mRNA could be the result from misregulation of microRNAs targeting ING1b or
ING2a mRNA [108].
Also certain mi-RNA‘s, such as miR-203, are epigenetically regulated by ING1b and
may repress cancer cell proliferation by downregulating CDK6, c-Abl and Src. [109]
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Using ING Family Genes in Gene Therapy
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Importance of ING Family Genes in Molecular
Diagnostics and Therapy
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As noted above, ING transcript levels are often down-regulated in cancer cells but
mutations are very rare. Despite the lack of mutations, it is known that the inactivation of ING
family genes at genetic and epigenetic levels plays a major role in the tumorigenesis of
various cancers. ING family genes may therefore be of value as a target for diagnosis and
molecular therapy in a variety of different tumors.
There are only a few in vitro studies in the literature in which gene therapy with ING
family members is investigated. First, the application of ING1 was reported as a pioneering
approach for the treatment of cancer in 1999 [110]. Adenovirus-mediated introduction of ING
1 was able to suppress the growth of glioblastoma cells, and when combined with p53
transduction, there was a synergistic enhancement of apoptosis in these cells [110]. It is
therefore suggested that ING1 may itself function as a pro-apoptotic factor as well as
enhancing the effect of p53.
Another study reported the combined usage of ING1 and p53 in esophageal cancer [111].
Co-expression of ING1 and p53 accelerated cell death as compared to each gene transcript
expressed separately in esophageal carcinoma cells. Hence, the synergistic effect between
p33ING1 and p53 in the induction of apoptosis has been demonstrated in two different human
cancers, i.e., esophageal carcinoma and glioblastoma. Considering these two in vitro studies,
combined gene therapy of one or more ING family members with or without p53 has
emerged as a potentially useful alternative therapy when the use of p53 alone does not elicit a
strong enough response.
Another recent study showed that the ING1 splicing isoform p47ING1a is differentially
up-regulated after cisplatin treatment in human glioblastoma cells (LN229). This may be part
of a DNA damage response triggered by this platinum-containing chemotherapeutic agent.
[112]. Increasing ING1 expression may promote tumor progression by enhancing the
progression of cells through G1 and thus promote apoptosis more rapidly in response to
cisplatin. LN229 cells express ING1 proteins and harbor mutated TP53. It was suggested that
ING1 expression levels, independent of p53 status, might predict the relative sensitivity of
glioblastoma to treatment with cisplatin and HDAC.
It is suggested that combined detection of p33ING1, p53, and Beclin1 genes and proteins
will be useful for early diagnosis and prognosis evaluation for NSCLC, and can give
experimental evidence for biotherapy of NSCLC [113]. It is found that the efficacy of 5azacytidine is increased by ING1b suggesting it could be used a therapeutic agent in breast
cancer [114]. Expression of p33ING1b is increased by Azidothymidine (AZT), regulating
possibly senescence and apoptosis of the TJ905 glioblastoma cells [115].
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Figure 2. Possible effects of ING genes in gene therapy.
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These results show that gene therapy withING1 could be added to chemotherapeutics in a
subset of human cancers (Figure 2).
It was recently demonstrated that ING4 may be effective as a novel anti-invasive and
anti-metastatic agent in gene therapy for human lung carcinoma [104]. Adenovirus-mediated
ING4 expression repressed tumor growth and cell invasiveness in A549 lung cancer cells.
These results suggest that ING4, as a potent tumor suppressing agent, may provide significant
therapeutic possibilities. Xie et al. reported an interesting finding when they showed that
ING4 could down-regulate the expression of MMP-2 and MMP-9, possibly by repressing the
NF-kB pathway, in lung cancer cells [104]. Moreover the inhibitory effect of ING4 on MMP2 and MMP-9 activities may help to repress melanoma cell invasion [92]. Overexpression of
lNG4 significantly reduced melanoma cell invasion by 43% and repressed cell migration by
63%. In A549 cells, treatment with Ad·ING4 caused down-regulation of the expression of
MMP-2 and MMP-9.These findings demonstrate that ING4 could repress the degradation of
ECM and basement membrane components. In this regard, the relationship between ING4
and the MMP pathway may present novel opportunities for molecular therapy of cancer.
Loss of ING4 may promote microvessel formation and play a role in facilitating the
development of ovarian cancer. One study showed that ING4 mRNA and protein were
significantly down-regulated in ovarian cancer patients as compared to normal controls. ING4
expression correlated negatively with stage and histological grade of ovarian cancers.
Although the specific mechanisms are not yet understood, our data suggest that ING4 may be
a promising target for the treatment of ovarian cancer [94].
Zhao et al. demonstrated that treatment with either ING4 or 125I radiotherapy could
trigger Panc-1 pancreatic cancer cell growth suppression and apoptosis in vitro. Importantly,
they also showed that in mouse xenograft models, both treatments could inhibit tumor growth
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and angiogenesis of Panc-1 pancreatic cancer cells. Furthermore, the combination therapy had
a synergistic effect [116].
It has emerged that, both in vitro and in vivo, the synergistic anti-tumor activity exhibited
by Ad-ING4-IL24 closely cooperative activation of extrinsic and intrinsic apoptotic pathways
and reduced production of the pro-angiogenic factors VEGF and IL-8. Hence, results show
that combining two or more tumor suppressors such as ING4 and IL-24incancer gene therapy
may constitute a novel and effective therapeutic strategy for cancers [117]. Zhu et al. showed
that in vitro adenovirus-mediated ING4 and IL-24 co-expression in A549 lung carcinoma
cells had an additive effect on growth suppression and triggered apoptosis. There was also a
combined effect on up-regulation of P21, P27, Fas, Bax and cleaved Caspase-8, -9, and -3 and
down-regulation of Bcl-2. In an in vivo xenograft experiment, A549 lung carcinoma cells
were transplanted subcutaneously into athymic nude mice. Subsequent Ad-ING4-IL-24
treatment had an additive effect on tumor growth inhibition and decreased CD34 and
microvessel density [118].
In SMMC-7721 hepatocarcinoma cells, Ad-ING4 plus CDDP (cisplatin) was shown to
trigger synergistic growth inhibition and accelerated apoptosis. Together they had an additive
effect on up-regulation of Fas, Bax, Bak, and down-regulation of Bcl-2 and Bcl-X (L). An
increase in cleaved Bid, cleaved caspase-8, caspase-9, caspase-3 and cleaved PARP was also
observed. Importantly, as with the above-mentioned lung xenograft model, when SMMC7721 cells were subcutaneously injected into athymic nude mice, a synergistic suppression of
tumor growth, reduced tumor vessel CD34 expression and decreased microvessel density was
observed. These results suggest that Ad-ING4 plus CDDP is a potential combination
treatment strategy for hepatocarcinoma [119].
Reduced ING4 nuclear and cytoplasm expression were both seen in lung cancer and
correlated with tumor grade. Interestingly, compared with normal tissues, tumors have higher
ING4 expression in the cytoplasm than in the nucleus. Nuclear ING4 inhibition correlated
with tumor stage and lymph node metastasis. Lack of nuclear ING4 correlated with tumor
stage and lymph node metastasis. ING4 expression was lower in grade III than in grades I-II
tumors and reduced ING4 mRNA correlated with lymph node metastasis. Overall reduction
of ING4 expression and high cytoplasmic ING4 expression (relative to nuclear expression)
may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4
expression may be useful as a clinical diagnostic and prognostic tool for lung cancer [119].
Li et al. demonstrated that Ad-ING4 gene transfer transfectiontomutantp53 MDA-MB231 breast carcinoma cells results in a G2/M phase cell cycle arrest and apoptosis. In this
model, P21, P27, and Bax were up-regulated andBcl-2, IL-8, and Ang-1 were downregulated. Furthermore, this promoted cytochrome c release from mitochondria into the
cytosol and caspase-9, caspase-3, and PARP were activated. Intratumoral injections of AdING4 in nude mice bearing mutant p53 MDA-MB-231 breast tumors markedly inhibited
tumor growth and reduced CD34 expression in tumor vessels and also reduced microvessel
density. Thus, ING4 is also a potential candidate for breast cancer gene therapy.
Borkosky et al. revealed that a significant relationship exists between LOH of D2S 140
(ING5 locus) and solid tumors. LOH of ING3MS (ING3 locus) was also high in solid tumors,
showing a near significant association. In addition, a notable tendency toward higher LOH for
half of the markers was observed in recurrent cases. LOH of ING family genes is a common
genetic alteration in solid ameloblastoma. In work published by Nozell et al. ING4was found
to interact physically with NF-kB, causing a decline in p65 phosphorylation, p300 protein
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levels, as well as reduced levels of acetylated histones and H3Me3K4, and an increased
amount of HDAC-l [120]. It was also demonstrated that ING4 interacts with p65 and can inhibit NF-kB-induced COX-2 and MMP-9 expression, genes which are known to be regulated
by NF-kB and play a role in gliomagenesis [120]. Some research has proposed the notion that
the transfer of ING4 into cancer cells by gene therapy could also target molecules that it
associates with, such as MMPs and COX-2. Hence, combined treatment with inhibitors of
MMP and COX-2 with ING4 gene delivery could be a potent treatment in some cancer types.
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HATs and HDACs have a role in regulating the activity of diverse types of non-histone
proteins, such as transcription factors and signal transduction mediators. The imbalance
between HAT and HDAC levels has been determined in tumors, with particular emphasis on
the activity of HDACs human cancers. This has led to the emergence of HDAC inhibitors
(HDACI) as a new class of molecular targets for cancer therapy [4, 12, 121, 122].
Acting through their PHD domains, ING proteins can alter chromatin structure by
acetylation or de-acetylation of HAT and HDAC complexes, respectively [2, 12, 55]. ING
proteins interact with these complexes, which are involved in key cellular processes such as
DNA repair, senescence, angiogenesis, apoptosis and tumorigenesis [4, 12, 55, 123]. Because
HDACs are part of a transcriptional complex that affects different tumor suppressor genes
and it was demonstrated that HDAC inhibitors trigger cell growth arrest and apoptosis in
cancer, ING gene products could act as a bridge to bring HAT/HDAC proteins into the
molecular targets for the development of enzymatic inhibitors to treat human cancer [21, 121,
122, 124].
HDACIs alter extracellular matrix and ECM related proteins and have a role in cell
migration, invasion and metastasis [124]. It has been shown that HDACIs up-regulate some
metastasis suppressor genes such as TIMP 1, RECK and tetraspanin and are also able to
down-regulate some metastasis activating genes such as MMP-2, MMP- 9 and metastasis
associated proteins MTAI, MTA2 and TGFB1.It is known that degradation of the ECM by
MMPs is a key step in tumor cell invasion and metastasis. Thus, combined therapy consisting
of HDACIs together with ING gene delivery could be promising for cancer therapy.
Nagahama et al.reported that [107] forced expression of run-related transcription factor
(RUNX3) in a gastric carcinoma cell line up-regulatedING1 and ING4, both of which have a
significant role in the regulation of apoptosis. RUNX3-inducedup-regulation ofING1 and
ING4 accelerated mitochondria-mediated apoptosis via the activation of p53 and suppression
of HIF-1. Down-regulation of TXN2 and HSPD1, which suppresses cytochrome c release
from the mitochondria, was also observed. This work indicates that ING genes are candidate
targets for treatment of cancer either through gene therapy or molecules such as HIF-1.
ING4 was proposed as a suppressor of HIF-l and repressor of angiogenesis [35, 98, 125].
HIF-1 is one of the major regulators of hypoxia-responsive genes such as VEGF, IL-8,
angiopoetins and OPN [98]. Reduced ING4 expression has been accompanied by upregulated expression of IL-8 and OPN in myeloma cells. Myeloma cells also produce OPN
via the activation of the Runx2/Cbfa1 gene [98]. This finding indicates the potential
involvement of the NF-kB pathways other than Runx2/Cbfa1 in OPN production. It was also
showed by Colla et al that ING4 interacts with the HIF- I -regulating critical enzyme, a HIF
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prolyl hydroxylase (HPH-2), which is responsible for the hydroxylation of HIF-1, enhancing
its proteosomal degradation. Both ING4 and HPH-2 are up-regulated under hypoxic condition
which indicates that both molecules are essential for the regulation of HIF-1 activity. The
inhibition of ING4 in myeloma cells also increasesHIF-1 activity and NIP-3 expression. This
finding indicates that ING4 has a role in the antigenic process and exerts an inhibitory effect
on the production of HIF-l, IL-8 and OPN. Besides this, one of the ING4/HAT complex
subunits, JADE1 (gene for apoptosis and differentiation in epithelia) is stabi1ized by von
Hippel Lindau (VHL) tumor suppressor activity [126]. The association of ING4 with HAT as
well as HIF-1 may suggest that we focus our attention on identifying the physiological
complex in which ING4 is found for investigating new therapeutic candidates.
Melanoma antigen A2 (MAGE-A2) was found to interact with and suppress p53 by
recruiting HDAC to p53 transcription sites [127]. The association between MAGE-A2 protein
expression and resistance to apoptosis was confirmed. The combined treatment of melanoma
cells with trichostatin A (TSA) and etoposide (ET) contributed to the p53 response and
chemoresistance was reduced.
A recent study reported an interaction between the lamin interaction domain (LID) of
ING proteins and lamin type V intermediate filament proteins [128]. Lamin type V
intermediate filament proteins are thought to play a role in nuclear stability. Lamins also
cooperate with oncogenes such as β-catenin. They demonstrated that laminA binds ING1,
thereby adjusting its cellular levels and activity, which suggests that ING proteins function as
bridges between chromatin and the nuclear lamina. As ING proteins are stoichiometric
components of HAT and HDAC complexes [3], this connection between ING and lamin A
provides a new clue for exploring the role of ING genes in cellular senescence and
tumorigenesis. Known roles for ING proteins in regulating apoptosis and chromatin structure
show that loss of lamin A-ING interaction may be an indicator of lamin A loss, thereby
contributing to laminopathies and tumor progression [21, 129-132]. Hence further elucidation
of this link may be helpful for the development of novel therapies.
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Using ING Genes as Cancer Biomarkers
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Sub-Cellular Localization of ING Genes as a Biomarker
In the regulation of cell cycle and proliferation, the shuttling of proteins between nucleus
and cytoplasm is a critical step. Many TSGs such as p53, BRCA1 and ING1 carry out some
of their functions through nucleo-cytoplasmic shuttling. Any impairment in the nuclear cargo
system abolishes the subcellular localization of the TSGs and causes cancer development
through the mis-localization and altered function of TSG proteins [123]. The mis-targeting of
tumor suppressors can lead to direct cellular consequences and potentially trigger initiation
and progression of cancer. These kinds of irregularities, which lead to the mis-localization of
tumor suppressors have been identified for p53, BRCA1, APC, and ING1.In the tumor
suppressor or their partners are generally present during the process of carcinogenesis [123,
133, 134].
Neuman et al. demonstrated that in the development and progression of melanomas,
translocation of p33lNG 1b from the nucleus to the cytoplasm of melanocytes may have an
important role [74]. Cytoplasmic ING 1b immunostained with newly developed monoclonal
antibodies (MAb) against GN1 and GN2 was related with malignant melanoma and may be
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an early sign of malignancy, thus indicating that ING1b Mab could be useful in diagnostic
approaches.
It was shown by Vieyra et al. that subcellular mis-localization of p33 ING1b is a
frequent occurrence in glooms and glioblastomas [75]. Overexpression and abnormal
localization of ING1b were identified in all 29 of the brain tumors studied. p33ING1b
includes a nuclear targeting sequence [12, 20]. It has been shown that altered subcellular
localization of p33lNG 1b abolishes its proapoptotic functions [135]. Loss of targeting factors
which ensure the correct intracellular localization of p33ING 1b or that are required for the
physical interaction between ING 1and p53 could be responsible for the aberrant localization
of p331NG 1b in tumors. New experimental observations, such as post-translational
stabilization of p53 by p33INGlb [136] and the discovery of the p53 cytoplasmic anchoring
parkin-like ubiquitin ligase (PARC) [137] and its p53-regulatory role shore up the possibility
that the interaction of ING1 with p53 could cause the observed aberrant localization.
Nuclear localization of ING1 is essential for its proper activity. ING 1b protein
phosphorylated at serine residue 199 will bind 14-3-3 proteins and subsequently be
transported from the nucleus to cytoplasm [138]. Some consequences of nuclear-cytoplasmic
transport of ING proteins are presented. Shifting of p33ING1b from the nucleus to the
cytoplasm, where the protein is tethered by 14-3-3, take parts in tumorigenesis and
progression in HNSCC [139]. It was determined recently by the Riabowol group that ING 1
binds karyopherin proteins and that interruption of this interaction influences localization and
activity of ING1 as a transcriptional regulator [138]. The karyopherin/importin family
fuctions as adaptors by binding directly to both the NLS of a cargo protein via one domain
and to karyopherin β via a second distal region.
A novel binding partner of ING4, liprin alpha 1, was recently identified [140]. Liprin
α1/PPFIA 1 (protein tyrosine phosphatase, receptor type polypeptide) is a cytoplasmic protein
necessary for focal adhesion formation and axon guidance interaction with ING4. This causes
suppression of cell spreading and migration. Liprin may function in guiding ING4 to its
cytoplasmic location. Cytoplasmic ING4 interacts with liprin α1 to control cell migration and
with its known anti-angiogenic function, may inhibit invasion and metastasis. All these
molecules that are interrelated with the sub-cellular localization of ING proteins could be
determined with different molecular and histopathological methods and may be used as a
biomarker to characterize the behavior of the tumor.
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ING Gene Expression Alterations as Prognostic Markers
Changes in expression of mRNA and/or protein of the ING family genes in various
cancers, paves the way to their potential use as biomarkers. A recent study investigated such
an association between p331NG1b protein expression and clinical outcome in colorectal
cancer. They found that patients whose tumors had normal p33ING1b protein expression
have a longer overall and metastasis-free survival rate as compared with patients with
decreased p33 ING1b protein expression, but the difference did not show statistical
significance [70, 141]. On the other hand, an important correlation between p53 mutation
status and overall and metastasis-free survival has been determined.
Another study demonstrated allelic loss of ING1 as a novel genomic marker associated
with progression to glioblastoma by using comparative genomic hybridization and DNA
microarray [141]. Also, it was reported by Takahashi et al. that low levels of ING1 mRNA
were significantly associated with poor prognosis in neuroblastoma [68]. Takahashi et al.
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also demonstrated that the expression level of ING1 was closely correlated to survival time.
In aggregate, these findings suggest that reduced levels of ING1 mRNA and/or protein
expression could be an indicator of poor prognosis in advanced stages and/or poor survival
for different human cancers.
Zhang et al. showed that reduced ING2 mRNA as well as protein expressions were
accompanied by tumor progression and shortening at survival in HCC. These epidemiological
studies proposed that ING2 loss or reduction may be significant for tumor initiation and/or
progression [61]. Also, our group demonstrated that high LOH frequency in ING2 locus at
4q35.1 was statistically associated with advanced T stage in HNSCC, suggesting that ING2
LOH might take place in later stages during HNSCC progression [62]. Thus, the importance
of ING2 in HNSCC carcinogenesis and its potential prognostic significance are compelling
results for future studies.
Our group recently measured mRNA expression of ING3 in HNSCC and compared this
with clinic-pathological characteristics in order to assess its prognostic value as a biomarker
[102, 142]. This study revealed that down-regulation of ING3 was more evident in late-stage
tumors as compared with early stage cases. Analyses have demonstrated that down-regulation
of ING3 may indicate an aggressive form of HNSCC.
The newly found connection of ING4 with survival rates and metastasis may prove to be
a promising prognostic marker in melanoma [92]. It has been determined that ING4
expression was significantly reduced in malignant melanoma compared with dysplastic nevi,
and overexpression of ING4 arrested melanoma cell invasion as compared with controls.
Using ING Genes as Chemosensitivity Markers
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One of the major therapeutic modalities for cancer is chemotherapy. Thus, identification
of the genes that predict the response of cancer cells to these agents is very important for
improving the efficacy of treatments [17]. Recent studies demonstrated that the ING genes
might play a role in regulating the response of cancer cells to chemotherapeutic agents. It was
found that there is a correlation between p33lNG 1b expression and resistance to vincristine, a
mitotic inhibitor, especially in brain tumor cells [143]. Thus, in brain tumors ING 1b mRNA
levels may be a useful marker to predict chemosensitivity. However, in melanoma cells, there
is no association between campthothecin-induced cell death and p33INGlb expression and
therefore would not be a useful predictor [143]. Subsequent studies have shown that over
expression of p33 ING1b in U2OS cells with wt p53 enhanced etoposide-induced apoptosis
[144]. This increase in apoptosis maybe p53-dependent since MG63 cells which are mutant
for p53 did not undergo clear apoptosis following treatment. Also, it was shown that using
taxol (paclitaxel) in the same cell types gave similar results [78, 145, 146], which indicate that
p331NG1b might be a significant marker or therapeutic agent for the treatment of metastatic
osteosarcoma. Conversely, down-regulation of ING1 in LN229, the p53-deficient
glioblastoma cell line, accelerated apoptosis following treatment with cisplatin, illustrating
that decreased ING1 expression could predict the sensitivity of some cancer cell types to
chemotherapy independent of their p53 status [112]. Although most findings showed that
expression of ING genes led to an increase in chemosensitivity, different conditions in
various tumors should be tested to predict accurate chemotherapy response. These differences
could also be linked to expression variations of the alternative splicing forms of ING genes.
31
Conclusion and Future Prospects
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Finally, curcumin , a chemopreventive agent, triggers ING4 expression during cell cycle
arrest by a p53-dependent manner in U251 glioma cells [147]. Thus, ING4 has been proposed
as a player in the signaling pathways of the chemotherapeutic agents.
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Approximately 15 years of studies on the ING1 gene has demonstrated that ING1
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[142] Wang J, Chin MY, Li G. The novel tumor suppressor p33ING2 enhances nucleotide
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ISBN: 978-1-62808-665-2
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Chapter 3
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Multifunction New Tumor Suppressor
Gene Family from Cancer to Metastasis:
A Disintegrin and Metalloproteinases
with Thrombospondin Motifs (ADAMTS)
Kadir Demircan1,, Yunus Emre Bilgen2, Tugrul Celik2,
Yudum Yaral2, Birsen Dogan3, Zisan Akcaaga2,
Zahide Nur Unal3 and Mehmet Gunduz3
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Departments of 1Medical Biology, 2Medical Biochemistry, 3Medical Genetics,
Faculty of Medicine, Turgut Ozal University, Ankara, Turkey
Abstract
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ADAMTSs are in the metallopeptidase family and are zinc-dependent matrix
enzymes. MMP (matrix metalloproteinase) and ADAM (disintegrin metalloproteinase)
are other zinc-dependent proteases are related to ADAMTSs. These proteases have roles
in the process of damage and repair of extracellular matrix (ECM). ADAMTSs exert their
affect by decomposing structural proteins of ECM like collagen, versican and aggrecan.
ADAMTSs are inhibited by TIMP (Tissue inhibitors of metalloproteinase), which are
known to be tissue inhibitors of metalloproteases and α2 macroglobulin, which is a serum
protein. In this chapter, functions and roles of ADAMTS in various biological events are
reviewed.
1. Historical Perspective and Introduction
In 1997, Kuno et al. discovered the ADAMTS proteinases (A Disintegrin and
Metalloprotinase with Trombospondin motif) that was upregulated in tumors [45]. The

Correspondence to: Kadir Demircan, PhD - Department of Medical Biology, Faculty of Medicine, Turgut Ozal
University, Ankara, Turkey. E-mail: [email protected].
46
Kadir Demircan, Yunus Emre Bilgen, Tugrul Celik et al.
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interest in ADAMTS is increasing day by day due to the role they play in the pathology of
many diseases. Another increasing interest in ADAMTS proteinases is that they are also used
in drug development studies for the treatment of diseases like osteoarthritis and that they
prevent tumorigenesis in cancer [24]. Together with 7 ADAMTS-like (ADAMTSL)
members, the 19 mammalian ADAMTS proteases have been discovered [4, 37]. Known
functions of ADAMTS proteases include cleaving of procollagens, cartilage proteoglycans
and von Willebrand factor [18]. Although ADAMTS are present in many tissues in the body,
they are synthesized in the embryonic period as well [38]. ADAMTS proteases play an
important role in physiopathological conditions such as coagulation, arthritis, cancer, tumor
cell invasion and metastasis diabetes, ovulation, tissue remodeling, angiogenesis and turnover
of extracellular matrix proteins [20]. A number of laboratories have focused on this newly
discovered proteinases family, providing new insight into the their roles in normal function
and disease. Many researchers have made efforts to identify target ADAMTS proteinases for
new therapeutic options and ultimately this effort will lead to new drug candidates in the near
future. The aim of this chapter is to provide a current knowledge about ADAMTS proteases
regarding cancer.
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2. ADAMTS Family
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ADAMTSs, which are from the metallopeptidase family, are zinc-dependent matrix
enzymes. MMP (matrix metalloproteinase) and ADAM (disintegrin metalloproteinase) are
other zinc-dependent proteases related to ADAMTSs (Figure 1). The roles of these proteases
in the process of damage and repair of extracellular matrix (ECM) is known [60]. ADAMTSs
exert their affect by decomposing structural proteins of ECM like collagen, versican and
aggrecan. ADAMTSs are inhibited by TIMP (Tissue inhibitors of metalloproteinase), which
are known to be tissue inhibitors of metalloproteases and α2 macroglobulin, which is a serum
protein. TIMP-3 is the only ADAMTS inhibitor currently known [11, 76, 87].
ADAM, ADAMTS and MMP are closely related proteases. ADAMTS and ADAM
proteinases are involved in the group of adamalysin. Generally, if we do not consider some of
the specialized modules, the adamalysin group of proteases [ADAM, ADAMTS and MMP]
consists of different modules [76]. MMP and ADAM proteases contain a transmembrane
region localized to the cell membrane, but ADAMTS doesn‘t have this region [83]. MMPs
are zinc- and calcium-dependent endopeptidases families that degrade the components of the
extracellular matrix. ADAM proteases play a role in the intracellular communication system
and cell-cell adhesion events such as the sperm-egg binding. 23 ADAM genes, and more than
30 MMP genes, have been identified in humans. 19 ADAMTS genes are cloned [4].
ADAMTS are classified according to their tasks (Table 1). For instance, ADAMTS1 and
ADAMTS8 are known to be antiangiogenic agents [63, 88]. These two proteases are currently
the targets in tumor suppression [41, 63]. Although ADAMTS1 take charge in processes like
ovulation, it is a protease that is strongly expressed in heart attacks [18]. In 2009, Hatipoglu et
al. demonstrated that hypoxia induces ADAMTS1 with HIF-1 [31]. While ADAMTS1 is
expressed in many organs, expression of ADAMTS8 has only been shown in the lung.
ADAMTS8, which cannot be expressed in cartilage tissue, is expressed in atherosclerosis in
Multifunction New Tumor Suppressor Gene Family from Cancer to Metastasis
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areas that are rich in macrophage [14, 88, 89]. We also demonstrated, for the first time, that
ADAMTS1, -4, -5 and -9, not -8, -15 were expressed in spinal cord tissues in mouse [15].
Figure 1. Domains and motifs of ADAM, ADAMTS and MMP.
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Table 1. ADAMTS classification and members of the group
CLASS MEMBERS
Aggrecanase family
ADAMTS1, 4, 5, 8, 9, 15, 16, 18
Anti-angiogenic ADAMTS
ADAMTS1, 8, 9
COMP-ADAMTS
ADAMTS7, 12
GON-ADAMTS
ADAMTS9, 20
Procollagen cleavage
ADAMTS2, 3, 14
von Willebrand Factor (vWF) cleavage
ADAMTS13
Orphan ADAMTS
ADAMTS6, 10, 17, 19
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ADAMTS CLASS
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Figure 2. Fibril organization in normal and abnormal stages.
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ADAMTS2, ADAMTS3 and ADAMTS14 are known to be procollagen processing
enzymes [4]. They cleave the collagen enzymatically and lead to the transformation of
procollagen into collagen. ADAMTS2 gene mutations lead to type VIIC Ehlers-Danlos
syndrome. In this syndrome, which was first diagnosed in cattle animals, the propeptide
domain of the Type I procollagen in the dermis cannot be cleaved by ADAMTSs and a
dysfunction in collagen production occurs. As a result, normal collagen fibril formation
cannot be completed and this leads to Ehlers-Danlos syndrome, which is characterized by an
excessively elastic and fragile dermis (Figure 2).
Some ADAMTSs (1, 4, 5, 8, 9, 15, 16 and 18) can enzymatically cuts aggrecan
proteoglycan, which is the main component of cartilage [18]. Therefore, this group is also
called aggrecanase. Aggrecanases cleave chondroitin sulfate proteoglycans such as aggrecan
and collapse ECM integrity. Aggrecanases also cut versican and brevican, which are the ECM
proteoglycans. ECM proteoglycans play important and critical roles in the pathogenesis of
musculoskeletal diseases e.g. osteoarthritis [24,]. In 1999, ADAMTS4 was called
aggrecanase-1 for the first time [84]. In studies on ADAMTS5 with knockout mice, it was
demonstrated that these mice acquire resistance against osteoarthritis. After these two studies,
ADAMTS5 (aggrecanase-2) came to be classified as a major aggrecanase in mice [26, 77].
ADAMTS13 cuts the von Willebrand factor (vWF), which is an important plasma protein
in hemostasis and clotting system. vWF, which enables thrombocytes to sticks to bleeding
point, is a great adhesion molecule that is synthesized from endothelium cells and
megacaryocytes [2]. The vWF, which is reduced to ideal size by ADAMTS13, interacts with
clotting factors like Factor VIII to play a role in clot formation [49]. In Thrombotic
Thrombocytopenic Purpura (TTP) disease, vWF becomes excessively large and sticky as a
result of ADAMTS13 mutations. In these mutations vWF cannot be cut by ADAMTS13 to
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optimal size and leads to hereditary TTP diseases characterized by the intravascular
breakdown of erythrocytes, which are called thrombocyte thrombi, embolisms, anemia and
thrombositopeny [50].
ADAMTS9 and ADAMTS20 are called as GON-ADAMTS proteases [8, 75]. They are
thus named since they have similarities with GON-1 protease of C. Elegans. GON is a
metalloprotease, which is expressed in gonad distal type cells in embryonic development. It
was shown that ADAMTS20 mutations formed belted white-spotting mutants [69]. In mice
with mutations, melanoblast development is defective as a result of defects in neural fissure.
GON ADAMTS have the longest TSP repeats in the ADAMTS family. They have 15 TSP
repeats [20].
Proteases that decompose cartilage oligomeric matrix protein (COMP) are ADAMTS7
and ADAMTS12 [53]. These two ADAMTS are known as COMP-ADAMTSs (Figure 3).
COMP is also known as thrombospondine-5. COMP that can bind calcium of 524 kDa is an
ECM glycoprotein, which is responsible for structural integrity of cartilage and its interaction
with other matrix molecules.
Figure 3. COMP degradation by ADAMTSs.
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3. ADAMTS-Like (ADAMTSL) Proteins
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ADAMTSs lacking proteolytic activity are called ADAMTS-like proteins, (Figure 4)
[46]. They play a role in ADAMTS regulation since they are structurally similar to ADAMTS
proteases and bound to extracellular matrix. Papilin and punctin are examples of ADAMTSlike proteins. It was shown that ADAMTSL2 mutations lead to autosomal recessive
geleophysic dysplasia characterized with high TGF-beta activity. In geleophysic dysplasia,
which is mapped in the 9q34.2 region brachydactyly, shortness, eye anomalies and heart
defects are seen. ADAMTS whose functions are not known or which do not have a known
substrate are called ‗orphan proteases‘. To date, they are ADAMTS6, -10, -16, -17, -18 and 19 [4].
4. Domain Organizations of ADAMTS
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The most distinguishing feature of ADAMTS proteins compared to other ADAM and
MMPs is that they have thrombospondin repeats (TSP) (Figure 1). ADAMTSs do not have an
epidermal growth factor domain and transmembrane module like ADAM proteases.
ADAMTSs are composed of a protease domain, which includes an active enzyme area and an
ancillary domain composed of thrombospondin repetitions. The protease part is composed of
signal peptide, propeptide, catalytic domain and disintegrated-like domains and the ancillary
domains are composed of thrombospondin repetitions, a cysteine-rich domain and a link
domain, which is called a ‗spacer‘. Thanks to propeptide, enzymes do not interact with the
substrate (Figure 5). Enzymes like furin are responsible for the cutting and removal of the
propeptide domain. This enzymatic cutting and activation process is known as the zymogen
activation process.
Figure 4. ADAMTS like proteins.
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Figure 5. Zymogen activation and furin cleavage for ADAMTS activation.
Figure 6. Domain organization of some of the ADAMTS proteases.
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The catalytic domain, which includes the zinc-binding domain, is responsible for enzyme
activity. All ADAMTS proteases have an active motif. Active motif is composed of a
HEXXHXXGXXHD sequence. Here X signifies any amino acid. If there is a mutation in this
motif, catalytic activity is completely lost. The disintegrin-like domain plays a role in matrix
and cell binding processes. They are called this because the amino acid sequence is similar to
the disintegrin motif in ADAM proteases. TSP is an extracellular matrix adhesion
glycoprotein released from thrombocytes. The TSP motif of ADAMTSs, bound to
extracellular molecules like fibronectine, collagen and laminin, play a role in cell-cell and
cell-matrix interaction [44]. The cysteine-rich domain and spacer domain are important for
the mechanisms of substrate specification and settlement in the matrix. In addition to these
domains, some ADAMTSs have unique domains such as GON, PLAC and CUB. For
example, PLAC motif is seen in close to half of ADAMTS family members (ADAMTS2, -3,
-10, -12, -14, -16, -17, -18 and -19). Another difference of ADAMTS13, compared to other
ADAMTSs, is that it has two CUB (complement C1r/C1s–urchin epidermal growth factorbone morphogenetic protein-1) motifs in the C- terminal domain, one being CUB-1 and the
other CUB-2 (Figure 6). In brief, ADAMTS proteins consist of two main parts; these are the
protease domain and the ancillary domain, which is composed of thrombospondin repetitions.
ADAMTS4 did not contain thrombospondin motifs and ADAMTS9 and ADAMTS20 contain
15 TSP motifs. ADAMTS13 has the shortest prodomain sequence. ADAMTS5 and
ADAMTS11 is the same enzyme. ADAMTS4 is the first found aggrecanase and called
aggrecanase-1. ADAMTS5 is aggrecanase-2. ADAMTS is activated by removal of the
propeptide part by the furin enzymes. Zinc is connected with active motif sequence
(HEXXH).
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5. ADAMTS in Health and Disease
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ADAMTS proteases play important roles for cells (Figure 7). Its deficiency or defect can
lead to serious diseases. For example, complete lack of the ADAMTS13 protease is fatal;
mice without the ADAMTS9 gene die in embryonic life, mutations in other ADAMTS genes
lead to serious diseases [1, 5, 13, 17, 19, 22, 23, 25, 30, 36, 40, 43, 52, 55, 64, 66, 68, 70, 72,
79, 85, 95]. When we look at the association between some ADAMTS members and diseases,
we can see that mRNA levels of ADAMTS1 and ADAMTS15 are linked to asthma and that
mRNA levels in these patients‘ pituitaries are low [32, 65, 74]. However, the level of TIMP-3
mRNA, an inhibitor of ADAMTS, is increased. Therefore, it is considered that ADAMTS1
can play role in the renewal of bronchial tissue in asthma. Shute et al reported that fibroblast
growth factor-2 (FGF-2), which is found in high amounts in patients with asthma, is an
important mediator in fibrosis pathogenesis. It is demonstrated that FGF-2 is controlled by
ADAMTS. Why are ADAMTS1 mRNA levels in patients with asthma low? Researchers
consider the hypothesis that the control of ADAMTS1 on FGF-2 is lost, ECM synthesis in
fibroblasts will speed up by FGF-2 stimulation, which will contribute to the renewal of
bronchial tissue. It was also reported that ADAMTS4, ADAMTS9 and ADAMTS15 are
induced in chronic asthma patients. In a recent study, a new role of ADAMTS5, ADAMTS9
and ADAMTS20 proteases were discovered. It was found that these three proteases play a
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role in the removal of cells with apoptosis and cleaning of ECM in embryonic development
[47, 57].
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Figure 7. ADAMTS associated cellular events and linked molecules. ADAMTS is a large enzyme
family, which is involved in many basic physiological processes like extracellular matrix remodeling,
angiogenesis, clotting, ovulation and morphogenesis as well as cancer and inflammation. ADAMTSs
exert various tasks such as a tumor suppressor gene in cancer, angiogenesis, hypoxia, morphogenesis,
apoptosis and inflammation by interacting with nidogen, fibuline, HIF, and NFKB. HRE: Hypoxia
response element, vWF: von Willebrand factor, TTP: Thrombotic Thrombocytopenic Purpura, FGF-2:
Fibroblast growth factor-2, TSG: Tumor suppressor.
Interruption of this process leads to syndactylia. As a result of the large-scale metaanalysis study by Zeggini et al. it was shown that the ADAMTS9 gene is a new locus of gene,
which has a tendency to lead to type 2 diabetes [94].
ADAMTS10 gene mutations lead to Weill-Marchesani syndrome, which is a rare
collagen disease [13]. WMS is characterized with brachydactyly, shortness, eye anomalies
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6. ADAMTS in Cancer Pathogenesis
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and heart defects. It is stated that ADAMTS10 plays an important role in the development of
skin, eye and heart.
As already mentioned, TTP can lead to damages in organs such as kidneys. As a result of
ADAMTS13 mutations there may be clots in the circulation system. Today, more than 65
mutations in the ADAMTS13 gene have been reported. Almost 60% of the mutations are
missense mutations [20, 37, 69].
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With functional studies it was shown that ADAMTS9 played a role as a tumor suppressor
gene in esophageal and nasopharyngeal cancers. However, the ADAMTS9 gene, which is
located at 3p14.2, has a significant relation with esophageal squamous-cell carcinoma
(ESCC) and nasopharyngeal carcinoma (NPC) [54, 92, 95]. It is known that the ADAMTS9
gene is induced with cytokines like IL-1 in human chondrocyte cells [16]. Although
ADAMTS9, discovered in 2000, is expressed in all fetal tissues, it is more expressed in heart,
kidney, lung, and pancreas tissues in adults.
It was reported that aortic anomalies occur in ADAMTS9 deficiency [40]. The
association of the ADAMTS9 gene with metastases was investigated and it was demonstrated
that ADAMTS9 mRNA expression decreased in metastatic tumors [17]. Zhang et al. showed
the significant correlation between ADAMTS9 methylation and loss of expression of
ADAMTS9 in gastric, colorectal, and pancreatic cancers [94].
It is known that ADAMTS18 protease has a close relation with various cancers. It has
been demonstrated that endothelium cells secrete ADAMTS18 and that thrombin induces this
secretion. Thrombin cuts down the ADAMTS18 enzymatically and forms a C-terminal
fragment of 45 kDa and this fragment is regulated in vivo bleeding time and prevents carotid
artery thrombus formation [51, 52].
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7. Mechanism of ADAMTS-Related
Tumorigenesis: Glioma
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Lectin-binding proteoglycans or lecticans such as versican and brevican organize the
central nervous system (CNS) extracellular matrix [2, 3, 9, 10, 33, 56, 58, 61, 91, 93]. These
chondroitin sulfate lecticans are highly expressed in the CNS and limit cell motility in the
CNS as a barrier. Brevican is abundant in the brain. Brain ECM, perineuronal net, is a netlike
structure between neurons and glial cells [2, 71, 78].
The Fosang group suggested that brevican in the perineuronal net may contribute to
neural plasticity. Upregulation of brevican is a hallmark of brain tumors [2, 78].
ADAMTS is responsible for cleavage of brevican, aggrecan and versican in CNS [48].
Brain ECM degradation by ADAMTS has a key role in malignant glioma invasion. In
gliomas, it is known that ADAMTS4 and ADAMTS5 are upregulated. Nakada et al. showed
that ADAMTS5 is overexpressed by glioblastoma and invading cells [61]. Induced
ADAMTS5 promotes invasion of glioma cells through degradation of ECM components.
Brevican is a molecular link between hyaluronan and tenascin-R of the brain ECM.
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Collapsing of the bridges by aggrecanases may facilitate glioblastoma cell invasion. Fibulin-3
is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion
[34]. So discovery of inhibitors of ADAMTS in brain has potential as effective therapeutic
options.
Figure 8. Acting ADAMTS on glioma cells matrix: One possible mechanism of glioma cell invasion by
ADAMTS proteinase activation
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8. ADAMTS Promoter Association with Cancer
and Metastasis
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Recently our group demonstrated that the promoter region of the ADAMTS9 is
associated with lymphatic metastasis [17]. We showed that the number of CA repeats in the
control group was significantly bigger than that in the non-metastatic group suggesting
expansion in CA repeats may not affect tumor formation but may potentially reduce the risk
of metastasis in the tumor micro-environment. ADAMTS9 locates to regions known to be
frequently deleted in breast cancer and expression of ADAMTS9 in breast cancer tissues was
down-regulated [8, 37, 67, 76].
Shimajiri and colleagues investigated CA repeat of the matrix metalloproteinase-9
(MMP-9) gene promoter in esophageal cancer cells (Discussed in the Turkish Journal of
Medical Sciences, In Press). In their study, the CA repeat length was thought to regulate
MMP-9 gene transcription and enzymatic function. MMP-9 plays an important role in tumor
growth, invasion, and metastasis. It is thought that a decrease in the number of CA repeats
induces down-regulation of the MMP-9 transcription [12].
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9. Prognostic Marker?
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Studies in the field of prognostic biomarkers that attempted to predict the course of the
disease have gained considerable importance due to their indisputable value in cancer
surveillance. Genetic factors are involved in carcinogenesis and are known to influence
prognosis. For this purpose, the MMP, ADAM, and ADAMTS gene families are of particular
interest for further study.
We found that a potential relationship exists between the number of CA repeats in the
ADAMTS9 promoter and lymphatic metastasis of breast cancer (Turkish Journal of Medical
Sciences, In Press). Therefore, we hypothesized that the ADAMTS9 gene may renew itself by
adapting to the host cell and imparting the tumor cell with the ability to metastasize through
CA repeat composition.
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10. Is ADAMTS a Player in the Signaling Cascade?
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The transcription factor nuclear factor-kB (NF-kB) plays a central role in regulating
inflammatory and anti-apoptotic responses. NF-kB is composed of homodimers and
heterodimers of the Rel family proteins including p65/RelA, RelB, c-Rel, p50/p105 and
p52/p100.4,5. The prototypical complex corresponds to a heterodimer of p65 and p50
subunits. The p65 subunit is also phosphorylated by the IKK complex during the process of
IkBa‘s degradation. Stimulus-dependent degradation of IkBa results in the translocation of
NF-kB into the nucleus, where it binds to specific binding sites within the promoter of target
genes. We recently demonstrated that NF-kB inhibitors, curcumin and BAY117085,
effectively inhibited ADAMTS9 gene expression at 10 mM concentration. Curcumin has
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been found to inhibit NF-kB-dependent gene transcription in articular chondrocytes (Figure
9) [18].
Conclusion
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Although ADAMTSs play role in proteolytic destruction of extracellular matrix and have
an active role in pathogenesis of many diseases, there is a need for studies for ADAMTS
proteinases. Gene mutation analyses like ADAMTS9, ADAMTS10 and ADAMTS13.
Promoter analyses of ADAMTS genes and gene polymorphism are fields waiting to be
researched.
Thanks to the increasing understanding of the relation of ADAMTS genes with
transcription factors like NF-kB, NFAT and RunX (Figure 10), our knowledge about
ADAMTS proteases will increase day by day. At the same time, many points about
ADAMTS proteinases have not been discovered yet. In this mini-review, we have tried to
help future studies, so we attempted to provide brief general information about ADAMTS and
recent studies on them.
Figure 9. Proposed signaling pathways of ADAMTS by cytokines.
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Figure 10. CA repeats in the promoter region of ADAMTS9. We recently found interaction between
CA microsatellite (cytosine and adenine) repeats in promoter region of the ADAMTS9 gene and breast
cancer lymphatic metastasis (Turkish Journal of Medical Sciences, In Press).
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Chapter 4
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Von Hippel–Lindau (VHL) Gene
and Protein (pVHL): A Member
of the Tumor Suppressor Gene Family
Ferah Armutcu*1, Kadir Demircan2 and Murat Oznur3
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Departments of 1Biochemistry, 2Medical Biology and 3Medical Genetics,
Faculty of Medicine, Turgut Ozal University, Ankara, Turkey
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The von Hippel-Lindau (VHL) tumor suppressor gene, responsible for the several
malignancies, encodes for a major regulator of the hypoxic response by targeting the
transcription factor hypoxia inducible factor (HIF). Inactivation of the VHL gene is
responsible for the development of renal carcinomas, pheochromocytomas and tumours
in several other organs. The gene product (pVHL) is a central component, and loss of
pVHL and subsequent up-regulation of HIF target genes has been attributed to the highly
vascular nature of these neoplasms. As such, systemic functions of VHL likely play
important roles in the development of VHL disease. The primary aim of this review is to
examine and evaluate the current knowledge regarding the pVHL, and the molecular
pathogenesis of VHL disease, with subsequent clinical implications.
1. Introduction and Historical Perspective
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Tumor suppressor genes (TGSs) protect a cell from one step on the path to cancer. Most
of the TGSs associated with familial cancer syndromes can be assigned specific cellular
functions. The von Hippel-Lindau TGS encodes a multifunctional protein, the mutations of
which underlie the genetic defect in the familial von Hippel-Lindau (VHL) syndrome
(Kapitsinou and Haase 2008). Germ-line mutations were detected in patients with VHL
* Corresponding to: Ferah Armutcu, Department of Biochemistry, Faculty of Medicine, Turgut Ozal University
Ankara, Turkey, E-mail: [email protected], or [email protected]
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disease, and it is characterized by the development of highly vascularized benign and
malignant tumors, including hemangioblastomas of the brain, spinal cord, and retina; renal
cell carcinoma; pheochromocytoma. Less frequent VHL gene-related tumors include those
pancreatic cysts and neuroendocrine tumors; endolymphatic sac tumors; and epididymal
cystadenomas (Barry and Krek 2004; Arjumand and Sultana 2012). In 1904, Eugen von
Hippel, a German ophthalmologist, first described retinal lesions in the eye, then Swedish
pathologist Arvid Lindau recognized the association between retinal and cerebellar
haemangioblastomas and also described the presence of visceral lesions in 1926 (Kim and
Kaelin 2004; Arjumand and Sultana 2012).
The term von Hippel-Lindau disease was first used by Charles Davison in 1936. Melmon
and Rosen reviewed the literature on what had come to be known as VHL disease and
suggested clinical diagnostic criteria in 1964, and has been in common use since the 1970s
(Maher, Neumann et al. 2011).
Discovering the VHL gene really started with the development of the tumor suppressor
gene theory and the earliest known mechanism for the pathogenesis of VHL disease is
explained by Knudson‘s two-hit model (Knudson 1971; Knudson 1993). The incidence of
VHL disease is estimated to be 1 in every 36,000 live births. It is inherited autosomal
dominantly trait with a high penetrance, and germline mutations of the VHL gene are
associated with VHL disease (Kim and Kaelin 2004).
2. Discovery and Identification of the VHL Gene
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It is found that VHL gene is located on the short arm of chromosome 3 at position 25.3.
The VHL TSG was characterized in 1993 following positional cloning studies in families
with the familial cancer syndrome VHL disease. More precisely, the VHL gene was located
chromosome 3 (3p25-26) from base pair 10,183,318 to base pair 10,195,353 (Figure 1). The
VHL disease, occurs as a consequence with new mutations accounting for 20% of cases, and
the majority of cases have a demonstrable germ-line mutation of the VHL TSG (Seizinger,
Rouleau et al. 1988; Latif, Tory et al. 1993). The VHL gene has three exons and encodes a
4.7-kb mRNA which is ubiquitously expressed in both fetal and adult tissues. Translation of
the VHL mRNA gives rise to two different protein products secondary to the presence of two
distinct in-frame ATG codons (codon 1 and codon 54), which can both serve as translational
initiation sites. The VHL mRNA encodes two VHL proteins; a full length 213 amino acid
protein (pVHL30) and a smaller protein (pVHL19) that lacks the first 53 amino acids
(Iliopoulos, Ohh et al. 1998; Woodward, Buchberger et al. 2000). In most biochemical and
functional assays, the two proteins behave similarly and unless otherwise noted are referred to
generically as pVHL. pVHL is primarily a cytoplasmic protein but can also be found
elsewhere, including the nucleus, the mitochondria and in association with the endoplasmic
reticulum (Kaelin 2002). Since its identification, considerable insights have been made
regarding the functions of the VHL gene that now appears as a crucial gene influencing many
cellular pathways. In the recent past two decades, the scientific data, not only identified
molecular basis for VHL disease, but also discovered that pVHL, through its oxygendependent polyubiquitylation of hypoxia-inducible factor (HIF), played a central role in the
human oxygen-sensing pathway (Kaelin 2002; Kim and Kaelin 2004). Firstly, in 1988 the
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Von Hippel–Lindau (VHL) Gene and Protein (PVHL)
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VHL gene was mapped to the short arm of chromosome 3 by linkage analysis and in 1993 the
gene was identified as a result of positional cloning strategies performed in VHL kindreds
(Seizinger, Rouleau et al. 1988; Latif, Tory et al. 1993). Subsequently in 1999, Maxwell et
al., showed that pVHL was pivotal for targeting the α-subunit of HIF for O2-dependent
proteolysis (Maxwell, Wiesener et al. 1999). HIF is a heterodimeric transcription factor
consisting of an unstable α subunit and a stable β subunit. Three HIFα genes (HIF-1α, HIF-2α
and HIF-3α) have been identified in the human genome (Semenza 2001).
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Figure 1. The VHL gene is located on the short arm of chromosome (3p25-p26), and protein VHL
product is encoded by three exons.
3. VHL Gene Pathway and HIF-Dependent
PVHL Functions
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The VHL gene pathway is involved in oxygen and energy sensing, and VHL complex
targets HIF for ubiquitin mediated degradation. This is an oxygen and iron sensing
mechanism; when the cell is low in oxygen or iron, the VHL complex cannot degrade HIF
and HIF over-accumulates (Bader and Hsu 2012). Hypoxia-inducible factors are oxygensensitive basic helix-loop-helix transcription factors, which regulate biological processes that
facilitate both oxygen delivery and cellular adaptation to oxygen deprivation. There are 3
HIFα genes in the human genome (HIF-1α, HIF-2α, and HIF-3α). HIF1α is ubiquitously
expressed whereas the expression of HIF-2α is more restricted. HIF-1α and HIF-2α can bind
to specific DNA sequences, hypoxia-responsive elements (HRE), and activate transcription.
Both HIF-1α and HIF-2α have two transcriptional activation domains, the N-terminal
transactivation domain and the C-terminal transactivation domain, which activate target genes
upon DNA binding (Sang, Fang et al. 2002). HIF-1α and HIF-2α share many target genes, but
it is also becoming increasingly clear that some genes are preferentially activated by one or
the other (Kibel, Iliopoulos et al. 1995; Li and Kaelin 2011). When oxygen levels are high, in
the pVHL pathway, HIF-α is hydroxylated on a crucial proline residue by HIF prolyl
hydroxylase (PHD); this process requires molecular oxygen, 2-oxoglutarate, ascorbate and
Fe+2 as cofactors, facilitating HIF-1α binding to pVHL, and proteasomal degradation by the
E3 ubiquitin ligase complex. Namely, presence of oxygen hydroxylates the HIF-α, and
hydroxylation of HIF permits recognition by the VHL complex, which therefore targets these
transcription factors for proteosomal degradation There are at least three PHDs identified to
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date: PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) (Kaelin 2008; Sudarshan,
Karam et al. 2013). Although PHD2 is believed to be the primary hydroxylase for both HIF1α
and HIF2α, other studies indicate that PHD3 may be mainly responsible for HIF-2α
hydroxylation (Li and Kim 2011). Hydroxylation of one or both proline residues within
HIF1α and HIF2α creates a high affinity pVHL binding site. HIF1-α, together with the
constitutively expressed HIF1-β subunit, bind to HRE in gene promoters to regulate the
expression of genes that are involved in energy metabolism, angiogenesis, erythropoiesis, iron
metabolism, cell proliferation, apoptosis and other biological processes (Kaelin 2008). HIF1α and HIF-2α mediate transcription of a number of downstream genes thought to be
important in cancer including vascular endothelial growth factor (VEGF), platelet derived
growth factor (PDGF), and transforming growth factor alpha (TGF-α). pVHL is part of a
multi-subunit ubiquitin ligase complex composed of elongin-B, elongin-C, Cullin-2 and ringbox1 (Rbx1) that targets HIF for ubiquitin-mediated degradation (Kibel, Iliopoulos et al.
1995; Iliopoulos, Levy et al. 1996). Under hypoxic conditions, the PHDs are enzymatically
inactive, and similarly, when the VHL gene is mutated, PHD can not hydroxylate HIF-α,
therefore inactive PHD or mutant pVHL does not bind to HIF-α resulting in HIF over
accumulation (Maxwell, Wiesener et al. 1999; Kaelin 2002), (Figure 2).
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Figure 2. Summary of the VHL functions, and in briefly HIF targets (Hsu 2012).
Early biochemical molecular studies revealed that pVHL forms a multiprotein complex
that includes elongin B and elongin C, and additional components of the complex Cul2 and
Rbx1. Normal pVHL binds to elongin C, which forms a complex with elongin B and cullin-2
and Rbx1 (Kaelin 2008). HIF is a heterodimer comprising the unique hypoxia-inducible
HIF1-α subunit and a second constituvely expressed protein termed HIF-1β (also known as
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the ARNT; aryl hydrocarbon nuclear translocator), and activate transcription of numerous
target genes involved in cell proliferation, angiogenesis, glucose metabolism, apoptosis and
other cellular processes (Woodward, Buchberger et al. 2000). Not binding and stabilized HIF1α, causes form active transcription factor complex with HIF-1β, and regulates gene
expression via the HRE. RNA polymerase II subunit POLR2G/RPB7 is also reported to be a
target of this protein (Maxwell, Wiesener et al. 1999; Arjumand and Sultana 2012). The most
investigated of these targets is HIF-1α, a transcription factor that induces the expression of a
number of angiogenesis related factors (Barry and Krek 2004). The HIF-α protein then
translocates to the nucleus where it dimerizes with HIF-1β and activates the transcription of
target genes. Although there have been over 100 genes identified downstream of HIF, HIF-1α
and HIF-2α are well characterized proteins regulated by pVHL (Maynard and Ohh 2007).
Ultimately, HIF-1α forms heterodimers with HIF-1β and activates transcription of a variety of
hypoxia-inducible genes (VEGF, EPO, TGF-α, PDGF-β) (Table 1). In the same way, when
pVHL is absent or mutated, HIF1-α subunits accumulate, resulting in cell proliferation and
the neovascularization of tumors characteristic of VHL disease (Maynard and Ohh 2007;
Roberts and Ohh 2008). Additionally, pVHL has many HIF-independent functions that are
also relevant to tumor development, regulation of extracellular matrix (ECM), and apoptosis
in certain cell types (Kaelin 2008), (Table 1 and Figure 3).
Table 1. List and examples of HIF-responsive gene products
(Semenza 2003; Li and Kaelin 2011)
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Angiopoietin-4
Angiogenic growth factors
Aurora kinase A
Carbonic anhydrase IX
C-X-C chemokine receptor type 4
Cyclin D1
Epidermal growth factor receptor
Erythropoietin
Folliculin
Fumarate hydratase
Glucose transporters
Hepatocyte growth factor and c-Met
Histone methylases and demethylases
Insulin-like growth factor 1 receptor
Insulin-like growth factor 2
Interleukin-6
Lactate dehydrogenase A
Lysyl oxidase
Platelet-derived growth factor
RTK-like orphan receptor 2
Stromal cell-derived factor-1
Succinate dehydrogenase
Transforming growth factor α
Vascular endothelial growth factor
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ANGPT4
ANGPT-1, -2
AURKA
CA9
CXCR4
CYCD1
EGFR
EPO
FLCN
FH
GLUT1,-3
HGF
HMT/HDM
IGF-1R
IGF-2
IL-6
LDHA
LOX
PDGF
ROR2
SDF-1
SDH
TGF-α
VEGF
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Figure 3. VHL protein interactions with HIF-dependent/independent gene products which play
important roles in tumorigenesis (Semenza 2000; Li and Kim 2011).
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4. Mechanism of VHL Defective Tumorigenesis
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Given the early onset of life of VHL-associated tumours such as retinal haemangiomas,
VHL inactivation is likely to be sufficient for their development. Biallelic VHL inactivation is
also common in nonhereditary hemangioblastomas and clear cell renal carcinomas (ccRCC),
in keeping with Knudson's two-hit model of carcinogenesis (Knudson 1971; Kaelin 2002).
Loss of pVHL function is the most common genetic event associated with sporadic sporadic
ccRCC, and HIF has a master regulator of renal cell carcinoma metabolism. As VHL loss is a
common event in ccRCC, a significant proportion of the current understanding of this tumor
type has been derived from the study of HIF biology. Although there is considerable overlap
in the genes that are transcriptionally regulated by HIF-1α and HIF-2α, in vitro and in vivo
studies have indicated that HIF-2α is the critical HIF for tumorigenesis in clear cell kidney
cancer (Maranchie, Vasselli et al. 2002; Sudarshan, Karam et al. 2013). Clear cell kidney
cancers that express both HIF-1α and HIF-2α exhibit enhanced signaling via the mitogenactivated protein kinase (MAPK) and serine/threonine-protein kinase mammalian target of
rapamycin (mTOR) pathways, whereas clear cell tumors that express only HIF-2α have
elevated c-Myc activity (Gordan, Lal et al. 2008). In addition to effects on angiogenesis and
growth factor expression, HIF has now been shown to have profound effects on cellular
metabolism (Sudarshan, Karam et al. 2013). Individuals with this disorder carry a defective
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copy of the VHL TSG, typically inherited from either parent but occasionally resulting from a
de novo mutation (Barry and Krek 2004). Mutations of the VHL TSG occur in patients with
VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/−RCC).
Furthermore, overexpression of HIF-1α accompanies p53 upregulation, angiogenesis and
metastatic progression in a variety on human tumors, and may occur secondary to loss-offunction muatations affecting the VHL gene in RCCs. Moreover, tumour-associated hypoxia
inhibits HIF degradation by inhibiting PHD directly or through TCA cycle generation of
ROS. HIF-α and its isoforms, HIF-1α and HIF-2α accumulate then translocate to the nucleus
and dimerize with HIF-β. Several clues exist as to why HIF-2α may be more oncogenic than
HIF-1α. First, HIF2α is less sensitive than HIF-1α to the inhibition by FIH-1 and is therefore
more transcriptionally active under normoxia. Second, HIF-1α more than HIF-2α, remains
susceptible to proteasomal degradation in VHL−/− cell lines (Kim and Kaelin 2006). Third,
HIF-2α appears to cooperate with MYC (a protooncogene) to activate MYC transcriptional
targets whereas HIF-1α antagonizes MYC transcriptional activation (Gordan, Lal et al. 2008).
Interestingly, a recent genome-wide analysis of copy number alterations noted that a region of
chromosome 8q encoding MYC is often amplified in both sporadic and VHL disease
associated tumours (Li and Kim 2011).
The HIF-α/HIF-β complex binds to HRE within the promoters of target genes and
thereby regulates transcription of genes involved in cell growth, angiogenesis, anaerobic
glucose metabolism, pH regulation, cell survival/apoptosis, cell proliferation, and other genes
that modulate various cellular functions (Kaelin 2007; Baldewijns, van Vlodrop et al. 2010)
(Figures 1 and 3). Examples of genes up-regulated by HIF in response to hypoxia include
VEGF, PDGF, TGF-α, GLUT1, carbonic anhydrase IX, erythropoietin (EPO), and others, all
of which are potentially important in the development of ccRCC (Levy, Levy et al. 1997;
Iliopoulos, Ohh et al. 1998; Kaelin 2002; Linehan, Pinto et al. 2007). Given the role of pVHL
in oxygen sensing, it is also interesting to note that mutations affecting fumarate hydratase
(FH) and succinate dehydrogenase (SDH) can, like VHL mutations, give rise to hereditary
renal cancer or pheochromocytoma (Baldewijns, van Vlodrop et al. 2010). Downstream HIF1α genes such as VEGF and GLUT1 would therefore be critically important to these cancer
cells for increasing vasculature and increasing glucose transport. The common endpoint
resulting from VHL, FH and SDH mutations is the stabilization of HIF through inactivation
of PDH, driving the transcriptional activation of genes that support tumor growth,
neovascularization, invasion and metastasis (Linehan, Srinivasan et al. 2010). As a result of
mutation or hypermethylation, biallelic VHL inactivation, is common in ccRCCs
(approximately 50% somatically mutated, 10-20% hypermethylated VHL) and sporadic
hemangioblastomas. In the kidney, VHL mRNA was differentially expressed within renal
tubules suggesting that the VHL gene product may have a specific role in kidney
development (Richards, Schofield et al. 1996; Corless, Kibel et al. 1997). On the other hand,
microRNAs (miRNA) regulate gene expression by resulting in direct cleavage of the targeted
mRNAs or inhibiting translation through complementarity to targeted mRNAs (Garzon,
Pichiorri et al. 2007). It has been shown that miRNAs are aberrantly expressed or mutated in
different malignancies, suggesting that they may play a role as a novel class of oncogenes or
TGSs (Fulci, Chiaretti et al. 2007; Baldewijns, van Vlodrop et al. 2010). Lately, miRNA
expression profiling studies identified different expression profiles in RCC. Certain ccRCC
oncogenes (mTOR, VHL, HIF-1α, PDGF-β) were detected as potential targets of miRNAs,
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which were up- or down-regulated in ccRCC (Petillo, Kort et al. 2009; Chow, Youssef et al.
2010). Already, the data obtained in a recent a study supporting a connection between
miRNA-binding site SNPs within the VHL-HIF-1α pathway and RCC risk (Wei, Ke et al.
2012). In brief, more than 100 direct HIF-responsive genes have been described with a
number of these genes active in carcinogenesis (Semenza 2001). Recent evidence has accrued
to indicate that pVHL has functions other than regulation of HIF-related pathways. The
majority of these alternate functions have been discovered through biochemical interactions.
However, gene expression studies also support the notion that there are HIF-independent gene
expression changes induced by VHL loss (Semenza 2001; Li and Kim 2011).
5. VHL Gene-Related Other Clinical Implications
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The VHL TSG encodes a multifunctional protein, the mutations of which underlie the
genetic defect in the familial VHL disease. In addition to VHL gene, hereditary forms of renal
cancers have been related to the following genes, FH, hepatocyte growth factor receptor (cMET), and Folliculin (FCLN) (Allory, Culine et al. 2011). Several alternative functions of
pVHL have been identified that are independent of its role as the substrate-recognition
component of an E3 ubiquitin ligase. These diverse functions also suggest that there may not
be a simple, unified pathophysiological mechanism that can explain the etiology of VHL
diseases. Although cell-autonomous mechanisms in VHL mutant tumors might be explained
by up-regulation of cyclin D1, increased Akt-mTOR signaling and, elevated FGF receptor
signaling and, disruption of cilia formation, down-regulation of p53, and, regulation of Ecadherin and stabilization Jade-1, among others, it is also well established that VHL mutant
cells secret a large repertoire of growth factors and cytokines, including EPO, VEGF, TGF-β,
PDGF, TNF-α, among many others (Bader and Hsu 2012), (Table 1). On the other hand,
cellular senescence is the phenomenon of irreversible growth arrest in response to DNA
damage but is also an important in vivo tumor suppressor mechanism (Kim and Sharpless
2008). Interestingly, it has been recognized that physiological oxygenation can extend the
replicative lifespan of cells in culture, which has typically been attributed to a relative
decrease in the amount of oxidative stress. Several reports have now confirmed that this
phenomenon is at least in part due to stabilization of HIF (Welford, Bedogni et al. 2006; Bell,
Klimova et al. 2007). Failure of pVHL to control these functions may also contribute to
tumour progression and metastasis (Nyhan, O'Sullivan et al. 2008; Baldewijns, van Vlodrop
et al. 2010). pVHL has a critical role in the regulation of the ECM. All VHL disease types
have impaired ECM assembly capabilities and sporadic ccRCC cases also show reduced
fibronectin staining, which highlights the importance of pVHL for this process (He, Liu et al.
2004). pVHL can bind directly to both fibronectin and hydorxylated collagen IV, and
interestingly all pVHL mutants studied to date are defective in this capacity. The inability of
VHL deficient cells to bind ECM components results in ineffective ECM organization that is
not mediated by HIF (Tang, Mack et al. 2006). Recent studies have reported that pVHL
interacts directly with collagen alpha-2(IV) protein (COL4A2) and indirectly with fibronectin
(Grosfeld, Stolze et al. 2007; Kurban, Duplan et al. 2008). It is proposed that fibronectin
interacts directly with COL4A2, which also binds to pVHL (Petrella and Brinckerhoff 2006).
pVHL has also been linked to the regulation of Matrix metalloproteinases (MMPs),
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particularly MMP-2 and MMP-9. MT1-MMP expression is mediated by HIF-2α. Therefore
loss of pVHL up-regulates the expression of MMPs and may promote ECM degradation and
possibly an increase in tumour invasiveness and progression (Struckmann, Mertz et al. 2008).
The primary cilium (microtubule-based structure) is important for sensing signals in the
extracellular environment and has been proposed to be a negative regulator of cell
proliferation. pVHL associates with and is able to stabilize microtubules. This function of
pVHL appears to be independent of its ability to either down-regulate HIF and its ubiquitin
ligase function. Moreover, pVHL's ability to stabilize microtubules is lost in VHL mutations
that predispose to the development of haemangioblastomas and pheochromocytomas, but not
those associated with the development of RCC (Hergovich, Lisztwan et al. 2003). However,
loss of cilia function in the kidney leads to excessive proliferation of tubular epithelial cells,
formation of fluid-filled cysts and kidney failure. pVHL has been localized to the primary
cilia in mouse and human proximal tubule epithelial cells and in mouse distal tubules, and
pVHL controls the orientation of microtubules and can interact with the Par3-Par6-aPKC
complex, which is important for maintenance of the primary cilium (Schermer, Ghenoiu et al.
2006; Thoma, Frew et al. 2007). Furthermore, pVHL's affects on microtubule dynamics is
negatively regulated by its phosphorylation by glycogen synthase kinase 3β (GSK-3β) and
appears to be HIF-independent, although some studies suggest that HIF dysregulation may
play at least a partial role in the loss of microtubule stability imparted by pVHL inactivation.
Interestingly, active GSK-3β itself can promote microtubule stability and cilium maintenance
in a pVHL-independent manner (Hergovich, Lisztwan et al. 2006; Li and Kim 2011).
Analysis of renal cysts from VHL disease patients and ccRCC cell lines demonstrated that
these cell types lacked primary cilium, implicating that pVHL may have an important role in
the regulation of primary cilium (Esteban, Harten et al. 2006; Lutz and Burk 2006). On the
other hand, pVHL has also been identified as a regulator of expression of E-cadherin. Loss of
expression of E-cadherin is a hallmark of the epithelial-mesenchymal transition and is
associated with loss of the cell-cell adhesion capabilities, which can promote tumour
progression. VHL-defective RCC cell lines and patient tissue samples display loss of Ecadherin expression in a HIF-α-dependent manner (Esteban, Tran et al. 2006; Nyhan,
O'Sullivan et al. 2008). Current evidence proposes that loss of pVHL results in the
stabilization of HIF-α, which leads to the transactivation of E-cadherin repressors that act on
the E2 boxes present in the promoter of E-cadherin. Knockdown of E-cadherin in human
RCC cells resulted in an increase in the invasive capabilities of these cells. Therefore it can be
envisioned that loss of pVHL and subsequent loss of E-cadherin could be critical for ccRCC
development and progression (Krishnamachary, Zagzag et al. 2006; Evans, Russell et al.
2007).
Interestingly, both HIF and pVHL appear to be able to influence p53 function. HIF can
directly bind to and modulate p53 activity, and pVHL is able to regulate p53 function in a
HIF-independent manner through suppression of MDM2 (also known as E3 ubiquitin-protein
ligase) mediated ubiquitination. Stabilization of p53 resulted in increased p53 transcriptional
activity, and loss of pVHL prevents this stabilization. Therefore pVHL loss appears to result
in p53 inactivation by both HIF-dependent and HIF-independent effects (Roe, Kim et al.
2006; Sendoel, Kohler et al. 2010) (Figure 3).
Jade-1 is a novel protein identified by yeast two-hybrid analysis as a pVHL-interacting
protein (Zhou, Wang et al. 2002; Roe, Kim et al. 2006). pVHL interacts with and stabilizes
Jade-1 protein, and mutations of the VHL gene disrupt this process and may be associated
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with an increase in renal cancer risk (Zhou, Wang et al. 2004; Kapitsinou and Haase 2008). In
addition to tumor formation, mutations in the VHL gene can result in the development of
polycythemia. At least 10 inherited mutations in the VHL gene have been found to cause
familial erythrocytosis, and it is often designated erythrocytosis (ECYT2) (Percy, Chung et al.
2012). Also, in support of a link between VHL and inflammation, it has recently been
reported that pulmonary hypertension, a complication of Chuvash polycythemia, is caused by
lung fibrosis (Hickey, Richardson et al. 2010). Accumulating evidence suggest that pVHL is a
negative regulator of nuclear factor-kappa B (NF-B). In the absence of a functional pVHL,
the expression and activity of NF-B are enhanced, which subsequently confer a drug
resistant phenotype to RCC by suppressing drug-induced apoptotic pathway. As NF-B is
over expressed in RCC, its inhibition as a potential treatment strategy has been a subject of
intense research using genetic or chemotherapeutic approaches (Morais, Gobe et al. 2011).
Developmental (cardiovascular, mammary gland and skeletal development; adipogenesis,
chondrogenesis and embryonic survival) and physiological (hypoxia-induced; -glycolysis, apoptosis, -erythropoiesis, -pulmonary vascular remodeling, -myocardial preconditioning, cell cycle arrest and angiogenesis) roles of HIF-1 as established by analysis of HIF-1α-null
mice and cell lines (Semenza 2004).
6. Therapeutic Targets and Future Prospects
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Over the past century, studies focusing on the structure and function of the VHL TSG and
its protein product, pVHL, have been highly informative with respect to the pathogenesis of
ccRCC as well as the molecular mechanisms of oxygen sensing. Many functions have been
attributed to pVHL; however, the one best characterized and most clearly linked to the
development of pVHL-defective tumours, is targeting of the HIF transcription factor for
proteolytic degradation. In addition to its direct role on tumor growth, HIF-1 has also been
implicated in modulating the tumor response to therapies (Arjumand and Sultana 2012).
There are a number of compounds reported with anti-HIF-1 activity and are mainly classified
as direct and indirect inhibitors based on their different modes of action. While direct HIF-1
inhibitors prevent HIF-1 from transactivation, DNA binding, and subsequently initiating
transcriptional activity, indirect HIF-1 inhibitors generally block the transcription or
translation of HIF-1α or promote the degradation of HIF-1α protein (Wang, Zhou et al. 2011).
It is supposed that antiangiogenesis therapy may enhance tumor hypoxia, and when the
hypoxia response is abrogated in cancer cells using HIF-1 inhibitors, a robust antitumor
activity may be observed. Inactivation of the VHL TSG is a frequent event in ccRCC. The
central role of HIF-1α, and particularly HIF-2α, in pVHL-defective ccRCC has spurred
interest in the development of HIF antagonists for this disease. Unfortunately, however, there
are very few examples of drug-like small organic molecules that are capable of inhibiting the
function of DNA-binding transcription factors, with the notable exception of the steroid
hormone receptors. Inhibition of mTOR kinase decreases the transcription and translation of
HIF1α, thereby lowering HIF-1α protein levels. This process might account for the
observation that rapamycin-like mTOR inhibitors (e.g. temsirolimus and everolimus) have
activity in the treatment of ccRCC (Li and Kaelin 2011). Today, currently, it is used many
agent in treatment of patients with advanced ccRCC. These agents work either directly on
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HIF-regulated targets and their receptors, such as VEGF, VEGFR, PDGFR and other tyrosine
kinase receptors, or on mTORC1, which controls cellular growth and increases HIF-1α
translation. However, knowledge of the VHL/HIF pathway provides the foundation for the
development of novel therapeutic approaches to the treatment of VHL-related tumor and
diseases (Linehan, Bratslavsky et al. 2010).
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The pVHL has many functions out of which the best recognized is pVHL ability to target
the HIF for ubiquitin-mediated degradation. Loss of pVHL function results in the stabilization
of HIF-α and activation of HIF responsive genes. Some of these gene products have been
shown to be protumorigenic in the renal cell carcinoma perspective. The product of the VHL
gene is a tumor suppressor protein, which targets several proteins for degradation by the
proteasomes, and both copies of the gene are inactivated in tumor tissues. The alpha subunits
of HIF-1 and HIF-2 are substrates for the product of the VHL gene. In the absence of VHLinduced degradation, HIF-1 and HIF-2 may contribute to increased levels of EPO, VEGF, and
other growth factors, providing a stimulus for tumor growth. HIF transcriptionally activates
several genes implicated in ccRCC pathogenesis, including VEGF. Multiple VEGF inhibitors
have now been approved for this disease based on randomized clinical trial data. Inactivation
of the VHL tumor suppressor gene is an early, causal event in the development of ccRCC,
and this gene is mutated or silenced in the majority of both hereditary and nonhereditary
ccRCC. In addition, VHL mutant tumor cells can secret a number of growth factors and
cytokines that can also activate the inflammatory and angiogenic components of the primary
tumors. Therefore, it is argue that in designing new treatments for the VHL disease, a
systemic approach including targeting the hematopoietic system and the inflammatory
response should be considered.
Developing knowledge of genetic and epigenetic changes implicated in tumor
development and behavior is becoming increasingly important for advancing the efficacy of
disease management. Pre-clinical and clinical investigations into HIF-associated pathogenesis
have led to the development of new FDA approved targeted therapies, which have produced
promising basis for new systemic therapies, including anti-angiogenic drugs and mTOR
inhibitors. In the future, a more complete understanding of the role of VHL, including its
HIF-independent functions, should provide novel therapeutics opportunities. Overall, these all
data suggesting a pivotal role of pVHL‘s in HIF-independent functions of tumor suppression,
development and normal tissue physiology. New additional genetic studies into the precise
mechanism and biological roles of VHL gene and protein are therefore highly welcome. To
discover new therapeutic intervention, more basic and more preclinic study is needed to better
understand the pathogenesis of VHL disease.
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ISBN: 978-1-62808-665-2
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Chapter 5
Application of Cancer Gene Therapy
Using Tumor Suppressor Gene p53
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Hiroshi Tazawa1,2, Shunsuke Kagawa2 and Toshiyoshi Fujiwara2,
Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan
2
Department of Gastroenterological Surgery, Okayama University Graduate School
of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Abstract
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The tumor suppressor gene p53 encodes a multifunctional transcription factor that
regulates diverse cellular processes, including cell cycle arrest, senescence, apoptosis and
autophagy; each of these processes suppresses tumor growth and progression. When
tumor cells with intact p53 function are exposed to genotoxic stresses, including
chemotherapy and therapeutic radiation, many kinds of p53-downstream target genes,
such as p21WAF1, BAX, and DRAM, are directly induced by p53; p21WAF1 then mediates
cell cycle arrest for the repair of the DNA damage, whereas BAX and DRAM activate
cell death pathways for the elimination of the damaged cells. Recent evidence
demonstrates that p53 induces not only protein-coding genes, but also small non-coding
microRNAs (miRNAs) such as miR-34 and miR-22, and that these miRNA act as
modulators in the p53-mediated tumor suppression system. The p53 gene is frequently
inactivated by genetic alterations in approximately half of all distinct types of human
cancers. Tumor cells with impaired p53 function are commonly refractory to genotoxic
stresses induced by conventional chemoradiotherapy. Therefore, restoration of p53
function by introducing exogenous p53 expression via a gene delivery system is a
promising antitumor strategy because it should result in suppression of tumor growth and
progression. This chapter focuses on recent advances in our understanding of the tumor
suppressive roles of the p53 gene, which include regulation of p53 target protein-coding
genes and of miRNAs. Furthermore, the potential application of p53-based cancer gene
therapy will be discussed for four types of p53 transfer systems — cationic liposome-

Address correspondence to this author at the Department of Gastroenterological Surgery, Okayama University
Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 7008558, Japan. Phone: 81-86-235-7255; Fax: 81-86-221-8775; E-mail: [email protected].
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microRNA
p53 reactivation and induction of massive apoptosis
human telomerase reverse transcriptase
internal ribosome entry site
vascular endothelial growth factor
dendritic cells
coxsackie and adenovirus receptor
histone deacetylase
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miRNA
PRIMA
hTERT
IRES
VEGF
DCs
CAR
HDAC
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Abbreviations
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Keywords: p53, gene therapy, microRNA, adenovirus, protein transduction
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DNA plasmid complexes, replication-deficient adenovirus vectors, replication-competent
adenovirus vectors, and protein transduction systems. A better understanding of the
precise molecular mechanism by which p53 mediates tumor suppression would provide
us with novel insights that may lead to improvement in p53-based cancer gene therapy. A
highly efficient system for inducing p53-mediated cell death may improve the clinical
outcomes of patients with any of the many types of p53-inactivated cancers.
Introduction
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The tumor suppressor gene p53 encodes a multifunctional transcription factor that
regulates diverse cellular processes such as cell cycle arrest, senescence, apoptosis, and
autophagy in normal tissues and in tumors [1]. When tumor cells with intact p53 function are
exposed to genotoxic stresses (including chemotherapy and therapeutic radiation), many
kinds of targets downstream of p53 are transcriptionally induced by activated p53, and these
p53 targets cooperatively regulate cellular processes that curb or reverse tumor progression
[2, 3]. In response to mild cellular stress, the main effect of p53 activation is the induction of
cell cycle arrest, which allows for repair of DNA damage and contributes to cell survival
(Figure 1). In contrast, severe cellular stress induces more p53 accumulation, which activates
several types of signaling pathways that lead to apoptosis, senescence, and autophagy;
therefore, severe stress results in the induction of cell death (Figure 1). Recent evidence
demonstrates that p53 regulates transcription of several small non-coding microRNAs
(miRNAs), as well as protein-coding genes, and that these miRNAs are important
components of the p53-mediated tumor suppression system [4]. A better understanding of the
precise molecular mechanisms in the p53-mediated tumor suppression network could lead to
novel insights useful for the development of p53-based antitumor therapies.
Analyses of the IARC TP53 database (http://www-p53.iarc.fr/) have shown that epithelial
and non-epithelial malignant tumors often harbor somatic mutations in the p53 gene and that
the types of p53 mutations and the types of tumors that harbor them vary widely [5, 6]. The
p53 gene is frequently inactivated by genetic alterations in approximately half of all distinct
types of human cancers. Patients with Li-Fraumeni syndrome, which is a cancer
predisposition disorder, each carry a germline mutation in the p53 gene, and they develop
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p53-based Cancer Gene Therapy
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early onset tumors [7]. Furthermore, inactivation of p53 via genetic engineering can induce
spontaneous tumor growth in mice; several different mouse models of p53 inactivation have
been developed, and spontaneous tumors develop in each model [8-10]. These findings
support the hypothesis that the p53 gene has a potent, critical role in the tumor suppression
network. Furthermore, tumor cells with impaired p53 function are often refractory to the
genotoxic stresses induced by conventional chemoradiotherapy [11]. Thus, restoration of
wild-type p53 function is a promising antitumor strategy because it could lead to suppression
of tumor growth and progression.
In principle, there are two ways to restore p53 activity in tumor cells that lack functional
p53 [12, 13]. One class of strategies involves the introduction of active, exogenous p53 via
any one of several gene delivery systems — such as liposome complexes with plasmid DNA,
replication-deficient or replication-competent adenovirus vectors, or protein transduction
tools (Figure 2). Among these delivery methods, adenovirus-mediated p53 gene therapy has
recently emerged in preclinical experiments and clinical studies as a promising antitumor
treatment for several types of human cancers [14, 15]. Another class of strategies involves the
reactivation of endogenous p53 expression via treatment with chemical compounds such as
Nutlin or PRIMA (p53 reactivation and induction of massive apoptosis) (Figure 2). For
example, the low-molecular-weight compound PRIMA reportedly induced apoptosis by
restoring DNA-binding activity and a functional conformation to mutant p53 protein in
human cancer cells that harbor p53 mutations [16]. A different small molecule, Nutlin,
induces p53 stabilization in tumor cells that overexpress MDM2 by interacting with MDM2
Fig.
1 thereby inhibit the MDM2-p53 interaction [17].
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Severe Stress
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Mild Stress
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Apoptosis
Autophagy
Cell Death
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p53
Figure 1. Conceptual diagram of the different p53-mediated cellular processes that are induce by mild
versus severe stresses. Mild stress induces a small amount of p53 activation and therefore cell cycle
arrest and repair of DNA damage; these process result in cell survival. In contrast, severe stress induces
a large amount of p53 accumulation and activation of three distinct cell death pathways — senescence,
apoptosis and autophagy — that result in elimination of the damaged cells.
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Fig. 2
Plasmid
Virus
PRIMA
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Protein
Nutlin
p53
p53
p53
p21
BAX
DRAM
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Autophagy
Cell death
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Figure 2. Schematic diagram of the p53-mediated tumor suppression network. Restoration of p53
function induces senescence, apoptosis, or autophagy via activation of three main p53 target genes p21,
BAX, or DRAM, respectively. Some chemical compounds, PRIM and Nutlin, reactivate endogenous
p53 expression; PRIM induces transcriptional activity of mutant p53 protein (mp53), whereas Nutlin
suppresses MDM2 expression. In contrast, some p53 delivery systems — DNA plasmid (plasmid),
adenovirus (virus), protein transduction tool (protein), or combinations thereof — can introduce
exogenous p53 activity.
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However, the therapeutic effects of PRIMA and Nutlin are limited to tumor cells that
carry specific p53 gene mutations or to tumor cells that overexpress MDM2, respectively.
The level of endogenous p53 expression reactivated by these compounds may be also less
effective for inducing tumor cell death when compared to ectopic expression of exogenous
p53 gene. Therefore, overexpression of exogenous p53 gene by introduction and
overexpression of p53 via any one of several gene transfer methods would be an effective
antitumor strategy for the induction of cell death in a variety of p53-inactivated tumor cells.
This chapter focuses on the recent advances in our understanding of the tumor
suppressive role of p53 through activation of several p53-downstream target genes including
protein coding genes and small non-coding microRNA genes. Furthermore, we will discuss
the potential application of p53-based cancer gene therapies to the treatment of patients with
various types of cancers; these therapies involve a liposome-DNA plasmid complex,
replication-deficient or replication-competent adenovirus vectors, or protein transduction tool.
p53 as a Guardian of Human Genome
p53-mediated Tumor Suppression System
The tumor suppressor gene p53 is a main mediator induced by various types of cellular
stresses. Activation of wild-type p53 influences the subsequent cell fate trajectories of normal
cells and of tumor cells with intact p53 function [2]. There are three cell death pathways—
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senescence, apoptosis, and autophagy — in activated by the p53-mediated tumor suppression
system. These cell death pathways are determined by the induction of several p53downstream target genes, such as p21WAF1 (p21), BAX, or DRAM (Figure 2). While p21
activation mainly induces cell cycle arrest [18, 19] and subsequent senescence [20], the p53induced BAX [21] and DRAM [22] lead to apoptosis and autophagy, respectively, resulting
in the cell death. In response to mild cellular stress, p21 gene, which is most rapidly and
strongly induced during the DNA damage response, mainly induces cell cycle arrest that
allows for the repair of DNA damage and consequently contributes to senescence. A recent
report suggests that p21-mediated induction of senescence is a promising antitumor strategy
[23]. In contrast, in response to severe cellular stress, p53-induced BAX activation induces
apoptotic cell death; similarly, p53-induced DRAM activation induces autophagic cell death
(Figure 2). Interestingly, p21 activation suppresses apoptotic and autophagic cell death
pathways [24, 25]. Therefore, induction of both the apoptotic and autophagic cell death
pathways via p53 transactivation would be a more effective antitumor strategy for the
suppression of tumor initiation and progression than induction of p21-mediated senescence.
Thus, p21 suppression may be an effective strategy for the induction of the apoptotic and
autophagic cell death pathways in tumor cells, particularly when the tumor suppressor gene
p53 is overexpressed in tumor cells in response to cancer gene therapy.
p53-MicroRNA Tumor Suppression Network
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Recent advances have shown that microRNAs (miRNAs), small non-coding RNAs
comprising 22 nucleotides, are novel potent modulators in the p53-mediated tumor
suppression network [4].
In fact, some kinds of miRNAs are induced by p53 activation in normal and cancer cells
exposed to various cellular stresses, such as chemotherapy and radiation. Among some p53inducible miRNAs, miR-34 was initially identified as a p53-regulated tumor-suppressive
miRNA; this discovery was made independently in several laboratories in 2007 [26-28].
Ectopic expression of miR-34 induces cell cycle arrest, senescence, or apoptosis in human
cancer cells because it can suppress many target genes, including E2F3 (Figure 3).
Downregulation of miR-34 expression by promoter methylation occurs in a variety of human
malignancies [29-31], suggesting that miR-34 may function as a tumor suppressor miRNA. In
contrast, miR-22, which is also a p53-inducible tumor suppressor miRNA, represses p21
expression and thereby induces apoptotic cell death (Figure 3) [32].
Interestingly, both miR-34 and miR-22 suppress the expression of SIRT1, which
negatively regulates p53 by inducing p53 deacetylation; these interactions result in a positive
feedback loop that enhances p53 activation (Figure 3) [33, 34]. Interestingly, several p53inducible miRNAs (e.g., miR-143/145 [35], miR-192/194/215 [36], and miR-605) suppress
MDM2, another negative regulator of p53 that induces ubiquitin-mediated p53 degradation
(Figure 3) [37]. Taken together, these findings suggest that targeting the p53-miRSIRT1/MDM2 axis as a p53 positive feedback loop may have therapeutic potential.
Furthermore, because p53 may be a master modulator of miRNA biogenesis [38], many kinds
of miRNAs may be directly or indirectly regulated by p53 and be involved in p53-mediated
tumor suppressor.
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Fig. 3
miR143/145/192/
194/215/605
miR-22/34
SIRT1
MDM2
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p53 Positive Feedback Loop
miR-22
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E2F3
p21
?
Cell Cycle
Arrest
Apoptosis
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Senescence
Cell death
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Figure 3. Schematic diagram of a p53-inducible miRNA-mediated tumor suppression network. p53inducible miR-34 mainly induces senescence by suppressing E2F3; in contrast, miR-22 induces
apoptosis by suppressing p21. Many kinds of p53-inducible miRNAs — including miR-22/34, miR143/145, miR-192/194/215, miR-605 — are involved in the induction of a p53 positive feedback loop
because they suppress two negative regulators of p53, SIRT1 and MDM2.
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Although the identities of the miRNAs that regulate p53-mediated autophagy induction
remain unclear, this relationship between p53 and miRNA suggests that p53-inducible
miRNAs positively regulate all three p53-mediated cell death pathways — senescence,
apoptosis, and autophagy — by enhancing p53 expression via a positive feedback loop.
Furthermore, miRNA-based cancer gene therapy may be a novel antitumor strategy;
specifically, introduction of one or more miRNAs could induce a p53-miRNA positive
feedback loop and consequently restore p53 function. Notably, miR-34-based antitumor
therapy has been emerging as a novel anticancer strategy [39-41]. We previously reported that
ectopic expression of miR-34 induced by miRNA mimics suppressed cell viability and
induced subsequent senescence-like growth arrest in human colon cancer cells that had either
wild-type or mutated p53 alleles [27].
Furthermore, a lentivirus vector-mediated miR-34 gene transfer efficiently induces miR34 expression, and this expression results in the suppression of cell proliferation and
suppression of tumor growth in human cancer cells [42-45]. Because miRNA-based antitumor
therapy holds real promise for future clinical application, development of efficient miRNA
delivery systems is warranted.
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p53-mediated Suppression of Stemness Property
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p53
Cancer Cell
Cancer Stem Cell
Normal Stem Cell
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reports have suggested that p53 plays critical roles not only in tumor suppression
Fig.Recent
4
in cancer cells, but also in the inhibition of self-renewal and other properties of normal stem
cells (Figure 4) [46].
Figure 4. Conceptual diagram of p53-mediated suppression of tumor growth and stem cell properties.
p53 induces suppression of proliferation and stem-cell-specific properties in cancer cells and normal
stem cells, respectively. In cancer stem cells, p53 activation suppresses both tumor growth and stemcell-specific properties.
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For example, p53 functions as a negative regulator of reprogramming [47] in normal
embryonic stem cells [48], induced pluripotent stem cells [49], and a variety of normal stem
cells [50-52]. Modulation of miRNA networks by p53 is an important factor in the regulation
of characteristic properties and differentiation of stem cells [53-55]. The emerging evidence
suggests that p53-mediated cancer gene therapy via miRNA modulation has significant
therapeutic potential with regard to cancer stem cells (Figure 4) [56]. In fact, ectopic
expression of the p53-inducible miR-34 inhibits the stemness properties of some cancer stem
cells and tumor progression [42, 57-59]. A greater understanding of the underlying molecular
mechanisms by which p53-mediated miRNA networks affect stem cell biology should
facilitate the development of cancer stem cell-targeting therapy.
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Overexpression of exogenous p53 gene by introduction and overexpression of p53 via
any one of several gene transfer methods is an effective antitumor strategy for the induction
of cell death in a variety of p53-inactivated tumor cells. We will next discuss the potential
application of p53-based cancer gene therapies that involve a liposome-DNA plasmid
complex, replication-deficient or replication-competent adenovirus vectors, or protein
transduction tool.
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In preclinical in vitro experiments, non-viral plasmid DNA expression vectors that
encode p53 are usually used to deliver an exogenous p53 gene into tumor cells (Figure 2).
Cationic liposomes have been shown to be useful delivery vehicles for transfection of DNA
plasmid vectors that encode ectopic p53 into human cancer cells [60-63]. Furthermore, to
increase transfection efficiency and tumor-specific delivery of plasmid vectors, an antibodyconjugated immunoliposome has been developed for cancer treatments [64-67]. However,
transfection efficacies using either liposome-based method are low and insufficient to induce
cell death especially in in vivo tumors.
Therefore, a transduction system that is more efficient than are liposome complexes is
required to induce exogenous p53 expression in tumor tissues that is sufficient to induce cell
death.
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To induce exogenous expression of p53 in vivo more efficiently than is currently possible
with p53-expressing plasmid DNA, a p53-expressing, replication-deficient adenovirus vector
(Ad-p53) is frequently used in preclinical in vitro and in vivo experiments (Figure 5) [4, 15,
68, 69]. We previously reported that adenovirus-mediated introduction of wild-type p53 into
human lung and colon cancer cells efficiently suppressed cell proliferation and tumor growth
[70-73].
Furthermore, infection with the Ad-p53 vector enhanced the in vivo antitumor effect of
chemotherapeutic agents because human cancer cells underwent apoptotic cell death [70, 72].
Moreover, Ad-p53-mediated p53 transduction enhanced the chemosensitivity of human
sarcoma cells [74-76]. These results suggest that Ad-p53-mediated p53 gene transfer is a
potential antitumor therapy that could be used as a monotherapy or in combination with
conventional chemotherapy.
Overexpression of p21, which is a downstream target of p53, may also have antitumor
effects and potential as a pro-senescence, antitumor therapy [23]. We previously compared
the antitumor effects of Ad-p53 with those of a p21-expressing replication-deficient
adenovirus vector (Ad-p21) in human tumor cells [71]. Ad-p53 infection induced apoptotic
cell death, whereas Ad-p21 infection mainly induced cell cycle arrest at G1. When Ad-p53
and Ad-p21 were co-transduced, Ad-p53-mediated p53 induction overcame the Ad-p21mediated cell cycle arrest and induced apoptotic cell death. Thus, Ad-p53-mediated gene
therapy is a promising antitumor therapy for induction of profound apoptotic cell death in
tumor cells.
In many clinical studies, Ad-p53 (Advexin; INGN-201; Introgen Therapeutics Inc.)
reportedly induces antitumor effects in patients with various types of cancers, including nonsmall-cell lung cancer (Figure 5) [77, 78], head and neck squamous cell carcinoma [79],
glioma [80], ovarian cancer [81], esophageal squamous cell carcinoma [82]. In these studies,
Ad-p53 infection exhibited a safe profile and clinical benefit as a monotherapy or in
combination with chemotherapy and radiotherapy [83].
Moreover, intratumoral Ad-p53 injection alone or in combination with cisplatin was
feasible and well tolerated in patients with advanced non-small-cell lung cancers [78].
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Figure 5. Schematic diagrams of DNA structures of several adenovirus vectors. Ad-p53 (Advexin,
Gendicine) is a p53-expressing replication-deficient adenovirus; the p53 gene expression is under the
regulation of the CMV promoter and this cassette is inserted into the E1 region; the E3 region is
deleted. In Telomelysin (OBP-301), the hTERT gene promoter element drives the expression of E1A
and E1B genes, which are linked with an IRES. OBP-702 is a p53-expressing telomerase-specific
replication-competent oncolytic adenovirus; p53 gene expression is under the regulation of the Egr-1
gene promoter, and this cassette is inserted into the E3 region.
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In the Ad-p53-treated tumor tissues, p53 transgene expression was confirmed by
quantitative PCR analysis. Of the 15 patients who received intratumoral injection of Ad-p53,
13 could be assessed to determine the efficacy of the treatment; one patient had a partial
response, 10 patients had stable disease during at least 9 months, and two patients had
progressive disease.
Furthermore, Ad-p53 (Gendicine, Shenzhen SiBiono GeneTech Co.) was approved by
the State Food and Drug Administration of China for clinical use in 2003 (Figure 5) [84-86].
Recent reports have demonstrated that Ad-p53 in combination with chemotherapy and
radiotherapy is clinically effective in patients with advanced hepatocellular carcinomas [8789]. Taken together, the accumulating evidence indicates that Ad-p53-mediated cancer gene
therapy is a promising anticancer therapy.
However, Ad-p53 is a replication-deficient adenovirus; therefore, it is impossible to
induce exogenous p53 expression in every tumor cell via this vector. The low transduction
rate of p53 gene transfer via the replication-deficient Ad-p53 vector is major problem that
needs to be overcome in order to improve clinical outcomes of patients with advanced
cancers. Based on findings from preclinical experiments, there are several potential
90
approaches by which Ad-p53-mediated p53 expression and therefore Ad-p53-mediated cell
death has been enhanced; here, we describe three such approaches (Figure 6).
E2F1
Ad-E2F1
ARF
Ad-ARF
MDM2
Ad-FHIT
E1A
Nutlin
Replication
p53
p53
p53
p53
p21
BAX
DRAM
Cell Cycle
Arrest
Apoptosis
Autophagy
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siRNA
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Fig. 6
Senescence
Cell death
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Figure 6. Schematic diagram of Ad-p53-mediated induction of two programmed cell death pathways
and strategies for enhancing the associated Ad-p53-mediated antitumor effects. Replication-deficient
Ad-p53 infection induces apoptosis and autophagy and therefore results in cell death, rather than cell
cycle arrest, when combined with replication-competent OBP-301, replication-deficient adenovirus
vectors (Ad-E2F1, Ad-ARF, Ad-FHIT), chemical compound (Nutlin), or p21 siRNA. Ad-p53 induces
apoptosis or autophagy through activation of BAX or DRAM, respectively. OBP-301 enhances Adp53-mediated p53 expression through E1A -induced replication of an E1-deleted Ad-p53. Infection
with Ad-E2F1, Ad-ARF or Ad-FHIT or treatment with Nutlin enhances Ad-p53-mediated p53
expression through MDM2 suppression. Transfection with p21 siRNA induces both Ad-p53-mediated
cell death pathways because p21 is downregulated.
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Ad-p53-mediated p53 expression is enhanced if combined with E1A-expressing
oncolytic adenovirus. We previously developed a telomerase-specific replication-competent
oncolytic adenovirus, OBP-301 (Telomelysin), that induces tumor-selective oncolytic cell
death in a telomerase-dependent manner; in this construct, the promoter from hTERT (human
telomerase reverse transcriptase) drives the expression of two adenoviral genes, E1A and
E1B, that are linked to an internal ribosome entry site (IRES) (Figure 5) [90-92]. A
combination therapy that involves co-transduction of Ad-p53 and oncolytic adenovirus OBP301 enhanced p53 expression; this combination therapy resulted in a more profound
antitumor effect and enhanced apoptotic cell death when compared to monotherapy with
either OBP-301 or Ad-p53 [93]. Adenoviral E1A expression induced by oncolytic adenovirus
supports the replication of Ad-p53 vector and subsequently enhance the Ad-p53-mediated
p53 expression (Figure 6).
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Another method is to inhibit the expression of MDM2, which functions as a negative
regulator of p53 via ubiquitin-mediated p53 degradation, and stabilize exogenous p53
expression induced by Ad-p53 (Figure 6). Suppression of MDM2 expression via treatment
with Nutlin [94] or via adenovirus-mediated overexpression of the tumor suppressor FHIT
gene [95] enhances Ad-p53-mediated p53 expression and apoptotic cell death in human
cancer cells.
Furthermore, overexpression of the ARF gene introduced via a recombinant adenovirus
vector, Ad-ARF [96] or Ad-E2F1 [97], induces enhancement of Ad-p53-mediated p53
expression and antitumor effects because the exogenous ARF downregulated MDM2 in
human cancer cells. MDM2 is downregulated by oncogenic stress-mediated ARF activation
in a mouse model of oncogene K-Ras-induced lung tumorigenesis, and this MDM2
downregulation is necessary for p53 overexpression to have a substantive antitumor effect in
this model of tumorigenesis [98, 99]; therefore, combining a MDM2-downregulating therapy
with Ad-p53 should be a more effective antitumor strategy than Ad-p53 monotherapy.
Ad-p53-mediated cell death in tumors could be enhanced via p21 suppression.
Suppression of p21 expression by genetic deletion [25] or an exogenous p21-targeted siRNA
[100] enhances Ad-p53-induced apoptosis (Figure 6). Since p53-downstream target p21
functions as a suppressor of apoptosis and autophagy [24, 25], p21 suppression may be a
critical factor for inducing both apoptosis and autophagy in response to p53 overexpression.
Each of these three strategies for enhancing Ad-p53-mediated cell death should improve the
clinical outcomes of Ad-p53-mediated cancer gene therapy.
Fig. 7
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Ad-p53
Suppression
of
Angiogenesis
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Infiltration
of
Neutrophils
N
VEGF
X
X
X
VEGF
X
p53
X
CD95L
N
CD95L
X
VEGF
N
p53
X
X
N
VEGF
VEGF
CD95L
N
CD95L
Figure 7. Conceptual diagram of Ad-p53-mediated bystander effects on neighboring, uninfected tumor
cells. When tumor cells are infected with Ad-p53, p53 overexpression induced cell death in the infected
tumor cells. In contrast, uninfected tumor cells are also eliminated via bystander effects, which can
include suppression of angiogenesis by VEGF downregulation and infiltration of neutrophils by CD95L
expression.
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Apparently, Ad-p53 transduction causes antitumor effects in not only Ad-p53-infected
cells, but also in uninfected tumor cells, because the exogenous p53 induces bystander effects
[101]. In fact, Ad-p53-mediated p53 gene transfer induces bystander effects to neighboring
tumor cells via multiple mechanisms in preclinical in vivo settings (Figure 7).
For example, Ad-p53 infection markedly inhibited the expression of an angiogenic factor
and vascular endothelial growth factor (VEGF) and increased the expression of a
antiangiogenic factor; together, the effects of p53 expression result in the suppression of
neovascularization in tumor tissues [73, 102]. Additionally, Ad-p53-mediated p53 transfer
induced overexpression of CD95 ligand (CD95L) in tumor cells and consequently massive
infiltration of neutrophils into tumor tissues [103]. Overexpression of CD95L was also
partially responsible for the Ad-p53-induced apoptosis that was mediated by the Fas
receptor/ligand system [104]. Furthermore, when bone marrow-derived dendritic cells (DCs)
infected with Ad-p53 were intratumorally injected into subcutaneous xenograft tumors, we
observed antitumor effects in both DC-injected and non-injected tumor tissues [105],
suggesting that the administration of Ad-p53-infected DCs caused a systemic immune
response. Natural killer cells may be the immunological mediators of some bystander effects
caused by Ad-p53-mediated cancer gene therapy [106]. These findings suggest that
adenovirus-mediated p53 overexpression is a promising antitumor therapy that has antitumor
effects because angiogenesis is suppressed and immune responses are induced within infected
and uninfected tumor tissues. Similarly, a combination therapy that involves Ad-p53 and
bevacizumab [107], a monoclonal antibody specific for VEGF-A, or FasL transduction [108]
may be effective in completely eradicating tumor cells.
Replication-Competent Oncolytic Adenovirus
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Tumor-specific, replication-competent oncolytic viruses are being developed as novel
vectors for anticancer gene therapies; in these vectors, the promoters of cancer-related genes
are used to regulate virus replication in a tumor-dependent manner [109-112]. OBP-301
(Figure 5) is a replication-competent oncolytic adenovirus that induces tumor-selective
oncolytic cell death in a telomerase-dependent manner [90-92]. A phase I clinical trial of
OBP-301 in patients with advanced solid tumors has been recently completed, and OBP-301
was well tolerated by the patients [113]. Sixteen patients with a variety of solid tumors were
enrolled. However, the antitumor effect of OBP-301 was limited in some of the OBP-301injected tumors; one patient had a partial response of the injected malignant lesion and seven
patients had stable disease at day 56 after treatment. Recently, armed oncolytic viruses that
express therapeutics transgenes have been genetically engineered to enhance the antitumor
effect of an oncolytic virus [114, 115]. Among the many possible therapeutic transgenes, p53
is an excellent candidate for such a novel approach because it is a potent therapeutic
transgene and can induce cell cycle arrest, senescence, apoptosis, autophagy, or some
combination thereof (Figure 1) [2]. Oncolytic adenoviruses that express p53 and are armed,
tumor-specific, and replication-competent induce higher p53 expression and stronger
antitumor effects than does Ad-p53 or a non-armed oncolytic adenovirus [116-121].
Moreover, we also generated an armed OBP-301 variant that expresses wild-type p53 (Figure
5); this construct, which is designated OBP-702, suppresses the viability of various types of
epithelial malignant cells more efficiently than does OBP-301 [122]. However, the molecular
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mechanism by which p53 enhances the antitumor effect of oncolytic adenovirus remains
unclear.
OBP-702 mediates profound antitumor effects because OBP-702-mediated p53
overexpression induces two types of programmed cell death, apoptosis and autophagy, in
malignant human cells, epithelial or non-epithelial, and thereby improves on the antitumor
effects of the OBP-301 precursor [122, 123]. The p53-mediated enhancement of the antitumor
effects of the oncolytic adenovirus could result from either or both of two possible
mechanisms (Figure 8). One possibility is that p53 activation is enhanced because of virus
replication and E1A-dependent MDM2 suppression in tumor cells. When human cancer cells
were infected with a similar dose of OBP-702 or Ad-p53, the level of p53 expression induced
by replication-competent OBP-702 was much higher than that induced by replicationdeficient Ad-p53 [122]. However, although OBP-702 induced high p53 expression, one of the
main p53-target genes, MDM2, was induced at a lower level in the OBP-702-infected tumor
cells than in the Ad-p53-infected tumor cells. This difference in MDM2 expression was due
to adenoviral E1A-mediated MDM2 suppression in cells infected with OBP-702.
Furthermore, this MDM2 suppression enhanced the adenovirus-mediated p53 expression and
the consequent apoptotic cell death. Another possibility is that E1A mediated p21
suppression. Activation of p21 often suppresses p53-induced apoptotic and autophagic cell
death pathways [24, 25]. Therefore, p21 suppression enhances the effects of the p53-mediated
induction of apoptotic and autophagic cell death pathways in tumor cells.
Fig. 8
OBP-702
E1A
ce
Rb
E1A
E2F1
p53
MDM2
en
E2F1
p53
p53
p53
p21
BAX
DRAM
Cell Cycle
Arrest
Apoptosis
Autophagy
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miR-93
miR-106b
Senescence
Cell death
Figure 8. Schematic diagram of OBP-702-mediated induction of programmed cell death pathways.
Replication-competent OBP-702 infection induces apoptosis and autophagy, resulting in the cell death;
these effects are dependent on p53-mediated BAX/DRAM upregulation and adenoviral E1A-dependent
p21 downregulation via E2F1-inducible miR-93/106b activation.
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Protein Transduction Method
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In fact, inactivation of p21 by adenoviral E1A can enhance apoptosis in human colon
cancer cells that overexpress p53 and have been treated with chemotherapeutic drugs [124].
Additionally, genetic deletion of p21 can induce autophagy in mouse embryonic fibroblasts
that have been treated with C(2)-ceramide or gamma-irradiation [24]. In contrast, p21
overexpression can inhibit the induction of apoptosis that is mediated by Ad-p53 [25].
Furthermore, E1A-dependent activation of the transcription factor E2F1 induces the
upregulation of two miRNAs, miR-93 and miR-106b, that efficiently suppress p21 expression
in OBP-702-infected tumor cells; this suppression of p21 leads to the enhancement of p53induced apoptosis and autophagy in these cells (Figure 8) [123]. Interestingly, E2F1 also
suppresses MDM2 expression by inducing upregulation of miR-25/32, which targets MDM2
[125]; therefore, cooperation between the MDM2-p53-p21 pathway and the E2F1-miRNA
pathway may be involved in the induction of the apoptosis and autophagy that is cause by
OBP-702. Thus, E1A-mediated downregulation of p21 and MDM2 would enhance p53induced apoptosis and autophagy in OBP-702-infected cells (Figure 8).
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Adenovirus-mediated p53 gene transfer systems are expected to induce exogenous p53
expression in various types of human cancer cells more efficiently than plasmid-based
delivery systems; nevertheless, adenovirus infection is mainly mediated by interactions
between virus particles and CARs (coxsackie and adenovirus receptors) that are expressed on
host cells. Therefore, CAR-expressing tumor cells are the main targets for any adenovirusmediated exogenous gene transfer system, and tumor cells that lack CAR can escape from
being killed by adenovirus-mediated p53 transduction. In fact, CAR expression can often be
downregulated as tumors progress [126, 127]. Furthermore, CAR-expressing tumor cells
often become refractory to Ad-p53-induced cell death because of decreases in CAR
expression [128]. In tumor cells with low CAR expression, a histone deacetylase (HDAC)
inhibitor can elevate CAR expression [129, 130]. However, CAR-negative tumor cells may
be less sensitive or insensitive to the CAR upregulation that is mediated by the HDAC
inhibitor. These findings indicate that development of a novel p53-based cancer gene therapy
targeted specifically against CAR-negative tumor cells is imperative.
Membrane-permeable peptides may be useful as tools for introducing exogenous,
therapeutic p53 protein into tumor cells. Recently, 11 polyarginine peptides have been used as
a delivery system to introduce the p53 protein into cells, and this exogenous p53 protein
induced the p21 gene promoter, a downstream target of wild-type p53, as efficiently as Adp53-mediated p53 transduction [131]. Modified isoforms of p53 that are resistant to MDM2mediated ubiquitination are more able to activate transcription of downstream targets and
induce profound antitumor effects than is the wild-type p53 protein [132]. Alternatively,
when pyrenebutyrate was used in combination with a fusion protein comprising three
polyarginine peptides and wild-type p53, the p53 fusion protein was efficiently taken up by
cancer cells and transported into the nucleus where it activated transcription of downstream
target genes [133]. Recently, this fusion protein transduction system has been used to show
that a fusion protein including only the carboxy-terminal region of p53 was able to efficiently
induce apoptosis and autophagy in human cancer cells [134, 135]. These results suggest that a
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p53 protein transduction method that involves polyarginine peptides and pyrenebutyrate is a
promising p53-based cancer therapy that is independent of CAR expression on target cells.
Conclusion
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Cancer gene therapy is defined as the treatment of malignant tumors via the introduction
of a therapeutic tumor suppressor gene or the abrogation of an oncogene. One of the most
potent therapeutic tumor suppressor genes is the multifunctional transcription factor p53,
which regulates diverse cell fates, including cell cycle arrest, senescence, apoptosis, and
autophagy. Recent advances in tumor biology indicate that p53-based cancer gene therapy has
substantial therapeutic potential because a p53-mediated miRNA network suppresses both
tumor growth and stem cell properties in cancer cells.
Gene replacement therapy that involves any one of several delivery systems to introduce
the tumor suppressor p53 is a promising antitumor strategy because active p53 can induce
tumor suppression in response to genotoxic therapeutic agents. For example, the replicationdeficient Ad-p53 adenovirus (Advexin, Gendicine) has antitumor effects against many types
of cancers in preclinical experiments and clinical experience trials. However, Ad-p53mediated p53 activation is insufficient for inducing cell death pathways in tumor tissues;
therefore, many kinds of strategies for enhancing Ad-p53-mediated p53 activation are
warranted. The enhancement of adenovirus replication, suppression of negative regulators of
p53 or of p53-related cell death pathways, activation of a miRNA-mediated p53 positive
feedback loop, or some combination of these strategies may be effective for improving the
clinical outcomes of p53-based cancer gene therapies. Given the underlying molecular
mechanisms of the p53-mediated tumor suppression system, we should endeavor to develop
safe and effective cancer gene therapies that are based on the potent tumor suppressor p53.
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Acknowledgments
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This study was supported by grants from the Ministry of Health, Labour, and Welfare of
Japan and from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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[5]
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ISBN: 978-1-62808-665-2
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Chapter 6
p63 and p73: Members of the p53
Tumor Suppressor Family
is
Zeynep Ocak1, Murat Oznur2, Ramazan Yigitoglu3, Esra Gunduz2,
and Mehmet Gunduz2
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Department of Medical Genetics, Faculty of Medicine,
Abant Izzet Baysal University, Bolu, Turkey
Departments of 2Medical Genetics and
3
Biochemistry, Faculty of Medicine, Turgut Ozal University, Istanbul, Turkey
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Abstract
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The p53 transcription factor plays a significant role in cellular homeostasis and
controls the transition between the G1 and S phases during cell division.
Thus, replication of damaged DNA and initiation of apoptosis are prevented.
Damaged DNA cannot be repaired in the absence of homozygous p53; thus, mutations
occur. However, some studies have tentatively reported that mice lacking the p53 tumor
suppressor protein can survive normally. It is thought that some proteins with functions
identical to those of p53 might be present; thus, the p53-related proteins p63 and p73
have been investigated.
The p63 and p73 proteins have high structural similarities as well as some common
functional properties. Nevertheless, p63 and p73 mutat distinct from p53 in human
carcinomas. P63 and p73 genes/proteins are members of the p53 gene family and show
some structural and functional similarities with p53.
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Keywords: p53, p63, p73, tumor suppressor, cell cycle

Corresponding author: Esra Gunduz, DMD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut
Ozal University, Turkey, Anadolu Bulvari 16A Gimat Ankara, Turkey, Email: [email protected].
106
Zeynep Ocak, Murat Oznur, Ramazan Yigitoglu et al.
1. Introduction
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p53 is one of the most-studied genes in human biology. The p53 gene is located on the
short arm of chromosome 17 at the 17p13-1 position and is a tumor suppressor gene. The p53
gene consists of 11 exons and is approximately 20 kb in length [1]. When the p53 gene was
initially investigated it was thought to be simply an oncogene; however, later it was
understood that only the mutated form plays an important role in abnormal cell growth and
that the normal p53 gene acts as a tumor suppressor [2].
Two genes that have prominent structural and sequence homology with p53 have been
identified. One of these, p73, is localized at 1p36, while the other is p63, which is localized at
3q27-29 [3, 4].
The p63 and p73 proteins encoded by these two genes have a high similarity in the Nterminal transactivation region, central DNA-binding region, and C-terminal oligomerization
region [5,6]. Furthermore, these two genes stimulate p53-sensitive promoter regions and
trigger programmed cell death when expressed excessively. Remarkably, these three genes
have similar intron and exon sequences [7].
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1.1. p63 General Information: Symbols, Location, Size, Cofactors,
Subunits
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HGNC approved gene symbol: TP63
Alternative names and symbols: tumor protein p73-like, TP73L, p53-related protein p63,
p63, KET
Cytogenetic location: 3q27-q29
Subcellular location: nucleus
Size: 680 amino acids; 76785 Da
Cofactor: Binds 1 zinc ion per subunit
Subunit: Binds DNA as a homotetramer. Isoform composition of the tetramer may
determine transactivation activity. Belongs to the family of homeodomain-interacting protein
kinase (HIPK) genes discovered 13 years ago. HIPK2, the most-studied member of the
family, acts as co-regulator of an increasing number of transcription factors and modulates
many basic cellular processes, such as apoptosis, proliferation, DNA damage response,
differentiation, and development. Most of these effects are mediated by phosphorylation and
activation of the p53 oncosuppressor protein [8]. Alpha and gamma isoforms of the p63 gene
interact with HIPK2. They also interact with structure-specific recognition protein 1, which
functions as a co-activator of the transcriptional activator p63. Isoforms 1 and 2 interact with
the WW domain containing E3 ubiquitin protein ligase 1 (WWP1). Isoform 5 (via activation
domain) interacts with nucleolar complex associated 2 homolog (www.genecards.org/cgibin/carddisp.pl?gene=TP63).
107
1.2. p63 Gene Structure
Figure 1. P63 gene structure.
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The p63 gene is found on chromosome 3q27-29 and is composed of 15 exons and six
isoforms [4].
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Structurally, it contains an N-terminal transactivation region, a central DNA-binding
region, and a carboxy-terminal oligomerization region (Figure 1). The p63 protein containing
the transactivation region is called Tap63 and the noncontaining p63 protein is called ΔNp63
[9, 10]. The p63 and p53 DNA binding regions have 60% sequence identity. The N-terminal
transactivation and C-terminal tetramerization regions are similar to the orders of 22 and 37%
(Figure 1). The TA and ΔN isoforms of p63 have opposite functions. The TA isoforms have
tumor suppressor activity, whereas the ΔN isoform has oncogenic activity. mRNAs of p63
yield three proteins known as α, β, and γ. These products have six forms that differ according
to transactivation region, as follows: Tap63α, Tap63β, Tap63γ, ΔNp63α, ΔNp63β, and
ΔNp63γ. These various p63 proteins have distinct features and functions [10].
P63 has a sterile alpha motive region (SAM) that is not in p53. Indeed, this region is
found only in α forms of p63. The SAM region is thought to play a significant role in cellular
lifespan, similar to apoptosis, transcriptional transactivation, focal adhesion, chromatin
formation, receptor tyrosine kinase stimulation, and SUMOylation [9]. Unlike p53, p63 is
transcribed under the control of two promoter regions. One is located in the internal region
and encodes the N-terminal transactivation noncontaining region protein. The other is
encoded as a normal N-terminal transactivation containing region protein [10].
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1.3. p63 Gene Function
Table 1. Mouse models of Trp63
Trp63−/− Brdm3
RU486-lunginducible ΔNp63α
RU486-lunginducible TAp63α
Trp63+/−
Lung SCC, K5/K14 expression, with hyperproliferation;
epidermal expression, accelerated chemical-induced tumor
development and progression, resulting in EMT to spindle cell
carcinomas and lung metastases
Higher tumor burden and metastasis compared with p53+/− mice
No defects found so far
No major phenotypic changes have been seen yet
Trp63−/− K5
TAp63α
TrΔNp63−/− knockdown
Trp63R279H
Embryonically lethal; no epidermis
ΔNp63α and
ΔNp63β
Conditional Cre/lox
TrTAp63−/−
Lung epithelium exhibit squamous metaplasia
Skin fragility and erosion with suprabasal defects
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Reference
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No major phenotypic changes have been seen yet
en
Conditional Cre/lox
TrTAp63−/−
K5 TAp63α
TAp63 essential for Ras-induced senescence; TAp63−/− increases
proliferation/oncogenesis
Normal epidermal development; UV-B challenge exhibit 45%
less apoptosis
Embryonically lethal; no epidermis
is
Conditional Cre/lox
TrTAp63−/−
Loricrin ΔNp63α
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Trp63−/− Brdm2
Phenotype
p63 -/- mice have major defects in their limb, craniofacial, and
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p63 -/- mice have severe limb and skin defects
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induce apoptosis [11]. P63 induces p53 target genes by binding p53, which stops the cell
cycle at G1 phase and triggers apoptosis.
Limb and cranofacial defects, ectodermal dysplasia
No limb defect, deterioration of hair morphology, ulcerated
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Koster et al.
2007
Lo Iacono et
al. 2008
Romano et
al. 2009
Su et al.
2010
However, this does not induce ΔNp63 transcription. The ΔNp63 isoform binds directly to
the p53 and TA isoforms or acts as a negative dominant by competing with the wild type
isoform. Thus, the p53 and TA isoforms are inactivated.
Therefore, ΔNp63 has anti-apoptotic specificity. Tap63 proteins ensure cell
differentiation, while ΔNp63 induces cell proliferation. ΔNp63 is a dominant isoform of p63
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1.4. Regulation of p63
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that is expressed on the epithelial basal layers of the skin, breast, prostate, and uterine cervix.
Expression of ΔNp63 might thereby contribute to the proliferation of basal or progenitor cells
by blocking the induction of apoptosis by p53 . TAp63 and ΔNp63 have distinct and
overlapping functions in normal and cancer tissues.
The development of epithelial tissues in humans is a highly complex process. p63 plays
an important role in embryogenesis, ectodermal differentiation, and packaging of epithelial
progenitor cells [12]. Studies of p63-null (p63-/-) mice, indicated that p63 plays a key role in
regulating epithelial proliferation and differentiation programs. Yang and Mills demonstrated
that p63 is required for ectodermal differentiation during embryogenesis [4, 5] (Table1). The
absence of p63 elicits skin, breast, and lacrimal glandular and prostate agenesis. The mice
were born with developmental abnormalities and died immediately after birth [13]. Hair
follicles, teeth, breast glands, and other epidermal mesenchymal originating structures do not
develop in mice lacking p63 [5] (Table1). Thus p63 is necessary for ectodermal
differentiation during embryogenesis.
The embryonic epidermis of p63-/- mice undergoes an unusual process of
nonregenerative differentiation, culminating in the striking absence of all squamous epithelia
and their derivatives, including mammary, lacrimal, and salivary glands [13]. p63 protein
localization and expression on the epidermis, hair follicles, sweat glands, cervix, tongue,
esophagus, breast glands, prostate, and urogenital canal have been identified by
immunohistochemical evaluation. p63 expression could be used as a stem cell marker of the
endometrial, cervical, breast, and prostate containing epithelial cell types. P63 is expressed
continuously on epidermal basal cell nuclei, germinative hair matrix cells, and the outer stem
sheath of the hair follicle.
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Expression of the p63 protein, including TAp63 and ΔNp63, occurs only during some
physiological processes in the body. ΔNp63 is expressed plentifully in basal epithelial cells,
such as in the basal layer of the skin and prostate, the myoepithelial cells of the breast, and
thymic epithelial cells [14, 15].
In addition, TAp63 is expressed at significant levels in the female germline cells, but not
in those of males [16].
However, TAp63 cannot be identified in basal epithelial cells by either Western blotting
or immunostaining techniques. TAp63 transcript expression in epithelial cells can be detected
by reverse transcription polymerase chain reaction, albeit at lower levels than ΔNp63
expression [17, 18]. ΔNp63 and TAp63 expression are regulated differently at the
transcriptional, post-transcriptional, and post-translational levels. ΔNp63α expression can be
induced in isolated dental epithelia and in the mouse lamboidal junction at the transcriptional
regulation level, which is a maturation feature during morphogenesis, and is under the control
of a binding element on the mouse Trp63 gene locus that is highly conserved in mammals.
Similarly, TAp63 expression can be induced in certain carcinoma cell lines [19, 20]. Micro
RNAs (miRNA) play a significant role in post-transcriptional regulation. Each type of
miRNA targets different expression patterns, and the effect of ΔNp63-TAp63 transcription on
skin development, epidermal stem cell proliferation and clonogenic capacity, myeloid cell
proliferation, G2 cell cycle progression and glioblastoma cells occurs at the post-translational
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level [21,22]. miRNAs act as sequence-specific DNA binding transcriptional activators or
repressors.
Previous studies have indicated that the co-activator and histone acetyltransferase (HAT)
p300 binds to TAp63 and stimulates TAp63-dependent transcription of the p21Cip1 gene.
The novel INHAT repressor (NIR) is an inhibitor of HAT.
Figure 2. Development Notch Signaling Pathway.
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The central portion of the NIR binds to the transactivation and C-terminal
oligomerization domains of TAp63. NIR is highly expressed during the G2/M phase of the
cell cycle and only weakly expressed during G1/S. Isoforms of the p63 gene contain a varying
set of transactivation and auto-regulating transactivation inhibiting domains, thus showing
isoform-specific activity [23, 24]. Isoform 2 activates RIPK4 transcription and may be
required in conjunction with TP73/p73 to initiate p53/TP53-dependent apoptosis in response
to genotoxic insults and in the presence of activated oncogenes. Isoform 2 is involved in
Notch signaling, likely by inducing JAG1 and JAG2. It also plays a role in the regulation of
epithelial morphogenesis (figure 2). The ratio of DeltaN- and TA*-type isoforms may govern
the maintenance of epithelial stem cell compartments and regulate the initiation of epithelial
stratification from the undifferentiated embryonic ectoderm. Isoform 2 is required for limb
formation from the apical ectodermal ridge and activates transcription of the p21 promoter
[23, 24].
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1.5. Role of p63 in the Cell Cycle
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The role of p63 in cell cycle regulation depends on the cell type and context. Exogenous
TAp63γ responds to genotoxic stress and upregulates p21 expression, stops cell cycle
progression in erythroleukemia cells, whereas exogenous ΔNp63α binds directly to the p21
promoter to inhibit reporter expression [25]. Endogenous p63 expression in human primary
keratinocytes increases p21 expression and leads to reduced proliferation and G1 arrest.
Cyclin D1, CDK4, and CDK2 expression in mouse primary keratinocytes diminishes when
endogenous ΔNp63α is reduced.
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Thus, this pattern prevents cyclin D1 and CDK4 downregulation and rescues the
proliferation defects caused by the p63 deficiency [26, 27].
1.6. Senescence and Aging
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Cellular senescence and aging is related to p63. For example, germline disruption of p63
expression results in a dramatic increase in senescence-associated β-galactosidase (SA β-Gal)
staining in mouse embryos, which is phenocopied by somatic disruption of p63 expression in
basal epithelial cells using a K5 promoter. Importantly, blocking p63 expression with the
ΔNp63 isoform is affected by the K5 promoter [28]. As mentioned previously, disrupting
TAp63 expression in the basal epithelial surfaces does not renew the skin defects that arise
after disruption in adults.
Furthermore, genomic instability and increased DNA damage emerge as a result of the
absence of TAp63 in dermal precursor cells. Nevertheless, exogenous expression of TAp63
isoforms in cell culture leads to increased cellular senescence independently of p53, as
evidenced by increased SA β-Gal staining and decreased proliferation. These observations
highlight the importance of both ΔNp63 and TAp63 in cell senescence and demonstrate that
positive or negative modulation of either p63 isoform may result in senescence by different
molecular mechanisms [28, 29].
2. Significance of the p63 Gene in Diseases
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Human p63 heterozygous germline mutations are strongly associated with human
autosomal dominant developmental diseases [30]. p63 gene mutations in humans are
characterized by lip anomalies and/or ectodermal dysplasia.
Figure 3. p63 are associated with diseases.
112
Syndrome
(Hay-Wells
he
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ADULT:
LMS:
UMS:
Ankyloblepharon-Ectodermal Defects-Clefting
sendromu)
Acro-dermato-ungual-lacrimal-tooth
Limb-Mammary Syndrome
Rapp-Hodgkin Syndrome
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AEC:
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Mutations in the p63 gene are known to cause at least five different syndromes and two
non-syndrome human disorders; these include isolated split hand/foot and non-syndromic
cleft lip [31] (figure 3).
2.1. Ectrodactyly, Ectodermal Dysplasia, and Cleft Lip/Palate Syndrome 3
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Ectodermal dysplasia (ED), as well as cleft lip and/or cleft palate, sparse hair, skin,
malformed ears, fingers and toes, and partial or complete syndactylyis, is an autosomal
dominant syndrome of ED [32]. Previous studies have identified frequent mutations of five
amino acids, including R204, R227, R279, R280, and R304 in the EEC population, all located
in CpG islands. These five mutations occur in almost 90% of patients with EEC syndrome
[33]. P63 mutation analyses have identified eight mutations that prevent DNA binding in 9
unrelated EEC3 families. These eight mutations are amino acid changes, while the ninth is a
frame-shift mutation. To date, 34 mutations involving 20 amino acids have been reported in
EEC. Only the following two mutations are outside the DNA-binding domain: one insertion
(1572 InsA), and one point mutation (L563P) in the SAM domain [31] (figure 3). The five
p63 arginine hotspot mutations, and probably the other DNA binding domain mutations, that
are found in EEC syndrome appear to impair p63 protein binding to DNA. In this syndrome,
slight mental retardation and liver malfunction have been reported in some cases.
Ectrodactylia is defined as a defect described as the fusion or the absence of the fingers in the
central part of hands and feet [34]. Thus, the description of syndactylia is included in the
description of ectrodactylia. 84% of the patients with EEC syndrome present ectrodactylia,
while 77% present ectodermic dysplasia, 68% present cleft palate and/or lip, 59% present
lachrymal duct anomalies, 23% present genitourinary tract anomalies, 14% present deafness
and 7% present mental retardation [35].
The major diagnosis criteria of the syndrome are: ectodermic dysplasia, ectrodactylia,
cleft palate and/ or lip, lachrymal duct anomaiesl. If the patient does not present with any
other findings suggesting some other syndrome, the presence of at least two of the criteria is
characteristic. Considering the diagnosis criteria of the syndrome, even without cleft palate
and/or lip and lachrymal duct anomaly, patients with ectodermic dysplasia and ectrodactylia
meet the criteria requirements of diagnosis [37].
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2.2. Split-Hand/Foot Malformation (SHFM) 4
SHFM is a congenital malformation characterized by deep median clefts of the hands and
feet. SHFM may occur in isolation or as part of a multisystemic syndrome. Transmission is
autosomal dominant. The non-syndromic from of SHFM4 is caused by several mutations
dispersed throughout the p63 gene, including a point mutation in the TA (R58C), a splice-site
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mutation in front of exon 4 (3‘ss intron 4), four missense mutations in the DBD (K193E,
K194E, R280C, R280H), and two nonsense mutations in the TI-domain (Q634X, E639X)
[31, 37]. Several SHFM4 mutations cause alterations in p63 protein activation and stability.
Q634X and E639X are known to disrupt the sumoylation site and increase the stability and
transcriptional activity of the p63a isoform.
Furthermore, amino acids K193 and K194 are required for ubiquitin conjugation by E3
ubiquitin ligase (Itch), and naturally occurring mutations in these amino acids result in a more
stable p63 protein [38] (figure 3).
he
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2.3. Ankyloblepharon-Ectodermal Defects-Clefting (AEC) Syndrome
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Hay–Wells syndrome, also known as AEC syndrome, is a rare autosomal dominant
disorder characterized by congenital ectodermal dysplasia, including alopecia, scalp
infections, dystrophic nails, hypodontia, ankyloblepharon, and cleft lip and/or cleft palate.
Analysis of the p63 gene in eight patients with AEC syndrome identified a missense mutation
in the TP63 gene in one patient. A duplication of 11 bp in the p63 gene has been identified in
a patient with Rapp–Hodgkin signs [32] (figure 3).
2.4. Acro-Dermato-Ungual-Lacrimal-Tooth (ADULT) Syndrome
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ce
ADULT syndrome is characterized by ectrodactyly, excessive freckling,
onychodysplasia, obstruction of lacrimal ducts, and hypodontia and/or early loss of
permanent teeth. Clinical presentation is variable, and transmission is autosomal dominant.
Fourteen cases have been described to date. All families and one of the sporadic cases had a
point mutation in exon 8, changing R298 in the DNA-binding domain into either a glutamine
or a glycine [39] (figure 3).
2.5. Limb-Mammary (LMS) Syndrome
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LMS is a rare disease, and <50 cases have been described in the literature. This syndrome
is characterized by severe hand and/or foot anomalies and hypoplasia/aplasia of the mammary
gland and nipple. The clinical presentation is extremely variable. LMS is an autosomal
dominant disease caused by loss-of-function mutations in exons 13 and 14 of the TP63 gene,
localized to the subtelomeric region of chromosome 3 (3q27) [40]. One large LMS family (29
affected members) has a point mutation in exon 4, causing a G76W substitution in the DNspecific putative TA2. One other point mutation (S90W) is also located between the TA
domain and DBD [41]. Other LMS mutations have been detected in the C-terminus, including
a TT deletion in exon 13 and an AA deletion in exon 14. These affect only the p63a protein
isoforms, in which they are predicted to cause a frame-shift mutation and a premature stop
codon [42].
Additionally, a stop mutation in the transcription factor inhibitory domain (TI) (K632X)
has been identified in a patient with sporadic LMS. The latter mutation is predicted to impair
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the suppressive effect of the TI domain on the TA domain, thus increasing transactivation
activity [31].
2.6. Rapp–Hodgkin Syndrome (RHS)
is
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RHS is characterized by the association of anhidrotic ectodermal dysplasia with cleft
lip/palate.
The syndrome is usually evident at birth but its prevalence is unknown, as < 100 cases
have been reported in the literature [43]. RHS mutations are located in the C-terminus of the
p63 protein, and RHS is transmitted as an autosomal–dominant trait. RHS mutations are
located in the C-terminus of the p63 protein. They are either point mutations in the SAM
domain or deletions in the SAM or TI domains. A mother and daughter with RHS syndrome
and corneal dystrophy revealed a deletion in the gene 1BP of the tumor protein p73-like
(TP73L) gene. A heterozygous missense mutation in the TP73L gene was identified in a 14year-old boy with RHS syndrome [31].
Pu
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3. Role of the p63 Gene in Cancer
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Researchers have identified that ΔNp63α, but not the ΔNp63γ or TAp63 isoforms,
modulate Erk2 signaling to inhibit mammary cell migration, invasion, and metastasis (figure
4). Furthermore, another study showed that Sharp-1 and cyclin G2 are two clinically relevant
p63 targets that inhibit cell migration and invasion. p63 inhibits RCP-mediated integrin α5β1
recycling to the plasma membrane via an unknown mechanism, thus downregulating
downstream signaling from these complexes to Akt and thereby inhibiting cell migration and
invasion. H1299 cells express mainly TAp63 isoforms, and TAp63α over-expression reverts
cell invasion induced by mtp53 back to control levels in this system [11].
There are many studies on the role of the p63 gene in neoplastic transformation. The p63
gene is widely investigated because it is thought to play a role in the regulation of neoplasm
proliferation and differentiation due to its determination in various solid tumors and epithelial
cells [44].
According to the analyses of the p63 gene in cancer studies, this gene is defined as an
oncogenous agent and not as a tumor repressing gene. The mutation of p63 is rare in human
cancers. Gene amplification and protein over expression are more frequent in skin, cervix,
oral cavity, bladder, nasopharynx, head and neck carcinoma [45,46]. As p63 gene expression
is limited to the epithelial cells, it can be involved in proliferation and differentiation of
premalignant and malignant lesions of epithelial origin. Di Como et al. studied the presence
of p63 expression in various human tumors using microarray technology. While a high level
of p63 expression is observed in basal and squamous cell carcinoma and transitional cell
carcinoma, no expression was observed in adenocarcinoma including in organs such as breast
and prostate [47,48]. High level of p63 gene expression has been observed in thymoma.
While this gene is expressed in some Non Hodgkin lymphoma, no expression has been
detected in tumors such as soft tissue sarcoma, mesothelioma or hepatocellular carcinoma.
115
Table 2. Major p63 expression and gene changes in cancer
Bladder cancer
Lung SCC
Prostate cancer
Prostate cancer
Urist et al.2002.
Massion et al.2003.
ΔNp63α predominant protein and reflects platinum
response and favorable outcome
Notes
Low p63 associates with higher TNM and low β-catenin;
p63 prognostic effect is independent of TNM
ΔNp63 associate with invasiveness and upregulation of Ncadherin
Correlation of podoplanin/CD44/ p63 in a hierarchical
manner
p63 marker of early neoplastic lesion (intraepithelial
neoplasia versus adenocarcinoma)
ΔNp63α more abundant than TAp63; 1 mutation exon 8;
Zangen et al.2005.
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a
Lung SCC
Lung SCC
Lymphoma
Thyroid
carcinoma
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Di Como et al 2002.
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Nylander et al.2002.
Sniezek et al.2004.
is
p63+ in dysplastic oral mucosa, with loss of E-cadherin
p63+ prognostic factor in ER+ Pt; no correlation with
standard parameters
p63 distinguish AC, (−ve) from SCC, (+ve)
ΔNp63 is the isoform that distinguish AC from SCC
ΔNp63 correlates with high MW cytokeratins in basal
cancer, more aggressive with poor prognosis
p63 improves identification of myoepithelial cells
MFG-E8 (ligand of integrin αvβ3-5) is a p63 target in vivo
NSCLC differential diagnosis: p63+ in 100% SCC, 10%
AC); p63 & cytokeratin5/6 allow accurate classification
77% cases
Cytokeratin5/7, TTF1, p63 allow accurate classification of
all NSCLC cases
Higher ΔNp63/TAp63 ratio in NSCLC indicating poor
outcome
TAp63 is the most expressed isoform
p63+ in 67% follicular adenoma, 41% papillary, 29%
follicular carcinoma
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Lung SCC
Lung SCC
Bladder
carcinoma
Breast cancer
Breast cancer
Lung SCC
Quade et al.2001.
nc
.
Loss of ΔNp63 associate with progression/invasion
Increased copy number in 88% SCC, 42% LCC, 11% AC;
ΔNp63α predominant protein, associated with better
prognosis
Head and neck SCC p63+ ΔNp63α predominant protein
en
Pre-cancerous
conditions
Breast cancer
Reference
Wang et al.2001.
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Head and neck
cancer
Head and neck
cancer
Cancer
Bladder cancer
Notes
p63 marker of squamous differentiation; HPV-p63
association
p63 preferentially expressed in immature squamous cells;
useful for differential diagnosis in early stages
p63 expressed in thymomas, non-Hodgkin's lymphoma,
basal cell/SCC, not in adenocarcinomas of breast/prostate
ΔNp63 is the most expressed isoform
ce
Cancer
Cervical
carcinoma
Cervical
carcinoma
Distinct
cancers
Distinct
cancers
Bladder cancer
Lung SCC
Reference
Barbieri et al.2006.
Fukushima et
al.2009.
Shimada et al.2009.
Hull et al2009.
Kellogg Parsons et
al.2009.
Das et al. 2010.
Hanker et al.2010.
Terry et al.2010.
Uramoto et al.2010.
Karni-Schmidt et
al.2011.
Aikawa et al.2011.
Yang et al. 2011.
Mukhopadhyay and
Katzenstein.2011.
Righi et al.2011.
Iacono et al.2011.
Iacono et al.2011.
Tan et al.2011.
116
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The p63 gene is known to be expressed in 93% of SCC of the lung, 10% of breast ductal
carcinomas, and 25% of ovary endometrial carcinomas. A low level of p63 gene expression
has been reported to be rare in breast, lung or prostate adenocarcinoma. The ΔNp63 gene has
been detected to be over expressed in neuroblastoma, colorectal carcinoma, bladder cancer,
nasopharynx cancer, head and neck SCC and hepatocellular carcinoma.
Figure 4. The p63 protein inhibits metastasis via multiple mechanisms.
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3.1. Role of p63 in Epidermis, Cutaneous Adnexal and Skin Cancer
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Ivan et al. have studied the relation between the p63 gene and primary cutaneous tumors
and Skin adenocarcinoma mestastasis. as‘According to their results, the p63 gene was found
to be a sensitive and specific determinant for benign or malignant adnexal tumors. There has
been observed no expression of p63 in metastatic carcinoma ,thus, they proposed that the p63
gene may be helpful in the differentiation of primary skin cancer and metastatic skin
adenocarcinoma [49]. Parsa et al. showed that the p63gene is expressed in vitro in normal
cells as well as neoplastic keratinocytes and that this gene constitutes an indicator for the
proliferation capacity of keratinocytes. It has also been postulated that the p63 gene can be a
diagnosis indicator of anaplasia in keratinosid tumors [50].
Laurikalla et al. studied the expression of 2 isoforms of the p63 gene in embryonic molar
teeth of mice; they observed that p63 gene expression is high at a specific step of the tooth
enamel epithelium.
The absence of Np63 expression in internal epithelial cells that differentiate into
ameloblasts has been reported. It has been demonstrated that Np63 gene expression continues
in the epidermis as well as in tooth and hair follicle epithelial cells and that it is down
regulated during the differentiation of keratinocytes and ameloblasts.. No transactivated TA
p63 isoform expression has been observed. Kumonoto et al. studied p63 and p73 gene
expression in tooth germ and ameloblasts (48 healthy and 5 malign). They observed that the
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p63 gene expression is high in desmoplastic ameloblastoma, acanthomatous and granular cell
ameloblastoma. They also found the TA p63 gene in 5 of 8 tooth germs andin 16 of 34
ameloblastomas (5 malignant ameloblastomas). Finally, Np63 has been observed in all
developing and neoplastic odontogenic tissues.
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3.2. Role of p63 in Bladder Cancer
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3.3. Role of p63 in Breast Cancer
is
he
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Urist et al. studied the p63 expression rate in 102 bladder variant epithelial cell carcinoma
(VECC). The expression rates of of p63 in the highly differentiated papillary surface tumors
was 93% and in the less differentiated surface cells it was 68%. The expression of p63
decreased in invasive tumors (16%). In relation with this, the phase and grade-based
urothelial differentiation in bladder VECC is proposed to be accompanied by p63 gene
expression [46]. In VECC, the decrease of p63 gene expression can induce the loss of p63
isoforms that inhibit growth and differentiation. It has been proposed that the p63 gene is not
necessary for the formation of bladder epithelium but is required for its differentiation [46].
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In a study about the relation between p63 and p53 gene expression in normal and
neoplastic breast tissue, no p63 gene expression was observed in intraductal carcinoma,
tubular carcinoma, lobular carcinoma, medullar carcinoma and grade I and II invasive ductal
carcinoma tumor cells. However, P63 gene expression has been detected in grade III invasive
ductal carcinoma. These results lead to the hypothesis that p63 gene is an indicator of cells
with pathological differentiation in breast cancer. P63 displayed expression in the
myoepithelial layer surrounding the normal ductal and alveolar epithelium [51]. In 21.17% of
breast carcinoma presented p53 expression, 11.76% of these carcinoma presented p63
expression. The expression of p63 is associated with poor prognostic parameters such as the
phase, size, histological differentiation, lymph node metastasis and estrogen receptor negative
status of the tumor. According to these results, the p63 gene is thought to be an indicator of
the aggressiveness of breast carcinomas [52].
3.4. Role of p63 in Cervix Cancer
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Based on some studies, it has been proposed that the p63 gene is expressed in basal and
immature cervical squamous epithelium. In a study including 96 cases of cervix SCC, Wang
et al. detected p63 gene expression in97% of cases SCC, while no expression was observed in
adenocarcinomas. They observed a significant relation between p63 gene expression and
squamous differentiation in the cervical transformation area. They did not detect any p63 in
normal mature endocervical epithelium [53]. It has been proposed that p63 may be used as a
marker for the basal cells of the ectocervix epithelium, for the identification of cervical stem
cells [53, 54].
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3.5. Role of p63 in Lung Cancer
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3.6. Role of p63 in Oral Cancer
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In a study of Pelosi G about the lung SCC, a high level of p63 immunoreactivity was
observed in precursor lesions such as squamous metaplasia and dysplasia, and in gradient
neuroendocrine tumors. In another study of p63 in lung tumors, expression was found to be
96.9%, 30%, and 37% in SCC, adenocarcinoma and in large cell carcinoma, respectively. In
neuroendocrine tumors, p63 expression increased with tumor grade, and an immunoreactivity
level of 1.9%, 30.8%, 50%, 76.9% was detected for classical carcinoids, atypical carcinoids,
large cell neuroendocrine carcinoma, and small cell carcinoma, respectively [71]. While p63
expression was observed in basal and suprabasal cells of the bronchial epithelium in lung
SCC, a loss of expression was seen in more superficial, differentiated cell layers. p63
expression increased with increasing intesity of bronchial epithelial dysplasia. The maximum
level of p63 expression was reported in SCC. In the same study [74], no significant difference
was observed between p53 and p63 expressions.
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One of the most frequently encountered malignant pathologies in the oral cavity is
squamous cell carcinoma. The relation between p63 expression and squamous cell carcinoma
and prognosis has been studiedextensively. The prognostic indicator of oral squamous cell
carcinoma is lymphatic tissue metastasis and 3q21-29 chromosome (p63 gene location)
amplification. Chen et al. observed that p63 expression is correlated with the level of oral
epithelial dysplasia. According to these observations, the increase of p63 expression has been
proposed as an important actor in early oral carcinogenesis [48]. In a similar study, Lo Muzio
et al. described the correlation between p63 expression in SCC and tumor differentiation.
Cases with diffuse p63 expression have been observed to be more aggressive and poorly
differentiated. The overexpression of p63 is found to be related to poor prognosis in SCC.
3.7. Role of p63 in Head – Neck Cancer
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The loss of expression of TAp63 has been related to tumor progression in laryngeal
carcinoma. In basal and suprabasal layers of the epithelium in normal laryngeal mucosa,
nuclear immunoreactivity with p63 has been detected in the myoepithelial cells of the
seromucinous glands. In laryngeal intraepithelial neoplasia, p63 immunoreactivity is not
limited to the basal layer. p63 expression has been observed in all layers of the epithelium.
p63 expression has also been observed in tumor cells of all larynx SCC at a rate varying
between 10% and 98%. Furthermore, tumor localization, lymphatic node metastasis, clinical
phase and smoking are found to be associated with tumor recurrence. As p63 expression has
been detected in laryngeal intraepithelial neoplasia, p63 is suspected to play a role in early
stages of the development of larynx cancer. Sniezek et al. studied the expression of p53 and
p63 in head and neck SCC. Out of a total of 36 cases, p53 expression was observed in 72%
and p63 expression was observed in 100% of patient samples. . In the 10 patients whose
cancers were not positive for p53 expression, a high level of p63 expression was detected.
The difference between p53 and p63 expression was statistically significant. In relation to
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this, it has been proposed that p63 may play an oncogenic role by antagonizing the p53
pathway in head and neck SCC [18,55]. In the same study, Western Blot analysis revealed
that the ΔNp63α is the most dominant isoform in head and neck SCC and that it is more
highly expressed in tumors when compared to normal contiguous tissue. It has been proposed
that the ΔNp63α isoform plays an anti-differentiation and anti-apoptotic role in mucosal
epithelium in head and neck SCC and that it may lead to the development of tumors.
Besides, higher p63 gene expression is associated with poor prognosis and disease free
survival [56].
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4. p73 General Information: Symbols, Location,
Size, Cofactor, Subunit
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Pu
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HGNC Approved Gene Symbol: TP73
Cytogenetic location: 1p36.3
Subcellular location: nucleus and cytoplasm.
Size: 636 amino acids; 69623 Da
Cofactor: binds one zinc ion per subunit
Subunit: found in a complex with p53/TP53 and CABLES1. The C-terminal
oligomerization domain binds to the ABL1 tyrosine kinase SH3 domain and interacts with
HECW2. The beta isoform interacts homotypically with p53/TP53, whereas the alpha isoform
does not. The gamma isoform interacts homotypically with all p73 isoforms. The delta
isoform interacts with the gamma and alpha isoforms homotypically. The alpha and beta
isoforms interact with HIPK2. The alpha isoform interacts with RANBP9, and the beta
isoform with WWOX. Interacts (via SAM domain) with FBXO45 (via B30.2/SPRY domain).
Interacts with YAP1 (phosphorylated form). Interacts with HCK (via SH3 domain); this
inhibits TP73 activity and degradation (http://www.genecards.org/cgi-bin/carddisp.
pl?gene=TP73).
4.1. p73 Molecular Structure
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The TP73 gene is composed of 14 principal exons. Primary transcripts generated from
two alternative promoters (P1 and P2) undergo differential splicing to generate multiple
isoforms. p73 (TA-p73) contains a N-terminal transactivation domain (TAD), followed by a
proline-rich sequence (PR), a central DNA-binding domain (DBD), and a C-terminal
oligomerization domain (OD), involved in the formation of tetramers (figure 5). The
sequences of the DBD, TAD, and OD regions of p73 and p53 exhibit 63%, 29%, and 38%
similarities [57].
4.2. p73 Gene Function
Evidence from in vitro studies indicates that p73 has a tumor suppressive role. Isoforms
containing the transactivation domain are pro-apoptotic, whereas those lacking the domain are
120
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nc
.
anti-apoptotic and block the function of p53 and transactivating p73 isoforms. P73 stimulates
the transcription of genes like p21WAF1/Cip1, RGC (Ribosomal Gene Cluster), mdm2, bax,
cyclin G, GADD45, IGF-BP3 (insulin-like growth factor-binding protein 3) whose
transcription are regulated by p53 and can pause the cell cycle in G1 phase.
Figure 5. Structure of the p73 gene and encoded proteins.
en
ce
Studies have demonstrated that p73 is required for the construction of specific neural
structures. One of these neural structures is a bipolar neuron named Cajal-Retzius. These
neurons are responsible for the cortex organization of the hippocampus. In p73 knock-out
mice, these neurons have been selectively lost thus leading to hippocampus dysgenesis. These
mice also present many limbic telencephalon malformations. In p73 knock-out mice (p73-/-),
there are neurological, pheromone and inflammatory defects but there is no spontaneous
tumor development [57].
Sc
i
4.3. Regulation of p73 Protein Levels
N
ov
a
TA-p73 and ΔN-p73 isoform protein levels might be regulated by other factors. The
intracellular p73 level does not change after applying DNA damaging agents such as
actinomycin D or UV. Degradation of p73 occurs through binding to p53 and by mouse
double minute 2 (MDM2), which directs the p53 protein to the ubiquitin-proteosome
pathway. MDM2 does not trigger p73 ubiquitination. However, it can catalyze p73
neddylation (the conjugation of the small ubiquitin-like protein NEDD8), which inhibits p73
transcriptional activity. These observations show that although p73 can perform the functions
of p53, it is not stimulated by DNA damage and is regulated in a manner different than is p53.
The NEDD4-like ubiquitin ligase Itch is an important regulator of the p73 protein level. It
recognizes a PY motif (the PPxY amino acid sequence) in the C-terminal region of p73 that is
not present in p53. c-Jun prevents degradation of TA-p73. Degradation of ΔN-p73 is
triggered at the same time by the nonclassical polyamine-induced antizyme (Az) pathway.
121
ce
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nc
.
The Az antizyme (a small protein initially identified as an inhibitor of ornithine
decarboxylase) is the first key enzyme in the polyamine biosynthesis pathway.
Az expression is promoted by c-Jun upon genotoxic stress, leading to proteasomemediated ubiquitin-independent degradation of ΔN-p73. The p73-induced RING 2 protein
(PIR2), a ring-finger domain ubiquitin ligase, regulates the TA- to ΔN-p73 isoform ratio.
Notably, PIR2 is a transcriptional target of TA-p73 that preferentially degrades ΔN-p73, thus
releasing TA-p73 and triggering apoptosis following DNA damage.
en
Figure 6. The p73 pathway and its regulators.
4.4. p73 as a Novel Target of Anticancer Therapies
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The p53 homolog p73 is frequently overexpressed in cancers. In particular, the
transactivation domain truncated isoform DNp73 has oncogenic properties, and its
upregulation is associated with poor patient survival. The p73 pathway is an attractive target
for cancer drug development. Experimental and clinical evidence demonstrates that TA-p73
isoforms have the potential to alter p53 functions in cancer cells by inducing apoptosis after
DNA damage. Conventional chemotherapeutic drugs can increase TA-p73 levels by
activating the E2F1-TA-p73 axis. TA-p73 activities are frequently dampened in tumors by
mutation or deregulated expression of p73 modulators or co-factors. The anticancer drugs
used in selection of the p73 target are presented in Table 3.
122
Table 3. List of Drugs Targeting the p73 Pathway
PML-RAR
Nutlin-3
MDM2
Reduction of N-p73 levels
and increase of p300mediated acetylation of
p73 in APL cell lines.
Increased expression of
both TA- and N- p73
expression in primary APL
cells.
Displacement of MDM2E2F1-p73 complex
PD9805,
PD184352
MEK1
37aa peptides
iASPP
RETRA
Mutant p53 (?)
Aptamers
Mutant p53
en
Unknown
SIMP peptides
Mutant p53
Disassembly of mutp53/p73 complex
Sc
i
Induction of TA-p73
expression
mTOR, NF- kB
Enzastaurin
(LY317615.HCL
PKCkinases
Forodesine
Unknown
Apoptosis in p53null or mut-p53
expressing cells in
combination with
chemotherapeutic
drugs (doxorubicin)
p73-mediated
apoptosis alone or in
co-administration
with ATO
Vassilev LT, et
al. 2004.Pierce
SK, et al. 2009.
p73-dependent
apoptosis in vitro
and in tumor
xenograft in vivo
p73-dependent
inhibition of cell
growth in vitro and
in vivo in tumor
xenografts
Apoptosis in mutp53 expressing cells
p73-dependent
apoptosis in p53null cells
p73-dependent
apoptosis in mutp53 expressing cells
in combination with
chemotherapeutic
drugs (doxorubicin)
p73-dependent
apoptosis
Bell HS, et al.
2007.
Pu
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ce
Disassambly of mutp53/p73 complex
Aurora kinase A
a
ov
N
Alteration of TA/ N-p73
ratio: reduction of N- p73
levels and accumulation
and tyrosine
phosphorylation of TA-p73
Disaasembly of iASPP-p73
complex
MLN8054
Curcumin
Reference
Bykov VJ, et
al. 2002.
NF-kB and mTOR
inhibition and TA-p73
accumulation and
activation
Accumulation of -catenin,
that promotes c-Jundependent induction of p73
Increased TA-p73
transcription
nc
.
Arsenic trioxide
(ATO)
Effects
Apoptosis in tumor
cells expressing
mutant-p53, alone or
in combination with
cisplatin
p73-mediated
apoptosis, alone or
by co-administration
with MEK1
inhibitors
(PD98059,
PD184352)
Lunghi P, et al.
2004.
,I
Mechanism of Action
Reactivation of mutant p53
through covalent binding to
their core domain
he
rs
Target
Mutant-p53
is
Drug
Prima-1, Prima1MET
(APR-246)
p73-dependent
apoptosis in multiple
myeloma cells
Apoptosis in CLL
cells, alone or in
combination with
bendamustine and
rituximab
Lunghi P, et al.
2004. Lunghi
P, et al. 2006.
Kravchenko
JE, et al. 2008.
Guida E, et al.
2008.
Dar AA, et al.
2008.
Di Agostino, et
al. 2008.
Chakraborty J,
et al.
2010.Beevers
CS, et al. 2009.
Raab MS, et al.
2009.
Alonso R, et al.
2009.
123
Unknown
Induction of CD154
expression, that trigger the
c-Abl-mediated activation
of p73
Panobinostat
(LBH589)
HDACs
Thymoquinone
Unknown
E2F1- and myc-mediated
transcription of miR-106b,
that targets the p73
ubiquitin ligase Itch
Increased TA-p73 protein
level
Reference
Rosenbluth
JM, et al. 2009.
Wong SW, et
al. 2010.
p73-dependent cell
cycle arrest and
apoptosis in acute
lymphoblastic
leukemia (ALL)
Jurkat cell line
Alhosin M, et
al. 2010.
nc
.
Lenalidomide
(CC-5013, or
evlimid)
Effects
p73-dependent cell
death, increased by
cisplatin coadministration in
basal-like triple
negative breast
cancer cells
CD95-mediated or
fludarabine-induced
c-abl/p73 dependent
apoptosis in p53deficient CLL cells
TA-p73 induced
apoptosis in CLL
cells
Lapalombella
R, et al. 2010.
,I
Mechanism of Action
Direct mTOR inhibition
and increased TA-p73
levels
Sampath D, et
al. 2010.
he
rs
Target
FKB12
Pu
bl
is
Drug
Rapamycin
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follicular adenoma. Ann. Diagn. Pathol., 15: 108-116.
[84] Terry J, Leung S, Laskin J, Leslie KO, Gown AM, Ionescu DN. 2010. Optimal
immunohistochemical markers for distinguishing lung adenocarcinomas from
squamous cell carcinomas in small tumor samples. Am. J. Surg. Pathol., 34: 1805-1811.
[85] Uramoto H, Yamada S, Hanaquiri T. 2010. Immunohistochemical staining with
deltaNp63 is useful for distinguishing the squamous cell component of adenosquamous
cell carcinoma of the lung. Anticancer. Res., 30: 4717-4720.
[86] Vera-Carbonell A, Moya-Quiles MR, Ballesta-Martínez M, López-González V,
Bafallíu JA, Guillén-Navarro E, López-Expósito I. 2012. Rapp-Hodgkin syndrome and
SHFM1 patients: delineating the p63-Dlx5/Dlx6 pathway. Gene, 15;497(2): 292-297.
[87] Visinoni AF, Lisboa-Costa T, Pagnan NA, Chautard-Freire-Maia EA. 2009. Ectodermal
dysplasias: clinical and molecular review. Am. J. Med. Genet. A, 149A(9): 1980-2002.
[88] Yang C, Hayashida T, Forster N, Li C, Shen D, Maheswaran S et al. 2011. The integrin
alpha(v)beta(3–5) ligand MFG-E8 is a p63/p73 target gene in triple-negative breast
cancers but exhibits suppressive functions in ER(+) and erbB2(+) breast cancers.
Cancer Res., 71: 937-945.
ISBN: 978-1-62808-665-2
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Chapter 7
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The Emerging Roles of Forkhead Box
(FOX) Family Proteins in Tumor
Suppression
Pu
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Pang-Kuo Lo
The Department of Biological Sciences, University of South Carolina,
Columbia, SC, US
Abstract
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The FOX gene family comprises gene members that encode forkhead box
transcription factors, which contain an evolutionarily conserved DNA-binding domain
termed the forkhead box or winged helix domain. FOX protein family members have
been recognized as pivotal transcriptional factors to regulate a wide spectrum of
biological processes, including metabolism, development, differentiation, proliferation,
apoptosis, cellular mobility, vascularization and longevity. Given that FOX proteins
control these imperative developmental and homeostatic processes, dysregulation of
expression and functions of FOX gene family members can lead to alterations in cell fate,
tumorigenesis and cancer progression. In this chapter, the emerging roles of FOX family
transcription factors in tumor suppression are discussed based on the growing evidence.
This comprehensive chapter covers topics regarding functional roles of these FOX tumor
suppressors in cancer, pathological mechanisms giving rise to down-regulation of their
expression as well as inhibition of their functions in cancer, and the potential of FOX
proteins as targets for therapeutic intervention in cancer. In addition to the main focus on
the tumor-suppressor roles of FOX proteins, the complexity of their dual roles as tumor
suppressors and oncogenes in tumorigenesis and cancer progression is also discussed.
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Keywords: Forkhead box transcription factors (FOXs), forkhead box-O (FOXO), forkhead
box-F (FOXF), forhead box-L (FOXL), forkhead box-P (FOXP), tumor suppressor

Correspondence to: Pang-Kuo Lo, Ph.D. Research Assistant Professor, Department of Biological Sciences,
University of South Carolina, 715 Sumter Street, CLS 601, Columbia, SC 29208, TEL: +1-803-777-7030,
FAX: +1-803-777-4002Email: [email protected]; [email protected].
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AMP-activated protein kinase
autophagy-related 12 homolog
ataxia telangiectasia mutated
ataxia telangiectasia and Rad3 related
B-cell lymphoma
BCL2/adenovirus E1B 19kDa interacting protein 3
cancer-associated fibroblasts
CREB-binding protein
cyclin-dependent kinase 2
cyclin-dependent kinase inhibitor
casein kinase 1
chronic myeloid leukemia
cAMP response element-binding protein
cancer stem cells
dual-specificity tyrosine-phosphorylated-regulated kinase 1A
eukaryotic translation initiation factor 4E binding protein
epithelial-to-mesenchymal transition
extracellular signal-regulated kinases 1 and 2
four and a half LIM 2
glucose 6 phosphatase
GABA-receptor-associated protein-like 1
herpesvirus-associated ubiquitin-specific protease
hypoxia-inducible factor 1
Immunohistochemistry
IkappaB kinase  InsR, insulin-like growth factor receptor
insulin receptor substrate-1
insulin receptor substrate-2
c-Jun N-terminal kinase
microtubule-associated protein 1A/1B-light chain 3
leukemia initiating cells
mitogen-activated protein kinases
RING-finger E3 ligase murine double minute 2
myeloid/lymphoid or mixed lineage leukemia
Mn superoxide dismutase
mammalian orthologue of the ste20-like protein kinase
mammalian target of rapamycin complex 1
mammalian target of rapamycin complex 2
nuclear export sequence
nuclear localization signal
ovarian granulosa cell tumors
phosphoenolpyruvate carboxykinase
peroxisome proliferative-activated receptor- coactivator 1
phosphoinositide 3-kinase
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AMPK
ATG12
ATM
ATR
BCL6
6BNIP3
CAF
CBP
CDK2
CDKI
CK1
CML
CREB
CSCs
DYRK1A
4E-BP
EMT
ERK1/2
FHL2
G6Pase
GABARAPL1
HAUSP
HIF1
IHC
IKK
IRS1
IRS2
JNK
LC3
LICs
MAPK
MDM2
MLL
MnSOD
MST1
mTORC1
mTORC2
NES
NLS
OGCTs
PEPCK
PGC-1
PI3K
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Abbreviations
The Emerging Roles of Forkhead Box (FOX) Family Proteins …
Introduction
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The forkhead box (FOX) gene family is composed of genes encoding evolutionarily
conserved transcriptional regulators with a common DNA-binding domain named the
forkhead box or winged helix domain [1, 2]. The forkhead box DNA-binding domain of
approximately 100 amino acids in length was originally discovered and defined as the
conserved protein region among Drosophila Fork head and mammalian FOXA family
proteins [3, 4]. Since the first forkhead box protein was identified in Drosophila
melanogaster, a transcription factor that promotes terminal rather than segmental
development, there are at least 41 genes currently identified in humans, which are classified
into 17 FOX gene subfamilies [1]. FOX gene family members are evolutionarily expanded
from worms to mammals to fulfill the needs of increased developmental and tissue
complexity. In spite of a conserved forkhead DNA-binding domain, different subfamilies of
FOX proteins have differential regulation and functional diversification, which are
attributable to protein sequence variations outside of the forkhead domain [1]. These nonDNA-binding protein regions are engaged in interaction with components of transcriptional
activators, transcriptional repressors, or DNA repair complexes to regulate gene transcription
and DNA repair [5].
FOX protein family members are imperative for a wide array of biological processes,
including developmental embryogenesis as well as organogenesis, metabolism, immune
responses, differentiation, proliferation, apoptosis, migration, invasion and longevity [2, 5-9].
Due to the essential roles of FOX proteins in regulating these developmental and homeostatic
processes, it is expected that dysregulating the expression and functions of FOX protein
family members can alter cell fate and give rise to tumorigenesis. Indeed, it is known that
many FOX subfamilies such as FOXO, FOXM, FOXP, FOXC, FOXA, FOXE, FOXQ,
FOXR and FOXF have been linked to tumorigenesis and the progression of certain cancers
[5, 9]. Genetic and epigenetic deregulation of the functions of these FOX proteins acting as
oncogenes, tumor suppressors or bi-functional factors are involved in initiation, progression
and metastasis of cancer [5].
Therefore, an understanding of dysregulating FOX protein expression and function is
crucial to addressing the potential that FOX proteins are employed as direct targets and/or
indirect effectors of therapeutic intervention.
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protein kinase B
phosphatase and tensin homolog
regulatory associated protein of mTOR
rapamycin-insensitive companion of mTOR
reactive oxygen species
S6 kinase
serum and glucocorticoid inducible kinase
S-phase kinase-associated protein 2, E3 ubiquitin protein ligase
regulatory T cells
tuberous sclerosis complex 1 and 2
von Hippel-Lindau tumor suppressor ubiquitin ligase
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PKB
PTEN
Raptor
Rictor
ROS
S6K
SGK
SKP2
Treg
TSC1/2
VHL
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Table 1. Dysregulation of FOX tumor suppressors in cancer
Chromosomal deletions,
somatic inactivating
mutations, alternative
splicing variants,
cytoplasmic
mislocalization
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Breast, colorectal and
prostate cancers
Somatic inactivating
mutations, loss of or
reduced expression
Ovarian Granulosa Cell
Tumors (OGCTs)
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Chromosomal deletions,
epigenetic silencing,
microRNA silencing,
cytoplasmic
mislocalization
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FOXL2
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FOXFs
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An X-lined tumor
suppressor in epithelial
cells acts as a
transcriptional repressor
for HER2, SKP2 and cMYC genes, but an
activator for the p21
gene.
Negatively regulates
E2F target genes to
inhibit the DNA
replication process and
activates DNA repair
genes.
Transcriptionally
activates target genes
with roles in cell cycle
arrest, apoptotic
regulation, ROS
detoxification and
inhibits metastasisrelated genes
Types of cancer with
dysregulation
Prostate cancer
(deletion), alveolar
rhabdomyosarcoma
(translocations),
leukemia
(translocations), various
types of epithelial
carcinomas with
deregulated PI3K-AKT,
MEK-ERK, and IKK
signaling (e.g. breast,
lung, gastric cancers)
Breast, prostate and
ovarian cancers
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FOXP3
Transactivates target
genes implicated in cell
cycle arrest, apoptosis,
autophagy, DNA repair,
ROS detoxification.
Mechanisms of
dysregulation in cancer
Chromosomal
translocations, deletions,
cytoplasmic
mislocalization,
proteasomal degradation,
microRNA silencing
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Tumor-suppressor roles
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Forkhead
subfamily
FOXOs
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Here, this chapter focuses on the recent advances in the emerging roles of tumorsuppressor FOX proteins in cancer, their cancer-specific dysregulation and therapeutic
implications in cancer therapy.
Therefore, this chapter encompasses FOXO, FOXP, FOXF and FOXL subfamilies due to
their roles in tumor suppression (Table 1). Since oncogenic FOX factors are not in the scope
of this chapter, the readers can refer to several recent review articles covering the functional
roles and dysregulation of oncogenic FOX proteins (e.g., FOXA, FOXM, FOXC and FOXQ)
in cancer if readers are interested in these topics [5, 9]. In addition, we discuss that tumor-
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A. FOXO Subfamily Genes
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1. The Biological Functions of FOXO Genes
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suppressor FOX proteins can exhibit bi-functional characteristics as oncogenes or tumor
suppressor genes in a context-dependent manner.
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The forkhead box-O (FOXO) subfamily genes comprise FOXO1 (FKHR), FOXO3
(FKHRL1), FOXO4 (AFX) and FOXO6. The RXRSCTWPL motif in the N-terminal protein
region and the RRRAXSMD motif in the forkhead box domain are conserved in all members
of the FOXO subfamily, whereas the longer motif with a sequence
RXRXXSNASXXSXRLSP in the middle protein region is only conserved among FOXO1,
FOXO3, FOXO4 proteins [5]. In line with this, according to the homology analysis of FOXO
subfamily protein members, FOXO1, FOXO3 and FOXO4 proteins are significantly
homologous to each other, but they are less related to FOXO6 in mammals [10]. It is known
that FOXO transcription factors can bind to the consensus DNA sequence, TTGTTTAC or
BBTRTTTTD [11-13], or interact with other transcription factors (e.g. SMAD proteins) to
regulate the transcription of target genes [14, 15]. Besides, transcriptional co-activators (e.g.
histone acetyltransferase and CREB binding protein CBP/p300) [9, 16] or repressors (e.g.
histone deacetylase SIRT1) have been found to interact with FOXO proteins [17-19], which
ultimately determine the outcome of gene-transcriptional regulation by FOXO proteins.
Currently there are numerous identified FOXO target genes functionally involved in
regulating cell cycle progression, DNA repair, apoptosis and homeostasis of reactive oxygen
species (ROS) [20] (Figure 1).
For example, activation of FOXO transcription activity leads to up-regulating the
expression of cyclin-dependent kinase inhibitor (CDKI) genes p21Cip1, p27Kip1, p15INK4b,
p19INK4d and retinoblastoma-like p130Rb2, resulting in cell cycle arrest at the G0/G1 phase
[21-24]. For apoptotic regulation, FOXO transcription factors enhance the transcription of
genes encoding the apoptotic inducers or mediators to elicit the onset of apoptosis, such as
Fas ligand (FasL), TRAIL, the Bcl-2-like protein BIM and B-cell lymphoma 6 (BCL6),
which mediates transcriptional down-regulation of the pro-survival factor BCL-XL [12, 2531]. Moreover, gene products implicated in DNA damage responses (e.g. GADD45 with the
functional abilities to arrest cell cycle progression and repair damaged DNA) and in
protecting cells from ROS-induced DNA damages (e.g. Mn superoxide dismutase MnSOD
and catalase) are transactivated by FOXO proteins [14, 32-34], which are cell survival
mechanisms mediated by FOXO proteins. Therefore, FOXO proteins control cell survival and
death in a context-dependent manner.
In addition to the well-know roles of FOXO factors in regulating cell cycle progression,
DNA repair and apoptosis, they also play crucial roles in the regulation of cellular
autophagy/atrophy and glucose metabolism (Figure 1). It has been initially highlighted that
FOXOs are important mediators of the muscle-related atrophy process. Forced expression of
a constitutively active form of FOXO3 in fully differentiated skeletal and cardiac muscle cells
results in atrophy [35-37]. Particularly, FOXO-triggered muscular atrophy results from a
reduction in cell size, not from apoptosis [35].
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Growth Factor
Stress
PIP3
PIP2
AKT
MST1
JNK
IRS1/2
PTEN
PI3K
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PDK
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S6K
mTORC1
TSC1/2
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RTK
4EBP1
AMPK
FOXO
mTORC2
Sestrin 3
14-3-3
FOXO
FOXO
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Rictor
FOXO
FOXO target genes
Ubiquitination-mediated
proteasomal degradation
Cell Cycle Arrest
Cyclin G2
p15
p19
p21
p27
p130Rb2
GADD45
Apoptosis
BIM
FasL
TRAIL
BCL6
Autophagy
& Atrophy
ATG12
Atrogin-1
LC3
BNIP3
BNIP3L
GABARAPL1
Metabolism
G6Pase
PEPCK
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Oxidative Stress
& DNA Repair
Mn-SOD
Catalase
GADD45
ATM
DDB1
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Figure 1. The regulation of FOXO activity by the growth factor-triggered RTK-IRS-PI3K-AKTmTORC and stress-induced JNK/MST1 signaling pathways. Upon activation of receptor tyrosine
kinases (RTK) by growth factors, activated PI3K increase the production of PIP3, which in turn triggers
activation of PDK1-AKT. PTEN inhibits the PI3K-mediated generation of PIP3. SGK is concurrently
activated with AKT by PI3K signaling in some circumstances to phosphorylate FOXO proteins (not
shown in this figure). Activated AKT phosphorylates FOXOs to create the docking site for 14-3-3. The
binding of 14-3-3 to FOXOs leads to nuclear export of FOXOs to the cytoplasm for ubiquitinationmediated proteasomal degradation. Activated AKT also inhibits TSCs to indirectly activate mTORC1,
which in turn phosphorylates the downstream substrates including S6K and 4EBP1. The mTORC1mediated phosphorylation stimulates S6K activity and activated S6K inhibits IRS1/2 to form the
negative feedback loop regulation. Upon activation of JNK and MST1 by cell stress, FOXOs are
activated by JNK- and MST1-mediated phosphorylation, which in turn transcriptionally activates the
expression of FOXO target genes involved in cell cycle arrest, apoptosis, autophagy/atrophy, oxidative
stress responses, DNA repair, metabolism, etc. FOXOs can transactivate the expression of Sestrin 3 to
inhibit mTORC1 activity. Transcriptional induction of Rictor by FOXOs leads to AKT activation.
The FOXO-induced reduction in muscular cell size is due to a decrease in overall cellular
protein levels, which is mediated by the FOXO-induced increased expression of atrogin-1, a
muscle-specific ubiquitin ligase that promotes protein degradation and muscle atrophy [35,
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37]. Consistent with these findings, a transgenic mouse model with the genetically engineered
overexpression of FOXO1 in muscle tissue manifests the atrophy symptom with the features
of a reduction in size of type I and type II muscle fibers [38]. Therefore, given the role of
FOXO factors in promoting cell atrophy, growth factor-induced PI3K-AKT/SGK signaling
(discussed in the next section) can increase cell size by negatively regulating FOXO factors to
enhance protein synthesis and decrease protein degradation. Furthermore, an increasing
number of reports indicate that FOXO factors can transactivate the expression of several
protein components involved in the lysosomal protein degradation pathway, such as LC3
(microtubule-associated protein 1A/1B-light chain 3), BNIP3 (BCL2/adenovirus E1B 19
kDa-interacting protein 3), ATG12 (autophagy-related 12 homolog), and GABARAPL1
(GABA-receptor-associated protein-like 1), to regulate the autophagy response [39].
Autophagy has long been regarded as a critical process to promote cellular survival under
nutrient deprivation by eliciting lysosome-mediated eradication of damaged or surplus
organelles [10]. Hence, the role of FOXOs in autophagy regulation is imperative for cells to
adapt to nutrient deprivation stress. In glucose metabolism, FOXO transcription factors are
implicated in facilitating gluconeogenesis by up-regulating the expression of glucosemetobolism-regulatory genes, such as glucose 6 phosphatase (G6Pase) which catalyzes the
conversion of glucose 6 phosphate to glucose and phosphoenolpyruvate carboxykinase
(PEPCK) which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate [40-43].
Thus, the effects of insulin on glucose metabolism are mediated in part by the activation of
PI3K-AKT signaling to negatively regulate FOXO function (discussed in the next section).
2. Regulation of FOXO Transcription Factors
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In normal mammalian cells, two modes of cellular signaling predominantly regulate
FOXO transcriptional activity; regulatory signaling pathways elicited by growth factors and
stress stimuli (Figure 1). Growth-factor-mediated signaling (e.g. insulin) is one of main signal
transduction pathways to regulate FOXO activity through the phosphoinositide-3-kinase
(PI3K)-AKT (protein kinase B, PKB)/SGK (serum and glucocorticoid inducible kinase)
signaling axis [20]. FOXO proteins are directly phosphorylated by AKT or SGK on their
three conserved Ser/Thr residues, giving rise to enhancing binding of the adaptor protein 143-3 to FOXO proteins [12, 20]. Binding of 14-3-3 elicits nuclear export of FOXO proteins
and consequently abrogates FOXO-mediated transcription [12, 20, 44] (Figure 1).
Intriguingly, AKT and SGK have their preferential phosphorylation sites on FOXO proteins
[45], indicating that external stimuli can regulate FOXO phosphorylation in a sophisticated
manner to modulate subcellular localization, degradation and transcriptional activity of
FOXO proteins through their differential effects on AKT and SGK activation.
The major outcome of FOXOs‘ phosphorylation by AKT and SGK is to facilitate the
relocalization of FOXO proteins from the nucleus to the cytoplasm through the enhancement
of their binding to 14-3-3 chaperone proteins [12, 44, 46] (Figure 1). There are several
mechanisms proposed for the inhibitory effect of the 14-3-3 protein on FOXO transcription
factors. It has been shown that 14-3-3 binding promotes the releasing of FOXO proteins from
a nuclear DNA anchor [47], indicating that binding of 14-3-3 to FOXO proteins alters the
FOXO protein conformation and in turn suppresses FOXO DNA binding ability. Regarding
the effect of 14-3-3 on promoting the nuclear exclusion of phosphorylated FOXO proteins,
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two lines of evidence suggest that 14-3-3 binding either leads to a conformational change in
FOXO proteins, which exposes their nuclear export sequence (NES) for allowing the
interaction with Exportin/Crm [44] or masks the nuclear localization signal (NLS) of FOXO
proteins to prevent their nuclear import [48, 49]. Another study also indicates that growth
factors (e.g. IGF-1) can elicit PI3K-PDK1-AKT-induced, CK1 (casein kinase 1)-mediated
phosphorylation at Ser322 and Ser325 of FOXO1, which accelerates FOXO1 relocalization to
the cytoplasm by facilitating the interaction between FOXO1 and the Ran-containing export
machinery [50].
Furthermore, through mutational studies of FOXO proteins, it has been unraveled that
each AKT/SGK-targeted phosphorylation site contributes to the nuclear export of FOXO
proteins [45]. Therefore, the regulation of subcellular FOXO protein localization by
phosphorylation is known to be an effective mechanism for modulating the transcriptional
activity of FOXO proteins in different cell types or in response to different signal stimuli
(Figure 1 and Table 2).
Besides regulating the nucleocytoplasmic shuttling of FOXO proteins, modulating the
degradation of FOXO transcription factors is found to be another layer of the regulatory
mechanism to alter their protein stability (Figure 1 and Table 2). Given that alterations in the
mechanisms to regulate FOXO protein degradation are highly associated with tumorigenesis
[51, 52], the elucidation of these regulatory mechanisms is crucial to the understanding of
their roles in initiating cancer development. To reveal these mechanisms, many research
groups worked on this issue and found that the ubiquitin-proteasome mechanism is
responsible for the degradation of FOXO proteins [51-56]. The followed studies further
demonstrate that both AKT-mediated phosphorylation and cytoplasmic localization are
required to be successfully ubiquitinated by E3 ubiquitin ligases and subsequently degraded
[52, 55, 56]. The ubiquitin ligase accounting for the ubiquitination of the FOXO1 protein has
been identified as the F-box protein SKP2 (S-phase kinase-associated protein 2), a component
of the Skp1/Cull/F-box (SCF) E3 ubiquitin ligase protein complex [52, 56] (Table 2).
Intriguingly, SKP2 only specifically interacts with FOXO1, not with FOXO3 and FOXO4,
raising the possibility that different FOXO family proteins are degraded by their specific E3
ubiquitin ligase machinery complexes. Besides the AKT-mediated degradation of FOXO
proteins, IB kinase  (IKK) has been found to mediate an AKT-independent mechanism
responsible for the proteasome-dependent degradation of the FOXO3 transcription factor [51]
(Table 2). IKK catalyzes the phosphorylation at Ser644 of the FOXO3 protein, which in turn
gives rise to the cytoplasmic retention and ubiquitination-mediated proteasomal degradation
of FOXO3 [51].
The role of the IKK-dependent degradation mechanism in tumorigenesis was revealed
by ectopic overexpression of either IKK or FOXO3 in cells. Constitutive expression of
IKK enhances cell proliferation and tumorigenesis, whereas the forced expression of
FOXO3 counteracts IKK‘s effect to suppress carcinogenesis [51]. Therefore, both AKTmediated and IKK-mediated degradation mechanisms are involved in the regulation of
FOXO protein stability. However, the exact physiological role of IKK in the regulation of
FOXO3 in normal cells is still unclear and needs further investigations.
In addition to growth factor signaling, cellular stress (e.g. oxidative stress, heat shock,
DNA damage) can elicit multiple signaling pathways to modulate FOXO transcriptional
activity (Figure 1).
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Table 2. The post-translational modifications of FOXO transcription factors and their
biological effects
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IKK
CK1
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JNK
Acetylation
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AMPK
CBP/p300
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PCAF
SIRT1/2/3
Ubiquitination
SKP2
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Deacetylation
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MDM2
Deubiquitination
The biological effect on FOXO proteins
Inhibition of FOXOs. FOXO proteins are subjected to
nuclear export and SKP2-mediated polyubiquitination
as well as proteasomal degradation.
Inactivation of FOXOs. FOXO proteins are subjected
to nuclear export and MDM2-mediated
polyubiquitination as well as proteasomal degradation.
Suppression of FOXO3. The FOXO3 protein is
subjected to nuclear export and polyubiquitinationmediated proteasomal degradation.
Inhibition of FOXO1. The nuclear export of FOXO1 is
increased.
increased.
increased.
Nuclear import of FOXOs and their transcription
activities are enhanced.
Upon stress, activated JNK phosphorylates FOXO4
and promotes its nuclear import as well as
transcriptional activity.
Upon stress, activated MST1 phosphorylates FOXOs
and promote their nuclear import as well as
transcription activities.
CBP-binding promotes FOXO activity, but CBPmediated acetylation of FOXOs inhibits FOXO
activity.
Similar to CBP, PCAF-catalyzed acetylation of
FOXOs inhibits FOXO activity.
SIRT-mediated deacetylation of FOXOs activates or
inhibits FOXO activity in a context-dependent manner.
SKP2-mediated polyubiquitination of FOXO1
promotes proteasomal degradation of the FOXO1
protein.
MDM2-mediated polyubiquitination of FOXOs
promotes proteasomal degradation of FOXO proteins,
but MDM2 also catalyzes monoubiquitination of
FOXO4 and enhances its activity.
HAUSP-mediated deubiquitination of
monoubiquitinated FOXO4 inhibits FOXO4
transcriptional activity.
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Enzyme
AKT/SGK
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The modification type
Phosphorylation
HAUSP
The c-Jun N-terminal kinase (JNK), a MAPK (mitogen-activated protein kinases) family
kinase that can be activated by stress stimuli, is implicated in the regulation of FOXO proteins
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in several organisms [57-59]. It has been reported that JNK can phosphorylate FOXO1,
FOXO3 and FOXO4 in vitro.
However, only FOXO4 has been reported to be phosphorylated by JNK at Thr447 and
Thr451 [57], and the JNK-targeted phosphorylation sites on FOXO1 and FOXO3 protein
remain elusive. In comparison to AKT-mediated phosphorylation, JNK-mediated
phosphorylation has an opposite effect to trigger the relocalization of FOXO4 from the
cytoplasm to the nucleus, which activates the FOXO4 transcriptional activity and induces
FOXO target genes such as MnSOD and catalase for detoxification of ROS in response to
oxidative stress [57] (Figure 1 and Table 2).
This regulatory mechanism is also conserved in worms and flies and implicated in
regulating longevity [58, 59]. Although the mechanism whereby stress stimuli and JNK cause
the relocalization of FOXO proteins to the nucleus is still under investigation, a line of
evidence indicates that JNK can phosphorylate the 14-3-3 protein and in turn release 14-3-3binding proteins such as FOXO factors [60]. Besides JNK, the mammalian orthologue of the
ste20-like protein kinase (MST1), which is also activated by oxidative stress, has been
reported to phosphorylate and activate FOXOs [61] (Figure 1 and Table 2). It has been
reported that the activation of FOXOs by JNK/MST1-mediated phosphorylation overrides the
inhibitory phosphorylation by AKT [17, 62].
Intriguingly, other MAPK members, extracellular signal-regulated kinases 1 and 2
(ERK1/2), are also implicated in the regulation of FOXO proteins but play an opposite role
compared with JNK‘s role (Table 2). Activation of ERK by Ras-Raf-MEK signaling
promotes the interaction between ERK and FOXO3 and ERK-mediated phosphorylation of
FOXO3, leading to cytoplasmic sequestration and proteasomal degradation of FOXO3 (63).
ERK-induced proteasomal degradation of FOXO3 is mediated by RING-finger E3 ligase
murine double minute 2 (MDM2) [63] (Table 2).
Therefore, the ERK-MDM2 mechanism is analogous to the AKT-SKP2 mechanism for
the negative regulation of FOXO factors. Regarding the MDM2 role in the regulation of
FOXO proteins, it has been known that MDM2 acts as a general E3 ligase for promoting
ubiquitination of various FOXO factors including FOXO1, FOXO3 and FOXO4 [63-65]. In
addition to promoting polyubiquitination of FOXO proteins, another study has shown that
MDM2 also catalyzes multiple monoubiquitination of FOXO4 rather than polyubiquitination
[64]. Intriguingly, monoubiquitination of FOXO4 promotes nuclear localization of FOXO4,
which is observed in cultured cells in response to oxidative stress [66] (Table 2). In the
nucleus, the deubiquitinating enzyme herpesvirus-associated ubiquitin-specific protease
(HAUSP/USP7) has been found to be involved in the deubiquitination of monoubiquitinated
FOXO4 [66], which acts as a mechanism to balance the effect of MDM2 on FOXO4 (Table
2). Moreover, it has been posited that monoubiquitinated FOXO proteins can be further
converted into polyubiquitinated forms by branching E3 ligases such as SKP2 [64].
As mentioned above, several kinases have been found to phosphorylate FOXOs for
repressing (e.g. AKT, IKK, ERK) or activating (e.g. JNK) the transcriptional activity of
FOXOs (Table 2).
In addition to these kinases, more kinase pathways have been identified to phosphorylate
FOXOs and regulate their transcriptional activity; the AMP-activated protein kinase (AMPK)
[67, 68] is involved in the activation of FOXOs‘ activity and the dual-specificity tyrosinephosphorylated-regulated kinase 1A (DYRK1A) is to inhibit FOXOs (69) (Table 2).
Therefore, the combined outcome from the relative ratio between active FOXO-targeting
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kinase levels (e.g. AKT, IKK, ERK, JNK, etc.), MDM2 levels, HAUSP levels and FOXOtargeting E3 ligase levels (e.g. SKP2) determines the subcellular localization and protein
stability of FOXO transcription factors.
Besides phosphorylation of FOXO proteins as the post-translational modification
mechanisms to regulate the subcellular localization and degradation of FOXO proteins, the
acetylation/deacetylation modifications also play critical roles in regulating FOXO
transcriptional activity (Table 2). It has been known that FOXO factors bind to coactivator or
corepressor complexes and the acetylation status of FOXO proteins are modulated by these
factors [19, 70, 71]. Transcriptional coactivators (e.g. PGC-1, peroxisome proliferativeactivated receptor- coactivator 1) have been reported to bind to FOXO proteins and
potentiate their transcriptional activity [43]. However, some of these coactivators (e.g.
CBP/p300 and PCAF) are also acetyltransferases and thus catalyze the acetylation of FOXO
proteins at several conserved lysine residues, which actually have the opposite effect to
inhibit their transcriptional activity [71]. Therefore, coactivator binding and coactivatormediated acetylation of FOXO proteins dynamically regulate the transcriptional activity of
FOXO factors.
It is well-known that protein acetylation can be reversibly regulated by deacetylasecatalyzed deacetylation. This acetylation/deacetylation bidirectional regulatory mechanism is
involved in modulating the transcriptional activity of FOXO proteins. Multiple lines of
evidence have shown that the Sir2 family deacetylases are responsible for the deacetylation of
FOXO proteins [17-19, 72-74] (Table 2).
The Sir2 family is classified as class III deacetylases that use NAD+ as a cofactor for the
enzymatic activity [75]. The studies have shown that Sir2 deacetylases extend the longevity
of organisms such as yeast, worms and flies (76-79). For the connection between FOXO and
Sir2, several research groups have demonstrated that SIRT1 is able to directly catalyze the
deacetylation of FOXO proteins in vitro and also to participate in the in vivo deacetylation of
these factors within cells [17-19, 72-74].
According to multiple studies, the consensus for the effect of SIRT1-mediated
deacetylation on FOXO function is to preferentially activate FOXO‘s transcriptional ability
for transactivating a subset of FOXO target genes, in particular stress resistance, cell cycle
arrest and DNA repair genes, but simultaneously inhibit FOXO factors to induce proapoptotic gene expression [17-19, 80].
Therefore, through binding to FOXO factors and catalyzing their deacetylation, SIRT1
can regulate FOXO protein function towards stress resistance but away from cell death, which
is in line with the role of Sir2 family proteins in extending longevity. However, the
underlying mechanisms for SIRT1-mediated regulation of FOXO factors remain unclear.
Therefore, the investigation of whether the binding of SIRT1 to FOXO factors and SIRT1catalyzed deacetylation of these proteins have differential regulatory roles in FOXO activity
will be critical to decipher the mechanisms. Although this chapter covers most of the key
aspects of the roles for post-translational modifications of FOXO family proteins in regulating
FOXO function, readers can find the more information in the recent comprehensive review
specific to this topic by Zhao et al. [81].
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3. The Roles of FOXOs in Coordinating the Activities of PI3K-AKT and
Targets of Rapamycin Complexes (TORCs)
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As indicated above, growth factors (e.g. insulin, IGF-1) trigger the activation of PI3KAKT signaling, which in turn inactivates the transcriptional functions of FOXOs. However,
multiple reports indicate that FOXOs are actually implicated in coordinating the negative
feedback circuit mediated by the mTORC1 (mammalian target of rapamycin complex 1)-S6
kinase (S6K) axis, which is the downstream target signaling axis of PI3K-AKT and activated
by active AKT (10, 62, 82) (Figure 1). There are two types of mammalian targets of
rapamycin complexes (mTORCs); mTOR complex 1 (mTORC1) is a conserved downstream
mediator of AKT and mTOR complex 2 (mTORC2) is a conserved upstream activator of
AKT. Each mTOR complex contains its defining subunit; the regulatory associated protein of
mTOR (Raptor) in mTORC1 and the rapamycin-insensitive companion of mTOR (Rictor) in
mTORC2 [83]. When growth factors are deficient, the mTORC1 activity is indirectly
inhibited by tuberous sclerosis complexes (TSCs). TSCs formed by the heterodimer of the
tuberous sclerosis complex 1 and 2 (TSC1/2) possess GAP activity and suppress Rheb
activity, a small GTPase indispensable for the activation of mTORC1 [84, 85]. The TSC2
activity is positively controlled by intracellular ATP levels and AMPK activity [86]. In
addition, it has been reported that AMPK phosphorylates Raptor to inhibit mTORC1 [87]. In
contrast, AKT can maintain intracellular ATP levels and attenuate AMPK activity by
activating mTORC1 [88]. The major physiological functions of mTORC1 are to increase
mRNA translation and fatty acid biosynthesis. To enhance mRNA translation, mTORC1
activates the downstream mediator S6K and inhibits the eukaryotic translation initiation
factor 4E binding protein (4E-BP), a repressor of mRNA translation [85] (Figure 1). Besides
the effect on protein synthesis, mTORC1 possesses anabolic activity to promote fatty acid
biosynthesis through the activation of the sterol-regulatory-element-binding protein
(SREBP1), a transcription factor engaged in transcriptionally activating the expression of
enzymes responsible for fatty acid synthesis (89). Furthermore, mTORC1 has an intrinsic
activity to inhibit the autophagy process via phosphorylating proteins essential for the
initiation of autophagy [90].
By using TSC1/2-null cell model systems, it has been demonstrated that there is a
negative feedback mechanism involving the inhibitory effect of the mTORC1-S6K signaling
axis on insulin receptor substrates (IRS1/2), which mediate PI3K-AKT activation by insulin
and IGF-1 [91] (Figure 1). The recent reports demonstrate that FOXOs play a central role in
coordinating this negative feedback mechanism through their downstream target genes,
Sestrin 3 and Rictor [82, 92-94] (Figure 1). Sestrin 3 belongs to the Sestrin family members
that were originally identified in mammals as antioxidants (95, 96). Sestrin 3 plays a dual
functional role downstream of FOXO signaling; it acts as a scavenger of ROS to mediate
FOXO-induced ROS detoxification, and as an activator of AMPK to inhibit the mTORC1S6K signaling axis. Thus, FOXO-induced Sestrin 3 expression relieves the mTORC1mediated negative feedback effect on IRS1/2 and in turn activates the PI3K-AKT signaling
axis [82, 92] (Figure 1). Intriguingly, the FOXO-Sestrin 3-AMPK-mTORC1-S6K signaling
axis is highly analogous to the reported p53-Sestrin 1/2-AMPK-mTORC1-S6K signaling axis
[97] and both regulatory mechanisms are implicated in negatively regulating mTORC1mediated cell proliferation and anabolic activity. Therefore, AKT activity is activated by the
FOXO-elicited inhibition of the mTORC1-S6K-mediated negative feedback loop circuit.
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However, besides this mechanism, FOXOs also can enact another mechanism that
transcriptionally induces the expression of Rictor, which increases mTORC2 activity and in
turn activates AKT activity [82] (Figure 1). Additionally, it has been shown that FOXOs can
elevate insulin-like growth factor receptor (InsR) and IRS2 mRNA levels to amplify growth
factor signaling [62, 98]. FOXO was also reported to induce the expression of HER2/HER3
tyrosine kinase receptors in several cancer cell lines (91). Therefore, the suppression of the
mTORC1-S6K signaling axis by FOXOs could further augment AKT activity by the FOXOmediated elevation of InsR and IRS2 expression.
The mechanisms mentioned above are not the sole ones to regulate AKT and mTORC1.
It has been shown that FOXO can inhibit the expression of the pseudokinase tribbles 3
(TRB3), an AKT inhibitor, to activate AKT [99, 100]. Moreover, FOXO has been reported to
elevate the expression of BNIP3 with activity to inhibit mTORC1 function [101, 102]. A
recent study also reported that FOXO3 transcriptionally elevates the expression of TSC1 to
suppress mTORC1 activity [103]. Therefore, FOXOs play a master role in coordinating both
InsR-IRS-PI3K-AKT-mTORC1-S6K and InsR-IRS-PI3K-mTORC2-AKT arms to regulate
AKT and mTORC1 activities for modulating the intracellular balance of energy metabolism,
ROS homeostasis, cell proliferation, cell survival and autophagy. When cells are under
physiological stress conditions, FOXOs are activated to keep AKT activation and
concurrently to inhibit mTORC1 activity. The inhibition of mTORC1 leads to a reduction in
its anabolic activity to consume cellular energy and the activation of AKT causes an increase
in cellular energy metabolism for maintaining cellular energy homeostasis.
4. The Roles of FOXO Proteins in Cellular Responses to DNA Damage,
Oxidative Stress and Hypoxia
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In addition to the critical role of p53 tumor suppressor protein in mediating the
downstream effects of ATM/ATR-dependent DNA damage responses, FOXO protein
members have been also found to be involved in ATM-triggered DNA damage responses
[104, 105]. FOXO3 has been reported to induce ATM gene transcription [105] and interact
with ATM to promote its kinase activity and downstream DNA-damage responsive signaling,
such as intra-S-phase, G2-M cell-cycle checkpoints and the repair of damaged DNA [104].
Furthermore, FOXO proteins are required for the expression of DNA repair genes such as
GADD45 and DDB1, which are essential for maintaining the integrity of DNA repair
machinery [20, 34]. Besides cell cycle arrest and DNA repair, induction of cellular apoptosis
by FOXO factors is also essential to maintain genomic stability especially when cellular DNA
is severely damaged. Therefore, this functional aspect of FOXO proteins in response to DNA
damage is well-accepted as one of their imperative tumor-suppressive properties.
A study has shown the important role of cyclin-dependent kinase 2 (CDK2) in the
regulation of FOXO1 in response to DNA damage. It has been known that the function of
CDK2 is frequently abrogated as cells respond to DNA damage [106]. In the normal
circumstance, CDK2-mediated phosphorylation of FOXO1 at Ser249 leads to cytoplasmic
localization and inhibition of FOXO1 [106]. This CDK2-mediated effect can be abolished by
the Chk1/2-dependent DNA damage response, which in turn activates FOXO1 to induce
cellular apoptosis for the elimination of severely damaged cells [106]. Therefore, this
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molecular interaction between CDK2 and FOXO1 provides a mechanism to regulate
apoptotic cell death during DNA damage responses.
Besides their roles in DNA repair and DNA damage responses, FOXO protein members
are known to play important roles in regulating cellular antioxidant capacity through upregulating the expression of free radical scavenging enzymes, including Mn superoxide
dismutase (MnSOD) and catalase, which function primarily to scavenge mitochondrial
respiration-derived reactive oxygen species (ROS) [14, 20, 32]. Thus, FOXO transcription
factors are responsible for two aspects of cellular resistance to stress: detoxification of ROS
and repair of DNA damages elicited by ROS. These FOXO-mediated protective mechanisms
for cell survival are responsible for the essential roles of FOXO proteins in cell longevity
[20].
Under the condition of low oxygen levels (e.g. insufficient blood supply due to rapid
growth of embryonic or tumorigenic tissue), the hypoxia-inducible factor (HIF) complex are
stabilized and become active to transactivate hypoxia-responsive genes, which are required
for tissue cells to adapt to hypoxia and to modulate angiogenesis. The well-known
mechanisms to regulate the stabilization and transcriptional activity of HIF1 proteins are posttranslational protein hydroxylation and ubiquitination-dependent protein degradation. Under
normal oxygenation conditions (normoxia), the HIF1 protein is consistently hydroxylated by
oxygen-dependent hydroxylases and subsequently degraded by the action of the von HippelLindau tumor suppressor ubiquitin ligase (VHL) [107, 108]. The hydroxylation-dependent
modification of HIF1 also prevents the association of HIF1 with the transcriptional coactivator p300, which in turn inhibits HIF1 transcriptional activity [109]. However, a VHLindependent mechanism to negatively regulate HIF1 activity has been identified, which is
mediated by the action of FOXO proteins. The first evidence is from the finding that FOXO4
negatively regulates the levels of HIF1 expression in a VHL-independent manner, thereby
compromising the responsiveness to hypoxia [110]. Another FOXO-dependent negatively
regulatory mechanism is achieved by competing with HIF1 for binding to p300, a
transcriptional co-activator required for HIF1 transcriptional activity. This competition is
mediated by binding of FOXO proteins [10, 111] or the FOXO-transcriptional target CITED2
(CBP/p300-interacting transactivator 2) to p300 proteins [109, 111], leading to the
suppression of HIF1 transcriptional activity. In addition to these mechanisms, the effect of
FOXO proteins on reducing the levels of ROS also negatively regulates HIF1 transcriptional
activity because ROS has been shown to be required for activating HIF1 [112]. Therefore, the
relative ratio between FOXOs and HIF1 determines the cellular response to hypoxia.
5. Are FOXO Transcription Factors Bona Fide Tumor Suppressors?
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Given that FOXO proteins possess tumor-suppressor characteristics that include the
blockade of cell cycle progression, the negative regulation of ROS production and the repair
of damaged DNA, their roles as tumor suppressor genes had been investigated. From in vitro
studies, it has been demonstrated that ectopic expression of FOXO proteins with mutations at
residues required for inactivation by AKT/PKB-mediated phosphorylation can inhibit colony
formation of cells with aberrantly activated PI3K/AKT signaling (e.g. RAS-transformed cells
or cells deficient for the phosphatase and tensin homolog PTEN) [22]. Furthermore, another
line of evidence has shown that a dominant-negative form of FOXO (dnFOXO) can replace
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RAS to collaborate with MYC for oncogenic transformation of cells [113], indicating that
PI3K/AKT-mediated phosphorylation to inactivate FOXO transcriptional activity is crucial to
cell transformation. Besides, several studies have shown that ectopic expression of FOXO in
human cancer cell lines attenuates their in vivo xenograft tumor formation in nude mice [51,
114]. Therefore, these lines of evidence, taken together, indicate that FOXO proteins
potentially function as tumor suppressors.
Recent in vivo animal studies have provided the solid evidence to support FOXO proteins
as genuine tumor suppressors. For example, Paik et al. performed broad somatic deletion of
all three FOXO genes in mice and found that inactivation of FOXOs resulted in the narrow
tumor spectrum, characterized as thymic lymphomas and hemangiomas [11]. In contrast, the
genetic deletion of only one or two FOXO genes leads to no or a moderate tumor-prone
phenotype [11, 115, 116], indicating that FOXO genes are a functionally redundant tumor
suppressor family. The evidence from their studies demonstrates that the mammalian FOXO
transcription factors are indeed bona fide tumor suppressors. However, it is unclear that
abrogation of FOXOs only induces tumorigenesis of hematopoietic lineages, which is
contradictory to the ubiquitous expression patterns of FOXO proteins in tissue. The possible
explanations for these findings are that hematopoietic cells more rely on FOXOs to maintain
genomic integrity and their high turnover rates might promote the tumorigenic progression
under the inactivation of FOXOs. Another line of evidence has shown that the sable
introduction of a dominant-negative FOXO (dnFOXO) gene moiety, which functionally
abrogates all of FOXOs, into Emu-myc transgenic hematopoietic stem cells enhances
lymphomagenesis in recipient mice [117]. This enhanced tumorigenesis is attributable to the
effect of dnFOXO to attenuate Myc-induced apoptosis [117]. Furthermore, forced expression
of dnFOXO in Emu-myc:p53 (+/-) progenitor cells overcomes the pressure to inactivate the
remaining p53 allele during lymphomagenesis by inhibiting the p19Arf signaling axis [117].
Given that p19Arf is known to mediate oncogenic stress-induced activation of p53 and in turn
to engender cell cycle arrest and/or apoptosis [118], this finding points the mechanistic
interaction between FOXO and p53. However, results from the studies using dnFOXOexpressing murine lymphoma models should be interpreted with caution because ectopic
expression of dnFOXO not only abrogates FOXO functions but also disturbs the normal
physiological functions of FOXO-interacting partners by protein-protein interaction, which
might be also involved in enhanced lymphomagenesis in the Emu-myc model. Nevertheless,
based on gathering evidence from the studies of genetic mouse models, FOXO proteins
indeed are bona fide tumor suppressors with in vivo activities to prevent both spontaneous
tumor formation and Myc-induced development of lymphoma. In addition, two molecular
mechanisms underlying FOXO-mediated effects to interfere with oncogenic functions of Myc
have been deciphered. According to the studies by Chandramohan et al., ectopic Myc
expression has a suppressive effect on the expression of cyclin-dependent kinase inhibitor
p27Kip1, whereas FOXO proteins counteract this inhibitory effect from Myc and promote the
transcriptional activation of p27Kip1 [119]. Another line of evidence indicates that the PI3KAKT-FOXO signal cascade can modulate Myc transcriptional function and in turn affect
Myc-regulated gene expression. Delpuech et al. found that FOXO3 can suppress the
expression of Myc target genes via transcriptional induction of MxiI, a member of Mad/Mxd
family transcriptional repressors [120]. MxiI is known to compete with Myc to bind to Max
for the formation of the MxiI-Max heterodimerized transcriptional repressor complex that has
a transcriptionally repressive effect on the expression of Myc target genes [120]. Although
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these two lines of evidence shed new light on how FOXO proteins counteract oncogenic
functions of Myc, it is not clear to what aspects of FOXO functions contribute to FOXOmediated in vivo tumor suppression. Hence further advanced studies are still mandatory to
address these issues.
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6. Genetic Alterations in FOXO Subfamily Genes
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It was initially identified that FOXO subfamily genes participate in chromosomal
translocations found in alveolar rhabdomyosarcomas (PAX3-FOXO1, PAX7-FOXO1) and
some forms of leukemia such as secondary leukemia and acute lymphoblastic leukemia
(MLL, myeloid/lymphoid or mixed lineage leukemia, MLL-FOXO3, MLL-FOXO4) [121127]. All of these translocations occur at a breakpoint in the intron 2 of FOXO subfamily
genes. These discovered chromosomal translocations predominantly lead to chimeric proteins
in which the C-terminal domains of FOXOs are fused to the N-terminal domains of other
transcription factors (e.g. PAX3, PAX7, MLL). Although the absence of a functional FOXO
allele may contribute to some extent of the tumorigenic effect derived from these
translocations due to the tumor-suppressive roles of FOXOs, it has been recognized that the
chimeric fusion proteins PAX3/PAX7-FOXO1 or MLL-FOXO3/FOXO4 mostly contribute to
tumorigenesis [128, 129]. However, a study has shown that the MLL-FOXO4 fusion has an
effect to transdominantly interfere with the expression of the remaining intact FOXO4 allele,
suggesting that this transdominant effect can potentiate the oncogenic effect derived from the
MLL-FOXO4 chimeric protein [130]. Notably, the subsequent knock-in animal studies
examining the oncogenic potential of the chimeric allele did not recapitulate tumor
phenotypes, raising the possibility that the disruption of the functional FOXO allele
independently causes or collaborates with the chimeric allele to elicit tumorigenesis [93, 131].
In addition to chromosomal translocations, the genetic deletion of FOXO genes has been
discovered. For example, the frequently deleted chromosomal region in prostate cancer
encompasses the FOXO1 gene, which is highly associated with the observations that FOXO1
mRNA expression is frequently downregulated in prostate cancer [132]. To date the identified
genetic aberrations in FOXO genes are not common in cancers and restricted to some types of
cancer mentioned above. The growing evidence has shown that the inactivation of FOXOs‘
tumor suppressive function in cancer frequently occurs at the protein level, which is discussed
in the next section.
7. Dysregulation of FOXO Gene Expression and Function in Cancer
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Since FOXO factors possess the ability to induce cell cycle arrest, DNA repair and
apoptosis, these tumor-suppressive features make FOXOs potential candidates as tumor
suppressors. A series of xenograft and genetic knockout studies also strongly indicates that
FOXOs are genuine tumor suppressors. Therefore, it is a well-accepted consensus that
dysregulation of FOXOs‘ expression and function is the crucial mechanism leading to the
development of a variety of cancers. In addition to genetic alterations in FOXO genes such as
chromosomal translocations and deletions as mentioned above, currently there are other four
different levels of dysregulation that can lead to the inactivation of FOXO transcription
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factors, including the dysregulation at the levels of mRNA synthesis, subcellular localization,
protein degradation and FOXO protein partners.
As mentioned in the prior section, the FOXO1 gene loci are within the commonly deleted
genomic region in prostate cancer, which is thought as a potential mechanism to cause downregulation of FOXO1 expression [132]. Chromosomal translocations found in alveolar
rhabdomyosarcoma and leukemia are also a mechanism leading to loss of half expression of
FOXO mRNAs [121-127]. Moreover, it has been reported that the EWS-Fli1 oncogenic
fusion protein functions as a transcriptional repressor to suppress the expression of FOXO1 in
Ewing‘s sarcoma cells [133], which inactivates the tumor-suppressive function of FOXO1 at
the mRNA synthesis level. Intriguingly, the recent evidence has revealed that miR-499-5p
targets the FOXO4 mRNA and negatively regulates its levels in colorectal cancer cells [134],
which is a novel tumorigenic mechanism to inactivate FOXO function via microRNAmediated silencing.
Multiple aberrant oncogenic mechanisms occurring in cancer have been found to cause
the activation of PI3K-AKT signaling, which in turn leads to the inhibition of FOXO function
by promoting cytoplasmic retention and protein degradation of FOXOs. The most known
mechanism is the frequent mutation or deletion of the phosphatase and tensin homolog
(PTEN) gene in a large spectrum of human cancer types such as brain, breast, prostate and
kidney cancers [135, 136]. PTEN is a lipid phosphatase which antagonizes the effect of PI3K
[137, 138] (Figure 1). Therefore, loss of PTEN gives rise to an increase in the levels of
phosphatidylinositol [3,4,5] trisphosphate (PIP3) in the plasma membrane, which in turn
results in activation of AKT [135, 136]. Activation of AKT by loss of PTEN leads to the
generation of phosphorylated FOXO proteins whose transcriptional functions are inactivated
by sequestrating phospho-FOXOs in the cytoplasm via 14-3-3 binding and by promoting the
depletion of phosphorylated FOXOs via SKP2-mediated ubiquitination and proteasomal
degradation (Figure 1). Besides loss of PTEN, the frequent mutation of the PIK3CA gene,
coding for the catalytic subunit p110 of class IA phosphatidylinositol 3-kinases (PI3Ks), in
human cancer is another pathological mechanism resulting in activation of AKT [139]. The
prevalent mutants of p110 (e.g. E542K, E545K, and H1047R) show a gain of enzymatic
function in vitro and are oncogenic in vitro and in vivo by activating AKT kinase activity
[139]. The in vivo tumorigenicity of PIK3CA mutants in an avian species strongly suggests a
crucial role for these mutated proteins in human malignancies [139]. Thus, activation of AKT
by the constitutively activated PIK3CA mutants in cancer cells leads to the inhibition of
FOXOs‘ tumor-suppressor function, which contributes to tumorigenesis induced by mutated
PIK3CA.
Cytoplasmic retention of the FOXO3 transcription factor has been found in breast cancer
tissue sections and correlates with poor survival of breast cancer patients. Intriguingly,
cytoplasmic FOXO3 detected in a subset of breast cancer cases is not correlated with the
active form of phospho-AKT and the further studies demonstrate that IKK is responsible for
the AKT-independent inactivation of FOXO3 [51]. IKK physically interacts and
phosphorylates FOXO3 at Ser644, and then subjects phosphorylated FOXO3 to the
cytoplasm for ubiquitination-dependent degradation by the proteasome complex [51]. In
addition to IKK signaling, Ras-Raf-MEK-ERK signaling that is frequently activated in
many types of cancer has been identified as another AKT-independent mechanism to
negatively regulate FOXO3 [63]. Activated ERK catalyzes the phosphorylation of FOXO3 at
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multiple residues, which in turn is subjected to MDM2-mediated ubiquitination and
proteasomal degradation [63]. Therefore, ERK-MDM2-mediated proteasomal degradation of
FOXO3 is also one of vital mechanisms for promoting cell proliferation and tumorigenesis.
Besides FOXO3, MDM2 also targets FOXO1 and FOXO4 and promotes their ubiquitination
and proteasome-mediated degradation [63-65]. In light of that gene amplification and
overexpression of MDM2 have been found in breast cancer, glioblastoma, osteosarcoma and
liposarcoma [140], the tumor-suppressor function of FOXO factors is inactivated in these
cancer types via the high expressed levels of MDM2 to enhance FOXO degradation.
To maintain cell survival and proliferation, it has been reported that cancer cells
specifically rely on some FOXO-interacting proteins to conquer the tumor-suppressive effects
of FOXO factors. For example, siRNA-mediated knockdown of SIRT1 in several types of
epithelial cancer cells is able to trigger FOXO-dependent apoptosis and/or cell cycle arrest,
whereas silencing of SIRT1 has no significant effect on cell survival and growth of normal
human epithelial cells and normal human diploid fibroblasts [80]. These findings indicate that
SIRT1 imposes restraint on FOXOs to maintain the survival and proliferation of epithelial
cancer cells. Moreover, another line of evidence has revealed that four and a half LIM 2
(FHL2) interacts with FOXO1 to promote the binding of FOXO1 to SIRT1, resulting in
SIRT1-mediated FOXO1‘s deacetylation and suppression of FOXO1-dependent apoptosis
and target gene expression in prostate cancer cells [74]. Particularly, the latent protein
LANA2 from the Kaposi-sarcoma-associated herpesvirus has the direct interaction with
FOXO3 and 14-3-3 proteins to enhance nuclear export of FOXO3 [141], which is the first
evidence that viral proteins can inhibit FOXO transcriptional activity as a mechanism to
trigger cell transformation and tumorigenesis. Therefore, the tumor-suppressor functions of
FOXO transcription factors are abrogated in cancer cells through the multiple mechanisms
that include chromosomal translocations or deletions of FOXO genes, miRNA-mediated
silencing of FOXO mRNAs, kinase-mediated cytoplasmic sequestration of FOXO proteins,
ubiquitination-mediated proteasomal degradation of FOXO proteins and protein-proteininteraction-mediated inhibition of FOXO transcriptional function.
B. FOXP Subfamily Genes
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The human FOXP subfamily is composed of four genes, FOXP1-4. All of FOXP genes
encode proteins that contain the highly conserved C-terminal tetramerization domain
comprising zinc-finger and leucine-zipper domains and a DNA-binding forkhead box domain
[142]. FOXP proteins function as sequence-specific transcription factors which are found to
participate in the development of speech and language regions of the brain during
embryogenesis, the development as well as function of regulatory T cells, tumor suppression
and oncogenesis. Among these four FOXP genes, FOXP1 and FOXP3 have been
convincingly connected to tumorigenesis. The FOXP1 transcription factor has been known to
potentially play a dual role as either an oncoprotein in a number of types of lymphomas and
hepatocellular carcinoma or a tumor suppressor in breast cancer [reviewed in [5, 143]].
However, molecular mechanisms underlying the roles of FOXP1 in tumorigenesis remain
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largely unknown. Therefore, this chapter section does not cover the details of the recent
advances in FOXP1 studies. The readers who are interested in FOXP1 can refer to these
recent review articles [5, 143]. In comparison to FOXP1, the FOXP3 gene at Xp11.23 has
been well-studied and defined as an X-linked tumor suppressor gene in epithelial carcinomas
(e.g. breast cancer, prostate cancer, ovarian cancer, etc.) [reviewed in [144-147]]. Besides its
critical role in tumor suppression, actually the Foxp3 gene was originally identified from the
scurfy mice bearing a lethal X-linked recessive immunodysregulation [148-151]. Since its
discovery, FOXP3 has been well-studied in immune cells and known as a master regulator in
the development and function of CD4+CD25+ regulatory T cells (Treg) which are involved in
immunosuppression [reviewed in [144, 147, 152]]. Although immunological studies have
shown that the role of FOXP3 in Treg is involved in the immunosuppression-related
tumorigenesis, this chapter section only focuses on the functional roles of FOXP3 in
epithelial cells and what pathological mechanisms lead to inactivation of FOXP3‘s tumorsuppressive function in epithelial carcinomas. The readers interested in the immunological
aspect of FOXP3 can refer to the recent review articles [144, 147, 152].
The expression of mouse Foxp3 in epithelial cells was first documented in Rag2-/- mice
which are a genetic mouse model with the Scurfy mutation and a notable lack of T
lymphocytes.
The study has shown that the expression of the Foxp3 mRNA and its encoded nuclear
protein has been found in epithelial cells of breast, lung and prostate, but not liver, kidney and
intestine [153]. Regarding the role of FOXP3 in epithelial cancer, there are multiple important
lines of recent evidence demonstrating that FOXP3 is a bona fide X-linked tumor suppressor
gene in breast, prostate and ovarian cancers [154-157]. By studying a female mouse model
with a heterozygous Scurfin mutation at the Foxp3 gene [Foxp3(sf/+)], Zuo et al. found that
the mutant mice developed mammary carcinomas at a high rate [154]. The further studies
have also shown that FOXP3 functions as a transcriptional repressor of HER2 and SKP2
oncogenes and a transcriptional activator of p21 gene in breast epithelial cells (154, 155, 158)
(Figure 2). HER2 is a well-known oncoprotein that triggers oncogenic signaling pathways
such as PI3K-AKT-mTORC1 and MEK-ERK [159]. SKP2 is aberrantly overexpressed in a
wide spectrum of cancers [160] and involved in ubiquitination and degradation of the cyclindependent kinase inhibitor (CDKI) p27 and tumor-suppressor FOXOs. Since overexpression
of HER2 and SKP2 is commonly present in breast cancer and accounts for or contributes to
breast tumorigenesis [154, 155], FOXP3-mediated suppression of the expression of these two
genes is critical for its tumor-suppressive effect. Consistently, there is a reverse expression
correlation between FOXP3 and these two oncogenes in breast cancer tumor specimens [154,
155]. Moreover, FOXP3 can transcriptionally activate the CDKI gene p21 promoter and
induce its mRNA expression [158].
The functionality of p21 has been demonstrated to be crucial to FOXP3-mediated tumor
suppression because shRNA-mediated silencing of the p21 gene rescues cell growth
inhibition induced by FOXP3 transduction [158]. Immunohistochemistry (IHC) analysis of
human breast cancer tissue microarrays has also deciphered the positive correlation between
the protein expression of FOXP3 and p21 [158]. The findings that genomic deletions,
functionally significant somatic mutations, and down-regulation of the FOXP3 gene are
commonly found in human breast cancer samples further strengthen its role as a genuine
tumor suppressor gene in female breast cancer [154].
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Somatic Mutations
Cytoplasmic
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Tumorigenesis and Cancer Progression
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Figure 2. A schematic view of the cancer-specific deregulation of FOXP3 function that has a
physiological tumor-suppressor role in epithelial cells. In epithelial cancers (e.g. breast, prostate,
ovarian cancer), FOXP3 is inactivated by multiple pathological mechanisms including genomic
deletions, somatic inactivating mutations, alternative splicing, cytoplasmic mislocalization, etc. In
epithelial cells, FOXP3 acts as a tumor suppressor to transcriptionally repress the expression of HER2,
SKP2 and c-MYC oncoproteins and to transactivate p21 (a CDKI) expression. SKP2 mediates the
polyubiquitination of p27 (a CDKI) and FOXO proteins for promoting their proteasomal degradation in
the cytoplasm. Therefore, FOXP3-mediated inactivation of SKP2 can restore the levels and functions of
p27 and FOXOs, leading to cell cycle arrest and apoptosis. Induction of p21 expression by FOXP3 also
results in cell cycle arrest. FOXP3-mediated cell cycle arrest and apoptosis in epithelial cells play a
protective role to prevent the occurrence of tumorigenesis and cancer progression. HER2 signaling can
activate both PI3K-AKT and Ras-Raf-MEK-ERK signaling cascades to enhance cell survival and
proliferation. The c-MYC protein functions as a promoter of cell proliferation. Suppression of HER2
and c-MYC expression by FOXP3 reduces cell survival and inhibits proliferation, which have the
effects to block cancer development and progression.
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In addition to its role in breast epithelial cells, the FOXP3 gene also plays an imperative
tumor-suppressor role in prostate epithelial cells. It has been reported that the X-linked
FOXP3 gene at Xp11.23 is frequently inactivated in human prostate cancer by genomic
deletions, somatic inactivating mutations and expression down-regulation [156]. By analysis
of lineage-specific ablation of FoxP3 in prostate epithelial cells in mice, Wang et al. found
that inactivation of Foxp3 resulted in prostate hyperplasia and prostate intraepithelial
neoplasia, suggesting the importance of Foxp3 in suppressing early prostate tumorigenesis in
male mice [156]. Importantly, in both human normal and neoplastic prostate tissues, FOXP3
is essential to transcriptionally repress the expression of the c-MYC gene, a most frequently
overexpressed oncogene in prostate cancer [156] (Figure 2). These findings demonstrate that
FOXP3 is an X-linked prostate tumor suppressor gene in males. Given that males have only
one X chromosome, these studies provide a paradigm of "single genetic hit" to inactivate Xlinked tumor suppressor genes [145, 156]. In the study of ovarian cancer, FOXP3 expression
has been shown to be down-regulated or lost in ovarian cancer cells compared to normal
ovarian epithelia [157]. Ectopic expression of FOXP3 in ovarian cancer cells displays the
tumor-suppressive effects to inhibit cell proliferation, migration and invasion [157]. Taken
together, FOXP3 principally acts as a tumor suppressor at least in breast, prostate and ovarian
epithelia to prevent tumorigenesis.
2. Alterations in FOXP3 Expression and Function in Cancer
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The aberrant mechanisms causing the inactivation of FOXP3 in epithelial carcinomas
involve genomic deletions, functionally significant somatic mutations, abnormal alternative
splicing, epigenetic gene silencing and unknown expression down-regulation [144-146]
(Figure 2). The prevalence of somatic mutations ( > 20 mutations found) at the FOXP3 gene
has been found in breast (36%) and prostate (25%) cancer cases (145, 146, 154, 156). The
frequency of the FOXP3 deletion has also been reported in breast (13%) and prostate (14%)
cancers [145, 154, 156]. IHC analysis of tumor samples was also performed to reveal the
frequency of FOXP3 protein expression in different types of cancer. However, the
interpretations of IHC results from different research groups are controversial, which might
have been complicated by the use of different anti-FOXP3 antibodies and the presence of
cytoplasmic FOXP3 in examined tumor tissues [145]. Nevertheless, the consensus from these
IHC studies is that normal epithelia commonly exhibit nuclear FOXP3 staining and
cytoplasmic localization of FOXP3 is predominantly present in FOXP3-positive epithelial
cancer cells, which is thought as an inactive form of the FOXP3 protein [144-146]. Although
it is still not completely clear, cytoplasmic localization of FOXP3 in tumor epithelial cells is
suggested to be attributable to somatic mutations and/or alternative splicing variants, which
inactivate or remove nuclear localization signals of the FOXP3 protein [144-146, 154, 156,
157, 161]. It is noteworthy that the alternative splicing forms of the FOXP3 protein have been
found to be predominantly expressed in some types of cancers such as breast and ovarian
cancers, malignant melanomas and malignant T cells of Sezary syndrome. These alterations
in alternative splicing of the FOXP3 pre-mRNA can lead to the truncation of the FOXP3
protein and inactivation of its function [146, 154, 157, 161, 162]. In spite of reported
deletions, somatic mutations and alternative splicing, these mechanisms can not completely
explain high frequencies of FOXP3 loss or down-regulation in examined tumor specimens
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1. The Biological Functions of FOXF Genes
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[145]. Exploring other potential mechanisms (e.g. DNA methylation, histone deacetylation
and the regulation by transcription modulators) is mandatory for future investigation.
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The FOXF subfamily has two member genes FOXF1 and FOXF2. The human FOXF
genes were first cloned and characterized by Hellqvist et al. and named as Forkhead RElated
ACtivator (FREAC)-1 and -2 [163]. In addition to the forkhead DNA-binding domain, the Cterminal portion of FOXF proteins possesses the characteristics of the transcriptional
activation domain [164, 165]. Intriguingly, FOXF2 has been reported to contain another
transactivation domain mapped in the central protein part [164]. The multiple studies have
shown that FOXF factors transcripitionally modulate the expression of tissue-specific genes
(e.g. lung, intestine, adipose, etc.) [163, 165-169]. According to published literature, the
expression of FOXF transcription factors is found in lung, placenta, breast, prostate, colon,
brain and head tissue [163, 170-175]. The in vitro hybridization studies have shown that Foxf
mRNA expression is predominately distributed in the mesoderm-derived tissues [176, 177],
suggesting their expression is mesenchyme-specific and their functions are important for
generation of descendants of splanchnic mesoderm. The studies of Foxf genetic knockout
mice have shown that the functions of Foxf transcription factors are essential for embryonic
development, organogenesis (e.g. lung, gut, liver, gall bladder) and vasculogenesis [167, 176,
178-183]. In line with these findings in mice, the germline deletions and inactivating
mutations of human FOXF1 gene have been discovered and associated with genetic disorders
in lung and gut organs such as alveolar capillary dysplasia (ACD), esophageal atresia and
VACTERL association (Vertebral anomalies, Anal atresia, Cardiac malformations, TracheoEsophageal fistula, Renal and Limb malformations) [184-186].
Although the roles of Foxf factors in mouse development have been extensively
documented, the biochemical roles of their functions in mammalian cells are largely
unknown. However, several studies have shown that the mammalian forkhead box-F1 gene
expression is regulated by the hedgehog (Hh) signaling pathway and Hh-downstream
mediators Gli transcription factors [167, 168, 179], suggesting that Foxf1 is a key
downstream mediator of hedgehog signaling that is critically implicated in modulation of
organ morphogenesis. Moreover, a further study has shown that inactivation of one allele
each of Foxf1 and Foxf2 results in defects in gut development, a decrease in stromal Bmp4
expression as well as an increase in stromal Wnt5a expression, and causing epithelial
hyperproliferation [167]. This study indicates that stromal Foxf factors are essential for
regulating the polarity and proliferation of epithelial cells during gut development. The
crucial role of the Foxf1 function in regulating the migration of normal fibroblasts has also
been reported [187]. Owing to the limited studies of FOXF2, its biochemical function remains
largely unknown. However, a recent study has deciphered a novel role of FOXF2 in the
regulation of growth factor signaling and the homeostasis of glucose metabolism in addition
to its well-known function in modulating the expression of lung-specific genes [169]. Ectopic
overexpression of Foxf2 down-regulates IRS1 mRNA and protein expression levels in
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adipocytes and concurrently lowers insulin-mediated glucose uptake compared with wild-type
adipocytes [169]. Therefore, these findings suggest that Foxf2 may be also implicated in
cellular and systemic whole body glucose tolerance via, at least in part, the down-regulation
of IRS1 expression [169]. Besides this role in glucose tolerance, the Foxf2-mediated
suppression of growth factor signaling (e.g. insulin, IGF-1) may have a tumor-suppressive
effect in cancer cells, which is worthy of further investigation.
2. Aberrant Regulation of FOXF Gene Expression and Function in Cancer
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There are several lines of recent evidence indicating that FOXF transcription factors are
potential tumor suppressors. In prostate cancer, the FOXF1 gene is located in the common
deleted regions of 16q23-qter [172]. In line with this, FOXF1 expression is markedly reduced
in prostate tumor specimens compared to normal prostate tissue [172] (Figure 3). We also
recently discovered that human FOXF1 plays a novel tumor-suppressor role in cell-cycle
regulation in breast epithelial cells, which is frequently silenced in breast cancer through
epigenetic mechanisms [174] (Figure 3).
Genomic Deletions
FOXF1
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DNA repair genes
BRCA1
MRE11A
G1-regulatory
genes
CDK6
Gene transcriptionregulatory genes
CCNT1 (Cyclin T1)
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CDC34
CDK5R1
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Figure 3. Dysregulation of FOXF1 expression and function in cancer. FOXF1 expression is lost or
down-regulated in cancer (e.g. breast and prostate cancer) through pathological mechanisms such as
chromosomal deletions and/or epigenetic gene silencing. FOXF1 expression is also underexpressed in
colorectal cancer; however, the underlying mechanism is unclear. Although it is still uncertain,
epigenetic gene silencing is a potential mechanism leading to the down-regulation of FOXF1 in
colorectal cancer. FOXF1 is also inactivated in colorectal cancer by cytoplasmic mislocalization.
FOXF1 is able to up-regulate and down-regulate the expression of genes functionally implicated in
multiple biological pathways for maintaining the stringency of DNA replication and genomic stability.
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Our functional studies have shown that ectopic overexpression of FOXF1 in FOXF1negative breast cancer cells inhibits the CDK2-Rb-E2F signaling cascade to block the G1-S
transition and inactivation of endogenous FOXF1 in FOXF1-expressing breast cancer cells by
siRNA knockdown leads to genomic DNA rereplication [174].
In addition, the expression profiling study indicates that inactivation of FOXF1 function
up-regulates the expression of E2F target genes implicated in DNA replication initiation and
S-phase cell cycle progression, suggesting that FOXF1 is involved in negatively controlling
DNA replication [174] (Figure 3). FOXF1 also up-regulates the expression of genes
functionally involved in DNA repair (BRCA1, MRE11A), mitosis (ANAPC2, ANAPC4, and
CUL3) and G1 progression (CDK6) (Figure 3), suggesting that FOXF1 may regulate multiple
biological processes to induce the tumor-suppressive effect [174]. Up-regulation of DNA
repair genes by FOXF1 further supported the tumor-suppressor role of FOXF1 in maintaining
genomic stability. Besides breast cancer, our very recent report has revealed that FOXF1
expression is predominantly silenced in colorectal cancer cell lines with the inactive p53 gene
[188]. The biological function of FOXF1 in colorectal cancer cells has been deciphered by the
siRNA knockdown study. Abrogation of FOXF1 function results in DNA rereplication (also
called over-replication, a mechanism known to cause genomic instability) in colorectal cancer
cells with a defect in the p53-p21 checkpoint [188]. Therefore, these findings, taken together,
imply that FOXF1 might play a tumor-suppressor role in colorectal carcinogenesis. Indeed,
our most recent clinical studies have shown that human colorectal adenocarcinomas exhibit
either a reduction in FOXF1 expression or cytoplasmic mislocalization of the FOXF1 protein
[175] (Figure 3). This is the first report revealing that the FOXF1 protein is overexpressed as
well as mislocalized in colorectal tumor epithelial cells and underexpressed/lost in tumorassociated stromal fibroblasts. Thus, in addition to the aberrant epigenetic mechanisms,
cytoplasmic mislocalization is another pathological mechanism to inactivate FOXF1 function
in cancer due to the fact that the functional FOXF1 protein is located in the nucleus.
However, the underlying mechanism causing FOXF1 mislocalization is unclear and thus the
future investigation is warranted to address this question. This study also indicates that
cytoplasmic FOXF1 is a promising prognostic marker owing to its statistically significant
association with the malignancy and metastasis of colorectal cancer. Regarding the role of
FOXF2 in cancer, a recent interesting report has shown that FOXF2 is the target of miR-301,
whose overexpression in lymph node negative (LNN) invasive ductal breast cancer has been
implicated as a poor prognostic indicator [189]. In this study, the authors posited that FOXF2
is an anti-proliferation factor by inhibiting WNT5A signaling, which is negatively regulated
by miR-301 in breast cancer [189].
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The FOXL subfamily is comprised of FOXL1 and FOXL2, which both are single-exon
genes located at 16q24 and 3q23, respectively. FOXL1 functions as a mesenchymal
transcription factor that has been linked to the regulation of the Wnt/-Catenin signal axis, a
vital pathway for gastrointestinal development and tumorigenesis [190, 191]. The in vivo
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animal studies of Apc(Min/+);Foxl1-/- mice indicate that loss of Foxl1 results in a significant
increase in tumor multiplicity in the colon of mice with the Apc(Min/+) genetic background
compared to Foxl1+/+ control mice [191]. In addition to promoting colorectal tumorigenesis,
Apc(Min/+);Foxl1-/- mice also develop gastric neoplasia that are not present in Apc(Min) control
mice. These outcomes are attributable to accelerated loss of heterozygosity (LOH) at the Apc
locus in gastrointestinal tissue [191]. These studies have revealed that Foxl1 is a potential
tumor suppressor playing a key role in gastrointestinal tumorigenesis. However, Foxl1-/- mice
only manifest abnormal intestinal epithelia, postnatal growth retardation and defective
intestinal glucose uptake, but do not develop gastrointestinal neoplasia [192, 193], suggesting
that single loss of Foxl1 is not sufficient for the neoplastic development in the gastrointestinal
tract. Furthermore, Foxl1 has been shown to collaborate with Foxf1 in mediating hedgehog
signaling and controlling epithelial proliferation in the developing stomach and intestine
[168]. It has also been reported that Foxl1 is involved in liver repair and the regulation of
bipotential hepatic progenitor cells [194, 195]. Despite these critical roles in mice, the
biological functions of human FOXL1 in cancer still remain unknown and need to be
deciphered by future investigation.
The forkhead transcription factor FOXL2 is emerging as a central transcription factor in
regulating the development of ovary and the growth as well as maturation of ovarian follicles
[reviewed in [196, 197]]. FOXL2 expression is mainly found in the periocular region, the
pituitary and ovarian granulosa cells [198, 199]. The FOXL2 gene was first identified and
cloned by Crisponi et al. from the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome
(BPES) region on human chromosome 3q23 [200], a genetic disorder with germline
mutations at the FOXL2 gene locus. Patients with BPES manifest a characteristic eyelid
dysplasia with associated symptoms such as premature ovarian failure (POF) and infertility in
affected females [201]. In line with these clinical findings in humans, the studies from two
independently created Foxl2-/- knockout mouse models have revealed that loss of Foxl2
impairs granulosa cell differentiation, follicle development and ovary maintenance [202,
203]. Therefore, the FOXL2 forkhead transcription factor is an essential ovarian transcription
factor crucial to ovarian development, female sex determination and the postnatal ovary as
well as follicle maintenance. The diverse transcriptional activities of FOXL2 are likely
modulated by posttranslational modifications (e.g. Sumoylation, phosphorylation, acetylation,
etc.) and binding to other key protein partners implicated in the regulation of granulosa cells
[196, 197, 204-206]. Besides germline mutations in BPES patients, somatic mutations and
down-regulation of the FOXL2 gene were identified in human ovarian granulosa cell tumors
(OGCTs) [reviewed in [204]]. These studies of FOXL2 in OGCTs suggest that it may act as a
tumor suppressor in ovarian tumorigenesis.
In line with clinical findings, the recent advances in the studies of FOXL2 biological
function also support a paradigm that FOXL2 is a potential tumor suppressor (Figure 4), at
least in ovary. FOXL2 are known to interact with several partner proteins that function as
tumor suppressors (Figure 4). For example, FOXL2 has been reported to be able to
heterodimerize with the transcription factor SMAD3, a crucial mediator responsible for the
TGF cytostatic effect. Their interaction is attributable to the binding of the C-terminal
portion of the FOXL2 forkhead domain to the SMAD3 N-terminal domain [207, 208].
Intriguingly, this heterodimerized transcriptional complex is involved in up-regulating the
expression of the Follistatin gene, encoding a cytostatic protein to inhibit BMP15-stimulated
granulosa cell proliferation [208].
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LATS1
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FST p16INK4A 
Apoptosis Regulation
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Figure 4. An illustrated view of the regulation of FOXL2 and FOXL2-mediated biological effects.
FOXL2 expression is positively regulated by the Rb protein. FOXL2 may interact with multiple protein
partners to execute its effects on cell cycle arrest and apoptosis. FOXL2 functions are implicated in cell
cycle arrest, apoptotic regulation, oxidative stress responses and cell adhesion via its downstream target
genes. Up-pointing arrows, up-regulation by FOXL2; down-pointing arrows, down-regulation by
FOXL2.
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Furthermore, it has also been shown that FOXL2 is able to interact with DDX20 (also
named as DP103 or Gemin3), a DEAD-box helicase that potentiates the ability of FOXL2 to
induce apoptosis in CHO cells [209]. Through the oncogenomics-based in vivo RNAi
screening, Ddx20 has been recently unraveled as a potential tumor suppressor in hepatic
carcinomas [210], implying that the interaction between FOXL2 and DDX20 may assist
FOXL2-mediated tumor suppression in ovary. Besides these two protein partners, FOXL2
can also interact with the serine/threonine kinase LATS1 (large tumor suppressor 1, also
named as WARTS) [204]. Lats1 has been demonstrated as a bona fide tumor suppressor gane
in Lats1-deficient mice, evidenced by suppressing the development of ovarian stromal cell
tumors and soft-tissue sarcomas [211]. Additionally, the cell model studies have shown that
LATS1 is involved in the G2/M checkpoint and can induce G2 arrest by inhibiting CDC2
kinase activity, a critical effector mediating the G2/M transition [212]. Thus, it will be an
interesting issue of whether the interaction with LATS1 plays a key role in FOXL2-driven
tumor suppression, which warrants future investigation. Another line of evidence also links
FOXL2 to a bona fide tumor suppressor, the Retinoblastoma (Rb) protein. Expression of
Foxl2 has been found to be significantly decreased in Rb-/- granulosa cells, suggesting that Rb
functions as an upstream modulator to positively regulate Foxl2 expression [213]. Since
inactivation of Rb is also found in ovarian cancer [214], it will be important to investigate
whether both molecules are synergistically implicated in preventing ovarian tumorigenesis.
Some of identified FOXL2 downstream target genes have been shown to have tumorsuppressive characteristics, which may mediate FOXL2-driven tumor suppression (Figure 4).
One of the examples is Foxl2-Smad2-upregulated Follistatin gene expression as mentioned
above [208]. Moreover, the p16-INK4a protein encoded from the CDKN2A locus is expressed
in a FOXL2-dependent manner [204, 215, 216]. The p16-INK4a protein is a well-known
CDKI with the ability to arrest cells in the G1 phase [217]. In line with the fact that FOXL2
expression is generally down-regulated or lost in the most aggressive OGCTs, FOXL2 has
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been reported to negatively regulate matrix metalloproteinases (e.g. MMP23), suggesting that
loss of or low FOXL2 expression will favor the metastatic progression. Through the
transcriptome perturbation study of a granulosa cell model, numerous apoptotic regulators
were identified as potential FOXL2 targets [215] (Figure 4). These identified FOXL2 targets
include pro-apoptotic CH25H, TNF-R1 (tumor necrosis factor-receptor 1) and Fas
(CD95/APO-1) [218, 219], anti-apoptotic TNFAIP3 [220], and modulators with dual roles in
the pro- or anti-apoptotic effect dependent on the cellular context such as BCL2A1, ATF3
and IER3/IEX-1 [215, 221, 222]. These discoveries suggest that FOXL2 might function as
either a pro- or anti-apoptotic effector in a context-dependent manner (e.g. the differentiation
status of granulosa cells, severity of cellular stresses, etc.). Finally, a recent study has shown
that oxidative stress activates the transcriptional ability of FOXL2 to induce the expression of
stress-responsive genes (e.g. MnSOD, SIRT1) [223] (Figure 4). Interestingly, its target SIRT1
deacetyase actually inhibits FOXL2-mediated transactivation, which forms a negativefeedback loop to counterbalance stress-induced FOXL2 transcriptional activity for restoration
of cellular status back to the steady state [223]. Taken together, by regulating the expression
of stress-responsive and apoptosis-regulatory genes, it has been proposed that FOXL2 is a key
actor to mediate cell stress responses in ovarian granulosa cells for the maintenance of cell
survival as well as genomic stability and the elimination of severely damaged cells [204].
2. Aberrations in FOXL Genes in Cancer
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FOXL1 expression has been shown to be elevated in low-grade fibromyxoid sarcomas
(LGFMS) [224]. However, its role in this type of tumors is completely unknown. As
discussed above, FOXL2 expression is lost or markedly reduced in more than half of juvenile
OGCT ovarian tumor cases [216]. A complete extinction of FOXL2 expression is further
observed in recurring juvenile OGCT cases [216]. The pathological mechanisms leading to
these alterations in FOXL2 expression in OGCTs remain elusive and are currently under
investigation. Through genome-wide next-generation sequencing analysis of the
transcriptome in OGCTs, Shah et al. identified the somatic mutation p.Cys134Trp in the
protein sequence of FOXL2 [225]. The prevalence of this mutation has been confirmed in
97% of examined adult OGCTs according to the published report [225]. The impact of this
somatic mutation on FOXL2 function has been studied, which has shown that the FOXL2
mutant protein is unable to trigger the full apoptotic response compared to wild-type FOXL2
[219]. In line with this result, this mutation attenuates the ability of FOXL2 to up-regulate
pro-apoptotic effectors TNF-R1 and Fas [219]. It is also worth noting that FOXL2 mutant has
been shown to be able to dimerize with the wild-type protein and has a dominant-negative
effect to interfere with wild-type FOXL2 to regulate its downstream target genes [226]. This
finding suggests that the FOXL2 mutant protein can impair the normal transcriptional activity
and target-gene specificity of the wild-type FOXL2 protein, a mechanism potentially leading
to ovarian tumorigenesis. Indeed, through ectopic overexpression of wild-type and mutant
FOXL2 in the granulosa cell tumor (GCT) line (COV434) with a lack of FOXL2 or the
silencing of mutant FOXL2 expression in the GCT line (KGN), mutant FOXL2 protein has
been shown to be able to differentially regulate the cellular transcriptome compared to the
wild-type protein [227]. Taken together, these findings indicate that in addition to loss of or
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reduced FOXL2 expression, this somatic mutation is another crucial mechanism accounting
for the development and aggressiveness of OGCTs.
E. The Bi-Functional Roles of FOX Transcription
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FOXO transcription factors are known to render cells resistant to oxidative stress,
premature aging and cellular senescence. Hence, FOXOs are critical to cell survival and
longevity. These surviving functions of FOXOs are mediated by their effects on ROS
detoxification, the enhancement of DNA repair, the induction of cell cycle arrest, the
inhibition of the mTORC1-S6K signaling axis, and the promotion of cellular atophy and
autophagy. Although these FOXOs‘ activities are commonly thought as their tumor
suppressor functions, the recent studies also indicate that cancer cells, especially cancer stem
cells (CSCs), can hijack these FOXO-mediated cell surviving mechanisms for the tumor
development and resistance to radiotherapy and chemotherapy.
A recent study reported that the expression of FOXO and anti-oxidant FOXO target
genes is elevated in the radiotherapy-resistant subset of the breast CSC population compared
with untreated control cells [228]. These findings indicate that FOXO-mediated ROS
detoxification renders CSCs resistant to oxidizing damage from therapeutics and potentially
contributes to relapse and the poor patient survival rate [228)]. In addition, FOXO1
expression has been found to be upregulated in paclitaxel-resistant ovarian cancer cells,
which protects cells by lowering ROS production and alleviating drug-induced cytotoxicity
(229). In doxorubicin-treated leukemia cells, FOXO3 has been shown to be responsible for
induced expression of the multi-drug resistant ABCG1 gene and PIK3CA gene, which is
considered as a potential mechanism leading to therapeutic resistance [230, 231]. Similarly, it
has also been reported that the expression of multiple drug resistant 1 (MDR1) in
doxorubicin-resistant breast cancer cells relies on FOXO1 function [232]. In light of multiple
studies demonstrating the role of FOXO proteins in the DNA repair pathway [34, 104], it is
plausible that therapy-triggered FOXO activation may be beneficial to cancer cell (or cancer
stem cell) survival by facilitating DNA repair in addition to ROS detoxification. These lines
of contradictory evidence raise issues of whether they are a general phenomenon in all kinds
of cancers as they become resistant to anti-cancer therapeutics, what cell subset in
heterogeneous cancer cell populations displays this phenomenon and whether this
therapeutics-resistant cell population must have CSC-like properties. These raised questions
warrants future investigation due to their importance to the development of therapeutics
targeting FOXOs.
As indicated above, through induction of multiple downstream target genes (e.g. InsR,
IRS2, TSC1, Sestrin 3, Rictor, HER2/3, BNIP3, etc.), FOXO factors can potentiate AKT
activity to maintain cell survival by inhibiting the mTORC1-S6K-mediated negative feedback
loop, activating mTORC2 and augmenting the upstream RTK and IRS input signaling.
Apparently, these FOXOs‘ effects contradict their roles as tumor suppressors. These
contradictory roles of FOXOs may give an explanation for the observations that overall AKT
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activity is decreased in FOXO-deficient cells and there are no epithelial origins of tumors
developed in FOXO-knockout mice [11]. Therefore, these FOXO-mediated effects may
potentially promote cancer cell survival in response to therapeutic treatments especially when
FOXO-mediated pro-apoptotic effects are abrogated in cancer cells. Indeed, a recent report
demonstrates that FOXOs are responsible for the development of the acquired resistance to
AKT inhibitors [91]. The AKT inhibitor (MK-2206) has been shown to inhibit mTORC1
signaling but to activate FOXO-dependent elevated expression of RTKs (e.g. HER2/3,
IGF1R) and RTK downstream signaling modules (91). Furthermore, through the downstream
autophagy-related target genes (e.g. LC3, BNIP3, ATG12, GABARAPL1) and the inhibition
of mTORC1 activity, FOXOs are known to promote the autophagy process, which is a
cellular mechanism to eliminate defective mitochondria by macroautophagy for increasing
cell survival.
Thus, transformed cancer cells can employ this aspect of FOXO function to escape from
a cellular energy crisis, increased ROS and DNA damage. Indeed, it has been reported that
autophagy is required for Ras-mediated tumorigenesis and the development of pancreatic
cancers [233, 234]. In light of all lines of evidence as mentioned above, it is warranted to
further investigate the functional significance of FOXO-mediated activation of ROS
detoxification, DNA repair, RTK signaling and autophagy in tumor maintenance and survival
upon metabolic, oxidative and DNA-damaged stress.
In mammals, cellular senescence is considered as one of the defensive mechanisms
against the in vivo tumor development. It has been reported that FOXOs are able to elicit
cellular senescence in melanoma cells harboring the oncogenic mutated BRAF gene, where
high ROS levels and activated JNK stimulate FOXO-induced p21 expression and p21dependent senescence [235]. On the other hand, other studies have shown the opposite role of
FOXOs in the regulation of cellular senescence. In cultured human endothelial cells,
constitutively activated AKT induces p53-p21-dependent cellular senescence, which is
mediated by the inhibition of FOXO activity and its effect on lowering ROS levels that are
required for activation of p53 [236]. It has been a consensus that hyper-activation of
oncogenic signaling can trigger p53-dependent cellular senescence. The hyper-activation of
mTORC1 has been found as one of such mechanisms that induce senescence by evoking
cellular checkpoint responses [237].
The hyper-activation of mTORC1 also leads to loss of a response to growth factor
stimuli, which promotes the occurrence of senescence [237]. A recent related study has also
shown that loss of PTEN induces senescence in prostate cancer cells. In particular, the
underlying mechanism for this type of senescence does not involve hyper-proliferation and
DNA damage response. In fact, it is mediated by the p53 action that is triggered by PTEN
loss-induced hyper-activation of mTORC1 [238]. In light of the fact that FOXOs inhibit
mTORC1 activity and induce cell cycle arrest upon cellular stresses, it is plausible that
FOXOs can block mTORC1-triggered senescence. Therefore, the investigations of whether
FOXOs possess intrinsic ability to antagonize cellular senescence in many cell types and what
role of this function in tumorigenesis are mandatory.
The reported role of FOXOs in maintaining leukemia initiating cells (LICs) in chronic
myeloid leukemia (CML) also indicates another side of FOXOs. The animal studies have
shown that FOXO3-deficient LICs fail to be successively transplanted in the syngenic mouse
model of Bcr-Abl-induced CML [239].
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The further study elucidates that abrogation of FOXO attenuates the TGF-FOXO
pathway, which is indispensable for the survival and maintenance of the LIC fraction [239].
Noticeably, the combined treatment with a tyrosine kinase inhibitor (Imanitib) and TGF
inhibitor (LY364947) more effectively depletes LICs and improves overall survival by
suppressing both AKT and TGF-FOXO [239]. Furthermore, it has been shown that FOXO
factors are essential for maintaining the long-term homeostasis of hematopoietic as well as
neural stem cells and regulating their responses to physiological stresses [240-242]. Whether
FOXOs have a parallel function in maintaining relatively quiescent cancer initiating cell
population in particular types of tumors is a critical issue that needs to be addressed by future
investigation. In prostate cancer, FOXOs also play a dual role to act as either tumor
suppressors to repress the expression of androgen receptor in androgen-dependent prostate
cancer cells [243] or oncoproteins to transcriptionally up-regulate VEGF expression
following androgen-ablation therapy, giving rise to the development of metastatic and
androgen-refractory tumors [244]. In conclusion, the exact roles of FOXO transcription
factors in cancer cells depend on the cellular context, disease stages, tissue types and
environmental cues.
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As discussed above, FOXP1 potentially functions as either an oncoprotein in various
types of lymphomas (e.g. Diffuse large B-cell lymphoma and mucosa-associated lymphoid
tissue lymphoma) and liver cancer or a tumor suppressor in numerous tissues and organs
[reviewed in [5, 143]]. These differentially functional aspects of FOXP1 are attributable to
tissue-type-specific and cancer-type-specific expression levels and/or alternative splicing
variants of the FOXP1 protein [5, 143]. In contrast, FOXP3 is a bona fide X-linked tumor
suppressor in breast, ovarian and prostate cancer. However, the immunosuppressive role of
FOXP3 in regulatory T lymphocytes has been known to be positively associated with tumor
development because FOXP3+ Treg cells are abundantly present in infiltrated tumor tissues
and experimental Treg reduction could dampen tumor growth and enhance the efficacy of
tumor immunotherapy [reviewed in [144, 147]]. Therefore, by being expressed in either
epithelial cells or regulatory T lymphocytes, FOXP3 has a bi-functional role in human
cancers.
3. The Bi-Functional Roles of FOXF Transcription Factors in Cancer
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As indicated above, our evidence supports the tumor-suppressor role of FOXF1 in breast
and colorectal tumorigenesis [174, 175, 188].
However, on the other hand, there are two lines of opposite evidence supporting that
FOXF1 acts as an oncoprotein. Saito et al. reported that FOXF1 is required for the
contractility of cancer-associated fibroblasts (CAF), CAF-mediated production of hepatocyte
growth factor and fibroblast growth factor-2, and CAF-mediated stimulation of lung cancer
cell migration and xenografted tumor growth [245]. In addition to its role in lung cancer,
another study has revealed that ectopic overexpression of FoxF1 in a mouse mammary
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epithelial cell line leads to the induction of epithelial-to-mesenchymal transition (EMT),
increased invasiveness of epithelial cells in vitro and enhanced growth of breast xenograft
tumors in vivo [246]. These findings are strengthened by the correlation between FOXF1
expression and a mesenchymal phenotype in human breast cancer cell lines [246].
Therefore, these two lines of evidence have deciphered the oncogenic aspect of FOXF1
that can enhance tumor-promoting properties of cancer-associated fibroblasts in lung cancer
and induce an EMT phenotype in mammary epithelial cells [245, 246]. Their studies suggest
that FOXF1 plays oncogenic roles in lung tumorigenesis and breast cancer metastasis. In
contrast, we identified another aspect of FOXF1 function that acts like a tumor suppressor to
negatively regulate DNA rereplication for maintaining the stringency of DNA replication and
genomic stability. According to expression analysis of FOXF1 gene in 20 breast cancer cell
lines [174, 188], FOXF1 expression is preferentially detected in basal-like (mesenchymallike) breast cancer cell lines (e.g. BT20, BT549, MDA-MB-157, HCC1937 and HCC1187),
but mostly silenced in luminal (epithelial-like) breast cancer cell lines. These results are in
line with observations from Nilsson et al. [246]. This raises a possibility that FOXF1 exhibits
the tumor-suppressive function in luminal breast cancer cells but displays tumor-promoting
features in basal-like (or metastatic) breast cancer cells. However, how basal-like breast
cancer cells can escape the FOXF1-mediated tumor-suppression control needs to be further
elucidated.
Thus, our and other studies conclusively support a paradigm that FOXF1 plays a dual
role in tumorigenesis as an oncoprotein or a tumor suppressor in a context-dependent manner.
Owing to the high homology between FOXF1 and FOXF2 protein sequences and their
functional redundancy, it will be interesting to decipher whether FOXF2 also has a dual role
in tumorigenesis and metastasis.
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In Apc(Min) mice, Foxl1 acts like a tumor suppressor to inhibit gastrointestinal
tumorigenesis [191].
However, Foxl1 collaborates with Foxf1 to regulate gastrointestinal morphogenesis by
mediating hedgehog signaling [168] and also positively modulates Wnt/-Catenin signaling
[190]. Both hedgehog and Wnt signaling pathways have been known to be implicated in
gastrointestinal stem cell regulation and tumorigenesis [247-249]. Therefore, it is possible
that FOXL1 may play a dual role in promoting either tumor suppression or tumorigenesis in
the gastrointestinal system depending on tissue cell types, tissue-environmental stimuli, tumor
stages, etc.
Similarly, in different tissue types, FOXL2 performs different roles that either activate or
inactivate tumorigenesis. In ovary, FOXL2 functions as a tumor suppressor to inhibit OGCT
tumor development [196, 197, 204]. Thus OGCTs exhibit the frequent inactivation of FOXL2
function via markedly reduced expression of FOXL2 or somatic mutations on its gene [204].
However, in breast cancer, FOXL2 is a transcriptional inducer of the aromatase CYP19A1
gene which encodes an enzyme required for the estrogen biosynthesis. FOXL2-induced
production of estrogen is essential for the growth of ER-positive breast tumors, but
concurrently renders breast cancer cells susceptible to tamoxifen therapy [250].
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1. Therapeutic Targeting of FOXO Proteins in Cancer
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F. The Potential of FOX Proteins as Therapeutic
Targets
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Among these FOX subfamilies discussed above, FOXO subfamily draws most attention
in recent targeting therapeutics. Inactivation of FOXOs‘ tumor-suppressor functions in
multiple cancer types is mediated by various mechanisms such as chromosomal translocation,
genomic deletion, miRNA regulation and post-translational dysregulation by a variety of
oncogenic signaling pathways (e.g. AKT, IKK and ERK). Reintroduction of FOXOs into
cancer cells defective of FOXO activity gives rise to in vitro growth inhibition of cancer cells
and in vivo tumor suppression. Therefore, due to the strong anti-tumor activities of FOXO
factors, anti-cancer agents that can reactivate FOXOs‘ functions in cancer cells become
compelling therapeutics for cancer treatments. For example, there are several preclinical/clinical chemotherapeutic agents reported to be able to reactivate FOXO3 and its
downstream pro-apoptotic (e.g. BIM) or CDKI (e.g. p27) target gene in various cancer types
(Table 3); (1) Paclitaxel activates FOXO3 in breast carcinoma cells by inhibiting AKT and
activating JNK [251, 252]. (2) KP372-1, a multiple kinase inhibitor, activates FOXO3 in
acute myeloid leukemia cells by reducing AKT activity [253]. (3) OSU-03012, a novel anticancer agent, activates FOXO3 and its target p27 expression in ER-negative breast cancer
cells by inhibiting AKT, which sensitizes ER-negative breast cancer cells to Tamoxifen
therapy [254]. (4) AZD6244 activates FOXO3 in lung and gastric cancer cells by suppressing
ERK [255, 256]. (5) Imatinib activates FOXO3 and its pro-apoptotic target gene (BIM)
expression in chronic myeloid leukemia cells via the inhibition of BCR-ABL fusion
oncoprotein [257, 258]. (6) Trastuzumab and cetuximab, antibodies to negatively target
epidermal growth factor receptors (HER family receptors), activate FOXO3 and
transcriptional induction of FOXO3‘s pro-apoptotic target BNIP3L gene in breast, prostate,
kidney, ovarian and lung cancer cells by interfering with activation of PI3K-AKT signaling
[9, 259].
In addition to these chemotherapeutic agents that target upstream kinases responsible for
negatively regulating FOXOs, there are several novel small molecules that have been
developed to inhibit the nuclear exclusion of FOXO proteins (Table 3). In light of the antitumor activity of FOXO1 in PTEN-null cancer cells, Kau et al. employed a high-throughput
chemical genetic screening to successfully identify novel general inhibitors to block FOXO
nuclear export [260].
These identified inhibitors either react with CRM1 (nuclear exporter) or inhibit
PI3K/AKT signaling [260]. Moreover, Schroeder et al. screened a collection of marine
natural product extracts for the identification of compounds that can compensate for loss of
tumor-suppressive functionality in PTEN-null cells [261]. They discovered the most effective
extract (psammaplysene A) that compensates for loss of PTEN by inhibiting the nuclear
exclusion of FOXO1 [261]. By using a cell-based imaging model system that tracks the
cellular translocation of FOXO proteins, Link et al. screened 33,992 small chemical
molecules and identified pyrazolopyrimidine derivatives as potent FOXO relocators [262].
These compounds actually are the biochemical inhibitors of PI3K. Based on virtual
161
Table 3. FOX tumor suppressors as anticancer therapeutic targets
Cancer
therapeutics
Paclitaxel
FOX factor
targeted
FOXO3
Tested cancer type
KP372-1
FOXO3
OSU-03012
FOXO3
Acute myeloid
leukemia
Breast cancer
AZD6244
FOXO3
Imatinib (Glivec)
FOXO3
Trastuzumab,
cetuximab, and
lapatinib
FOXO3
Psammaplysene A
FOXO1
The underlying therapeutic
mechanism
Inhibition of AKT and activation
of JNK (251, 252)
A reduction of AKT activity
(253)
Inhibition of AKT and
sensitizing ER-negative breast
cancer cells to Tamoxifen (254)
Inhibition of ERK (255, 256)
Inhibition of BCR-ABL fusion
oncoprotein, leading to FOXO3
dephosphorylation and BIMdependent apoptosis (257, 258)
Inhibition of EGFR and HER2
signaling, suppression of PI3KAKT, activation of FOXO3 and
induction of pro-apoptotic
FOXO3 target BNIP3L (9, 259)
Inhibiting the nuclear exclusion
of FOXO1 (261)
Inhibition of PI3K activity (262)
FOXO3
FOXO3
en
Adenovirusmediated transfer
of constitutively
active FOXO3
Anisomycin
FOXP3
Sc
i
Doxorubicin
Kidney cancer
FOXP3
Osteosarcoma,
breast carcinoma
Melanoma
Breast cancer
Breast and
colorectal cancer
Activation of FOXO3 and
induction of apoptosis (263)
Induction of FOXP3 expression
and apoptosis (269)
p53-dependent induction of
FOXP3 expression (270)
a
These studies provide examples to screen and identify active therapeutic agents to target
oncogenic PI3K/AKT signaling based on their effects on relocalizing FOXO to the nucleus.
Besides reactivation of FOXO by chemical agents, reintroduction of constitutively active
FOXO3 into melanoma cells via the adenovirus-mediated transfer was also reported and this
strategy induces apoptosis in melanoma cells [263]. This therapeutic strategy may be
beneficial to the killing of FOXO-deficient cancer cells with or without the combination with
other therapeutics. Owing to the frequent occurrence of FOXO inactivation in cancers, the
combination with therapeutic agents that reactivate FOXO activity may potentially enhance
the treatment efficacies of traditional anti-cancer agents. These combined therapeutic
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kidney, ovarian
and lung cancer
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ETP-45658
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Chronic myeloid
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Breast cancer
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screening and molecular modeling, they synthesized a most potent PI3K inhibitor ETP-45658
with a strong effect to suppress tumorigenicity in vitro and in vivo [262].
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regimens are also potentially beneficial to the treatment of drug-resistant cancers derived
from the inactivation of the FOXO signaling pathway. It is noteworthy that ionizing radiation
has been reported to activate FOXO3-induced BIM up-regulation and concurrent apoptosis in
p53-null osteosarcoma cells [264]. This suggests that the combination of radiotherapies with
chemotherapies targeting FOXOs may be a potential therapeutic regimen to sensitize radioresistant tumor cells to radiation therapy. Additionally, previous reports have shown that the
resistant tumor cells developed in treatment with MEK inhibitors manifest hyper-activated
AKT activity [256, 265-267]. It has been known that inhibition of MEK-ERK signaling can
reactivate FOXO activity for inducing tumor-suppressive effects (e.g. BIM, p27). However,
AKT is hyper-activated during the development of drug resistance, which in turn inhibits
FOXO activity. Therefore, reactivation of FOXO by therapeutic agents is a promising
strategy to conquer cancer resistance to the MEK inhibitors [255]. Conclusively, these studies
implicate that FOXO reactivation may be a prospective therapeutic target and
pharmacological indicator to potentiate and predict the efficacies of MEK inhibitors in
clinics. Although most of studies emphasize the beneficial effects of FOXO reactivation on
cancer therapy, the roles of FOXOs in negative feedback regulation loop complicate the
applications of targeting FOXO to cancer treatment. The inhibitory effect of FOXO activation
on mTORC1 signaling could potentially benefit cancer therapy. Despite these benefits from
FOXO-mediated tumor-suupressive effects, it could not be ignored that the effects of FOXOs
on the coupling to the activation of AKT and upstream RTK signaling may raise the question
of whether the abrogation of FOXOs is preferred over their activation for cancer therapy.
Therefore, more future investigations are warranted for the understanding of what signaling
context in cancer cells is suitable for which therapeutics based on either activation or
inhibition of FOXO activity.
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X-chromosome inactivation occurring during embryogenesis is a vital mechanism to
silence gene expression from one of the two X chromosomes in females. Given that the
majority of the somatic mutations and deletions of the FOXP3 gene are heterozygous in
human female cancers, it is possible to reactivate the remaining wild-type FOXP3 allele for
cancer therapy [145, 146, 268]. Indeed, it has been reported that anisomycin treatment
triggers an unknown cellular response to induce the expression of FOXP3 (Table 3). Induced
FOXP3 elicits apoptosis in various breast cancer cell lines and inhibits xenograft mammary
tumor formation in vivo [269]. Intriguingly, the FOXP3 gene is the downstream target of the
p53 transcription factor and the chemotherapeutic agent doxorubicin, a DNA damaging drug
to activate p53, has been reported to induce the p53-dependent transcription of the FOXP3
gene in vitro [270] (Table 3). In light of these two lines of evidence, a paradigm that anticancer therapeutics can be implemented by the restoration of FOXP3 function in cancer cells
might have great potential to be translationally applied to effective therapy of human
malignancies.
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Conclusion
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It is convincing that FOX transcription factors are crucial regulators in a variety of
biological processes, embryonic development, organogenesis and tissue regeneration. Given
that the expression of a wide spectrum of direct and indirect target genes is under FOX
regulation, dysregulation of FOX expression and function can significantly contribute to or is
sufficient for tumorigenesis and metastasis. As mentioned in this chapter, the functions of
FOX genes with tumor-suppressor characteristics are inactivated in cancer through multiple
mechanisms such as chromosomal translocations, genomic deletions, somatic inactivating
mutations, aberrant alternative splicing, cytoplasmic mislocalization, protein degradation,
epigenetic gene silencing, microRNA deregulation, etc. For those pathological mechanisms
involving cytoplasmic mislocalization and protein degradation, there are therapeutic
opportunities to target these aberrant mechanisms to correct these alterations and in turn
restore the functions of FOX tumor suppressors (e.g. FOXO3, FOXP3) in cancer cells. For
those FOX genes (e.g. FOXF1) silenced by the epigenetic mechanism, their expression can be
potentially restored by current clinically used epigenetic drugs (e.g. demethylating agents and
histone deacetylase inhibitors) [271]. Based on these viewpoints, FOX-targeted therapies look
promising in the future. However, the complicated natures of these FOX proteins, being able
to act as either oncoproteins or tumor suppressors in a context-dependent manner, render the
clinical implications of FOX-targeted therapeutics questionable.
Therefore, before the translational applications of FOX-targeted therapies to clinics, the
context-dependency of FOX functions should be comprehensively investigated and defined in
various types of human cancers and also in different stages of cancers (e.g. non-metastatic
and metastatic). Such information will be crucial to further rationally optimize the
combination of FOX-targeted therapies with current therapeutic regimens. In light of recent
innovations in both next-generation sequencing and bioinformatics technologies [272], the
genetic and epigenetic landscapes of FOX family genes as well as FOX-binding sites within
the regulatory regions of FOX-target genes in human cancers can be characterized and
defined in a genome-wide, high-throughput manner.
These future efforts could promote understanding of the mechanisms underlying FOXregulated tumorigenesis and the development of novel cancer diagnostics, prognostics and
anti-cancer therapeutics.
a
I thank Dr. Saraswati Sukumar at the Johns Hopkins University for previously supporting
the FOXF studies and Drs. Vicki Vance and Richard Showman at the University of South
Carolina (USC) for supporting and assisting my research at USC. This review chapter was
supported in part by the Advanced Support Program for Innovative Research Excellence-I
(ASPIRE-I) Grant 13010-A026 to P. K. Lo, awarded by the Office of the Vice President for
Research at USC.
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Acknowledgments
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Pang-Kuo Lo
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ISBN: 978-1-62808-665-2
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Chapter 8
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Molecular Basis of BRCA1 and BRCA2
and Clinical Approaches to BRCA1/2
Mutation Carriers
Department of Medical Genetics, Istanbul University, Cerrahpasa Medical School,
Istanbul, Turkey
2
Department of General Surgery, Breast Division, Istanbul University,
Cerrahpasa Medical School, Istanbul, Turkey
3
Department of Pathology & Immunology, Baylor College of Medicine,
Houston, TX, US
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Kemal Keseroglu1,† , Fatih Aydogan2,† and Mustafa Ozen1, 3,
Abstract
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The human BRCA1 and BRCA2 genes are tumor suppressor genes. Whereas BRCA1
is mainly expressed in the cells of thymus and testes, BRCA2 has the highest expression
in mammary glands and thymus. Both BRCA1 and BRCA2 proteins are nuclear proteins
and whilst BRCA1 protein has functions in DNA repair of double-stranded breaks,
ubiquitination, transcription and transcriptional regulation; BRCA2 has functions in
homologous recombination and DNA repair.
Due to their essential functions in the cell, BRCA1/2 mutations lead to tumor
formation, especially breast and over cancer. Lifetime risk of BRCA1/2 mutation carriers
is 35-87% for breast cancer and 16-60% for ovarian cancer and the lifetime risk for other
cancers are also elevated. Genetic counseling must be given according to
recommendations for risk assessment to hereditary breast and over cancer.
There are three possible results of the BRCA1/2 testing: (i) Negative, which shows
that no mutation was detected (ii) Positive, which means that the risk of developing
breast and ovarian cancer for the client is increased (iii) and inconclusive result.

Correspondence: Mustafa Ozen M.D. Ph.D. Department of Medical Genetics, Istanbul University, Cerrahpasa
Medical School, Istanbul, Turkey, Email: [email protected], [email protected].
†
These authors contributed equally.
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The recommendations for increased cancer risk in carriers of BRCA1/2 mutations
are: (i) surveillance, which includes clinical exams, mammography and such blood tests,
(ii) prophylactic mastectomy, where the incidences of breast cancer and ovarian cancer
decrease up to 90% and 80%, respectively, in women that carry BRCA1/2 mutation, and
(iii) chemoprevention. Two agents, Tamoxifen and Raloxifen, which are the selective
estrogen modulators, are approved up to now to decrease breast cancer risk.
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Keyaords: BRCA1, BRCA2, breast cancer, ovarian cancer, BRCA mutation carriers, risk
assessment
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BRCA1 and BRCA2 Structures, Expressions
and Functions
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The human BRCA1 and BRCA2 genes are tumor suppressor genes [1, 2], which produce
the breast cancer type 1 susceptibility protein (BRCA1) and breast cancer type 2
susceptibility protein (BRCA2), respectively. The human BRCA1 gene spans 81189 base
pairs (bps) on chromosome 17q21. It contains 23 exons and it is transcribed into 7094 bps
product; BRCA1 has 207721 Da weight and 1863 residues in translation length. The human
BRCA2 gene, on the other hand, spans 84193 bps on chromosome 13q12.3; the transcript of
the gene has 27 exons, 10930 bps; the protein has 384201 Da weight and 3418 residues in
translation length (Figure 1). BRCA1 is widely expressed in a variety of cells and tissues such
as thymus, testes, breast, ovary, uterus, spleen, liver and lymph nodes [3], however, as seen in
Table 1, the majority of the mutations of the gene causes breast and ovarian cancers where
damaged DNA is failed to be repaired. The subcellular localization of the encoded protein is
reported to be primarily in nucleus [4], but alternatively it is also in cytoplasm [5],
centrosome [6], perinuclear region [7] and mitochondrion [8]. The BRCA1 protein contains
four major protein domains; (i) the Zinc finger Cys3HisCys4-RING domain where it interacts
with a homologous region of BARD1 [9] and is an essential region for ubiquitination [10],
(ii) the BRCA1 serine domain where the phosphorylation sites are concentrated in [11] and
(iii-iv) two BRCA1 C Terminus (BRCT) domains those interact with each other to control
cellular responses to DNA damage [12] (Figure 1b). Thus, the human BRCA1 protein has
multiple functions in cells such as DNA repair of double-stranded breaks [13], ubiquitination,
transcription and transcriptional regulation [14]. The highest expression levels of the human
BRCA2 gene are detected in mammary glands and thymus [15] and its subcellular localization
is primarily seen in nucleus [16], whilst alternatively its cytoplasmic [16] and extracellular
[17] presence is also documented. It has six phosphorylation sites and can directly bind the
single strand DNA [18] (Figure 1d). BRCA2 is involved in different molecular pathways
including homologous recombination [19], Fanconi anemia pathway [20], pathways in
pancreatic cancer [21] and other cancers [22]. BRCA2 has several motifs for interaction with
the recombinase RAD51 [23], which functions in homologous recombination and DNA repair
[24]. Therefore, the mutation of BRCA2 causes mismatch repair, which leads to loss of
stability of the human genome, dangerous gene rearrangements and so tumor formation [22].
After DNA damage is occurred, both BRCA1 and RAD51 proteins accumulate at the
damaged region and BRCA1 has shown to be necessary for subnuclear Rad51 cluster [25].
During this process, BRCA1 is phosphorylated by active ATM, ATR and/or Chk2 and its
Molecular Basis of BRCA1 and BRCA2 …
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phosphorylation has demonstrated to either directly or indirectly regulates homologous
recombination, non-homologous recombination and S/G2 phase checkpoint mechanisms [2629].
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Figure 1. Structures of BRCA1 and BRCA2 genes (a, c), transcript (b, d) and tertiary conformations of
BRCA1 and BRCA2 proteins (e, f), respectively.
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Figure 2. Molecular pathways of BRCA1 and BRCA2 proteins.
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Table 1. Number of mutations of BRCA1 and BRCA2 according to disease
Breast and/or
Ovarian
Cancer
Ovarian
Cancer
Prostate
Cancer
Others
BRCA1
643
455
110
5
11
BRCA2
516
308
52
10
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Gene
Breast
Cancer
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Besides, BRCA2 directly interacts with RAD51, which keeps it in inactive form and
facilitates Rad51 filament formation [25, 30] Contrary to BRCA1, BRCA2 only acts in
homologous recombination (Figure 2).
BRCA1 is also involved in ubiquitination of proteins via interaction and making
heterodimer with BARD1. Both BRCA1 and BARD1 have RING-finger motif close to their
amino termini, which are documented to be associated with their ubiquitination activities
[31]. Ubiquitination function of BRCA1 has shown to be related to its DNA damage repair
activity, due to occurring as a consequence of replication stress [32]. Meanwhile,
ubiquitination activity of BRAC1 has shown to be related to Fanconi anemia pathway through
BRCA1-associated helicase, BACH1 (also known as FANCJ) and BRCA2 (also known as
FANCD1) [33, 34].
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Clinical Management of BRCA Mutation Carriers
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a. Risk Assessment
5–10% of familial breast cancers and 7-10% of familial ovarian cancers stem from
inherited gene mutations [35]. Among these genes, in many cases, BRCA1 and BRCA2 are
responsible for the hereditary breast and ovarian cancer. Although the BRCA mutations are
not common (1/800 – 1/1000) in the most populations, its prevalence varies in different ethnic
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groups and geographical regions. Founder effects have been reported in multiple populations,
most notably in Ashkenazi Jewish, Icelandic, Dutch, and French Canadian [36]. It has been
published that if a BRCA1/2 mutation carrier woman was once affected by breast cancer,
propensity for another independent breast or ovarian cancer development is notably increased
[37-39]. Thus, for the BRCA1/2 mutation carriers, identification of the mutation is highly
important not only for herself, but also for her family members. Lifetime risk of BRCA1 and
BRCA2 mutation carriers is 35% to 87% for breast cancer and 16-60% for ovarian cancer
(40-42). BRCA mutation carriers also have high risks for developing other cancers, such as
prostate cancer, pancreatic cancer and colon cancer (Table 1, 2) [42, 43]. Genetic testing is
recommended by The American Society of Clinical Oncology when these criteria are met: (i)
an individual who has a personal or family history that suggests an inheritable cancer
situation, (ii) insufficient interpretation of the test and (iii) results that will help to diagnose or
find the effect of the medical or surgical approaches for the affected individual or family
members [44]. Many countries and organizations have proposed their own criteria for that a
patient should be referred for genetic counseling and testing (Table 3, 4) [45, 46].
BRCA2 Mutation
Breast cancer (initial) ......... : 45%
Breast cancer (second) ....... : 3% per year
Male breast cancer.............. : 6%
Ovarian ............................... : 11%
Prostate ............................... : 7.5-39%
Pancreatic ........................... : 2-7%
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BRCA1 Mutation
Breast cancer (initial) ......... : 65%
Breast cancer (second) ....... : 3% per year
Male breast cancer .............. : 1%
Ovarian ............................... : 39%
Prostate cancer ................... : None to 2- to 3fold increase
Pancreatic ........................... : 1-4%
Colon cancer ....................... : slight increase
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Table 2. BRCA1 or BRCA2 Mutations and Associated Cancer Risks (by age 70)
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Table 3. Recommendations for genetic testing of hereditary breast and ovarian cancer
according to American Society of Breast Surgeons
Early onset breast cancer (diagnosed before age 50)
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Two primary breast cancers, either bilateral or ipsilateral
Family history of early onset breast cancer
Male breast cancer
Personal or family history of ovarian cancer (particularly nonmucinous types)
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Ashkenazi (Eastern European) Jewish heritage in the setting of a newly diagnosed breast
cancer or family history of breast cancer
A previously identified BRCA1 or BRCA2 mutation in the family
―Triple negative‖ (ER-, PR-, Her2 normal) breast cancer diagnosed prior to age 60.
Pancreatic cancer associated with a family history of hereditary breast and ovarian related
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Two breast cancer cases <40 years
Male breast cancer and ovarian cancer or early onset female breast cancer
Young onset bilateral breast cancer
Breast and ovarian cancer in the same patient
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b. Genetic Counseling
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Ashkenazi Jew with breast cancer of <60 years
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Three or more breast and/or ovarian cancer cases, at least one <50 years
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Table 4. Recommendations for genetic testing of hereditary breast and ovarian cancer
according to European Society of Medical oncology
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Genetic counseling is a communication process, which considers the genetic-related
health problems of the patient and helps both the individual and family according to
occurrence or incidence of a genetic disease, carried out by one or more health professions
[47].
National Society of Genetic Counselors has reported a guideline about recommendations
for risk assessment and genetic counseling in hereditary breast and over cancer [48]. Genetic
counseling must be guided by these recommendations and must be carried on by educated
people. The first step in genetic testing for the cancer patient or his/her relative is giving
adequate information and preparing a family tree. Information given to the person must
include all the aspects like the meaning of genetic counseling, the way it is done, the
limitations, the benefits, the cost, probable test results and the alternative approaches. The
information given must be suitable with the patient's educational and cultural degree and if
necessary must be supported by visual materials. For the persons who are not thought to be
psychologically ready to be informed about the test results, especially for the young women,
psychiatric consultation can be requested.
Genetic testing must be applied if the clinical approach for the person and the family is
going to change. Patients without a known mutation may be candidates for analysis of large
rearrangements in BRCA1/2. The mutation must be searched for in the cancer patient first; if
it is found, then the other members of family must be screened too.
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c. Interpreting Genetic Test Results
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There are three possible results of the BRCA1 and BRCA2 testing: (i) Negative (ii)
Positive (iii) Inconclusive result.
A negative test shows that no mutation was detected in the BRCA1 or BRCA2 gene.
Additionally, having no mutation as has been previously reported in the family for an index is
called a ―true negative‖ test result, although it does not mean that the person will not be
affected by cancer; contrary, it is thought that the risk of developing breast and ovarian cancer
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d. Prophylactic Surgery
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would be same as that of the general population. It is essential to share the possibilities of a
false-negative test with clients receiving a negative test result,
A positive result gives an explanation for the inheritance of the cancer that is observed in
a family and means that the risk of developing breast and ovarian cancer for the client is
increased. Current risk estimates for BRCA1 and BRCA2 mutations are 35-87% lifetime risk
for breast cancer and 16-60% lifetime risk for ovarian cancer [40-42]. Further tests are
recommended for the relatives who have high risks, however, the health and social
consequences for family members, even for future generations, should be cared.
Thirdly, finding a variation in BRCA1 or BRCA2 that has not been defined previously as
a reason for cancer, the test result is accepted as inconclusive. Having a variant of uncertain
or unknown clinical significance is reported in 10-15% individuals who have done genetic
testing for BRCA1/2 mutations [49].
Recommendations for BRCA1 and BRCA2 mutation carriers who have increased cancer
risk are: surveillance, prophylactic mastectomy and chemoprevention.
The aim of surveillance is following the mutation carriers closely and making early
diagnosis. The surveillance methods for breast cancer include clinical breast examination,
mammography and magnetic resonance imaging; for ovarian cancer, the methods consist of
clinical exams, blood tests for CA-125 antigens and transvaginal ultrasound.
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Prophylactic bilateral mastectomy: Prophylactic bilateral mastectomy was shown to
decrease the occurrence of breast cancer up to 90% in women carrying BRCA1/2 mutation
[50]. After the surgery in the same session some reconstructive attempts are made for these
patients to promote the life quality. But it is impossible to determine the psychological and
physical deprivation of the patients, which is caused by the operation. Because of that reason,
a preventive surgical intervention like this must be offered to the patient only after a full
psychological assessment [51].
Prophylactic bilateral salpingho-ooferektomy: It decreases the occurrence of breast
cancer 50% and the ovarian cancer 80% in women who are having BRCA1/2 mutations. The
preventive effect is shown to be the same in both BRCA1 and BRCA2 mutations [52].
Because this surgery is recommended for premenopausal women, the menopausal symptoms
and osteoporosis, which may occur after surgery, must be taken in consideration. This kind of
preventive surgery is only recommended for women who have BRCA1/2 mutation [53].
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e. Chemoprevention
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Two agents received approval up to now to decrease breast cancer risk. Tamoxifen and
Raloxifen are the selective estrogen modulators, which bind the estrogen receptors
competitively showing both agonistic and antagonistic effect. The most of the cancers
occurring with BRCA1/2 mutation are known to be estrogen negative. There isn't any prove
of chemoprevention to decrease the incidence of breast cancer in women with BRCA1 and
BRCA2 mutation. In NSABP (National Adjuvant Surgical Breast and Bowel Project)-P1
study, while use of tamoxifen was shown to decrease the frequency of occurrence of breast
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cancer in women with BRCA2 mutation up to 62%, it didn't show any significant effect in
women with BRCA1 mutation [54].
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ISBN: 978-1-62808-665-2
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Chapter 9
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On The Verge of Being a Tumor
Suppressor Gene or an Axonal
Guidance Molecule: DCC
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Omer Faruk Karatas,1,2 Betul Yuceturk1 and Mustafa Ozen1,3,
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Department of Medical Genetics,
Istanbul University Cerrahpasa Medical School, Istanbul, Turkey
2
Molecular Biology and Genetics Department,
Erzurum Technical University, Erzurum, Turkey
3
Department of Pathology & Immunology Baylor College of Medicine,
Houston, TX, US
Abstract
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Towards the end of the 1980s, the efforts to reveal putative tumor suppressor genes
in colorectal cancer were mostly concentrated on loss of heterozygosity (LOH) studies,
which have been shown to be a crucial mechanism in inactivation of tumor suppressor
genes. As a consequence of these studies, the DCC (deleted in colorectal cancer),
localized on 18q21, has been found to be a tumor suppressor gene in colorectal cancer.
Further studies confirmed the reduced level of DCC in several cancers. However, rare
detection of point mutations within the coding region of DCC raised the doubts about the
candidacy of DCC as a tumor-suppressor gene and the tumor suppressor status of DCC
has been challenged since its discovery.
In the mean time, additional roles and functions are attributed to DCC such as axon
guidance in the following years. DCC is involved in axon attraction through mediating
neuronal growth cones towards Netrin-1 expressing regions. Besides, DCC behaves as a
dependence receptor, which is defined as having the functional property of inducing cell
death when its ligand is detached.
In this chapter we have reviewed and summarized the most current evidences that
implicate the importance of DCC in cellular functioning in association with its tumor

Correspondence: Mustafa Ozen M.D. Ph.D. Department of Medical Genetics, Istanbul University, Cerrahpasa
Medical School, Istanbul, Turkey, E-mail: [email protected]
196
Keywords: Tumor suppressor, cancer, DCC, Netrin-1, axonal guidance
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Introduction
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suppressive role through unraveling the recent insights into its expression, function and
activity in normal and pathological states. We believe that this chapter will help
investigators not only working in cancer research field, but also studying DCC in
neuroscience, to delineate the functions of DCC.
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The DCC (deleted in colorectal cancer) is a tumor suppressor gene that is so called due to
its identification firstly in colorectal cancer. Towards the end of 1980s, the efforts, to reveal
putative tumor suppressor genes in colorectal cancer, was mostly concentrated on loss of
heterozygosity (LOH) studies, which has been shown to be a significant mechanism in
inactivation of tumor suppressor genes [1]. These studies identified several chromosomal
regions such as 5q, 17p and 18q that are affected through LOH [2, 3]. Among them, loss of
18q region, as a promising marker detected in almost 70% of primary colorectal cancers
especially in advanced ones with hepatic metastasis, was preferentially attracted the attentions
knowing the fact that no known tumor suppressor genes has been localized in this region [4].
Further studies revealed the DCC gene localized in the 18q21 region, as a candidate tumor
suppressor gene involved in colorectal carcinogenesis [5]. Thereafter, LOH of chromosome
18q have been evaluated as causing haploinsufficiency at DCC [6] and being responsible for
reduced level of DCC, which is detected in a large percentage of colorectal tumors [7, 8].
Moreover, sequencing analysis revealed point mutations of the DCC gene in about 6% of
sporadic colorectal cancers [9]. On the contrary, the tumor suppressor function of this gene
has been called into question in the following years [4, 10] because of several reasons that
will be discussed in the chapter, however, additional roles and functions are attributed to DCC
such as axon guidance [11].
This chapter summarizes the importance of DCC in cellular functioning in association
with its tumor suppressive role through unraveling the recent insights into its expression,
function and activity in normal and pathological states.
a
Having a homolog in mammals, called neogenin [12], DCC has conserved orthologs in C.
elegans (UNC40), Drosophila (Frazzled) and Xenopus [13-15]. In human, DCC gene spans
about 1.2 Mb on chromosome 18q21 and includes 29 exons. It encodes for a 1447-amino-acid
transmembrane protein with several protein isoforms produced as a consequence of
alternative splicing [16, 17]. All characterized isoforms are reported to be type I
transmembrane glycoproteins of approximately 175 to 190 kDa with a single membranespanning domain. DCC is a member of the immunoglobulin superfamily of cell adhesion
molecules, and its extracellular domain, which is made up of approximately 1,100 amino
acids, contains four immunoglobulin-like domains and six fibronectin type III-like motifs
(Figure 1). This domain shares a high level of structural similarity with the extracellular
domains of certain types of cell adhesion molecules, such as NCAM [9]. The cytoplasmic
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Structure of DCC
On The Verge of Being a Tumor Suppressor Gene …
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DCC Expression and Function
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domain, however, that is made up of approximately 325 amino acids, shares very little
similarity with other proteins [4]. Nonetheless, P1, P2 and P3 domains within the cytoplasmic
domain have shown to be more specifically conserved throughout the DCC orthologs and are
proposed to be involved in DCC activity [18].
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DCC encodes for a receptor protein that is targeted by the axon guidance molecule
Netrin-1 (Figure 1). Its expression is detected in many of the developing and adult tissues [16,
19]. Most of the tissues, especially the basal lamina of several epithelia including the
gastrointestinal tract, skin, lung and bladder exhibit low levels of expression in both mRNA
and protein levels [19-22]. The highest level of DCC expression is observed in the nervous
system during development [23]; and in the brain in adults [16]. Development of the visual
and olfactory system has been shown to involve DCC expression [24-26].
Figure 1. A schematic representation of DCC receptor and its ligand Netrin-1.
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Particular axon populations, especially those forming large tracts of fasciculated axons,
like the lateral olfactory tract, the internal capsule, corpus callosum, anterior commissure, the
fimbria/fornix complex, the fasciculus retroflexus and the estria medularis has been reported
to express DCC [27]. Moreover, its expression is detected in the components of peripheral
nervous system; and developing spinal, segmental and sciatic nerves, sensory ganglia and
their axonal projections, and the early developmental phase of the enteric nervous system has
been reported to exhibit high levels of DCC expression [28]. DCC is involved in axon
attraction and it mediates axon guidance of neuronal growth cones towards Netrin-1
expressing regions [29, 30]. Netrin-1 is a laminin-related diffusible protein, which has both
chemo-attractive and chemo-repulsive potential for axons and neurons via interaction with its
receptors [23, 31-33]. DCC, being the main receptor for Netrin-1, is a chemo-attractant for
commissural axons in the vertebral spinal cord [34] and it accomplishes axon attraction upon
ligand binding and through interaction of its cytoplasmic tail with the tyrosine kinases Src
and focal adhesion kinase (FAK, also known as PTK2) [35-37]. It is also proposed to play
role in axon repulsion through interaction with UNC5 receptors via forming heterodimers [38,
39]. Besides, in the absence of its ligand, DCC behaves as a dependence receptor, which is
defined as having the functional property of inducing cell death when its ligand is detached.
DCC has shown to accumulate in the lipid rafts and cause apoptosis, when Netrin-1 is
disengaged from DCC [40, 41].
DCC and Cancer
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LOH of chromosome 18q, which contains DCC, as well as other genes, has been found
frequently in colorectal cancer [5]. Allelic losses of 18q have been detected mostly in primary
colorectal carcinomas and in almost all of their hepatic metastases, however, very rarely seen
in early stage tumors such as small adenomas. This suggests that allelic loss of 18q primarily
contributes to the progression of colorectal cancer rather than its initiation [4]. In more than
90% of the 18q LOH occasions in the primary colorectal cancer, DCC is involved in the
allelic loss region [42]. Further studies revealed the reduced level of DCC in several cancers,
including neuroblastoma [43], hematologic malignancies [44], gastric [45], prostate [46],
endometrial [47], ovarian [48], esophageal [49], breast [50], testicular [51], and glial cancers
[52]. DCC mutations detected in different cancer types are summarized in Table 1. Besides,
restoration of DCC expression through introduction of an intact copy of chromosome 18 into
a colorectal cancer cell line that has reduced endogenous DCC expression resulted in
inhibition of tumor growth [53]. As a consequence of these observations, DCC was accepted
as a tumor suppressor gene. Nevertheless, sequencing analysis resulted in rare detection of
point mutations within the coding region of DCC [7], raising the doubts about the candidacy
of DCC as a tumor-suppressor gene. Additionally, no tumor predisposition phenotype has
been observed in heterozygous mice with an inactivating mutation of the DCC gene [54] and
other novel candidate tumor suppressor genes such as deleted in pancreatic cancer 4 (DPC4,
also known as SMAD4), and mothers against decapentaplegic homolog 2 (MADH2, also
known as SMAD2) have been identified in the allelic loss region of chromosome 18q [18,
55]. Their nonsense, frame-shift and missense mutations have been found in a variety of
cancers [18, 42].
199
Homozygous Mutations
Somatic Mutations
Epigenetic Alterations
References
[5]
[71]
[72, 73]
[74]
[46]
[51]
[61]
[61]
[50]
[58]
[75-77]
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Loss of Heterozygosity
CancerType
Colorectal
Lung
Prostate
Endometrial
Bladder
Testicular
Pancreatic
Biliary
Breast
Gastric
Head and Neck
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MutationTypes
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Table 1. DCC mutations detected in different cancer types
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Among these genes SMAD4 has shown to be also associated with cancer progression
[56]. Taking above-mentioned reasons into account, the tumor suppressor status of DCC has
been challenged since its discovery.
However, it should be kept in mind that low levels of DCC might stem from the
mutations, out of the coding region, that would result in the reduction of DCC expression. For
instance, as a common somatic mutation in colorectal cancer, in roughly 10-15 % of all cases,
a 120 to 300 bp expansion is found in a dinucleotide repeat region in the intron residing
between exon 7 and exon 8. DCC level was reported to be low in all of the tumors where
dinucleotide expansion is observed. Although there is no clear evidence between the
expressional reduction of DCC in these tumors and nucleotide repeat expansion in the intron,
with further confirmation experiments, it can be proposed that the mutations are not
necessarily confined to the coding regions of DCC to cause loss of expression [57].
Besides, aberrations in epigenetic regulation might be involved in the DCC inactivation,
which might also explain the low frequency of somatic mutations of DCC. As an example,
DCC expression was inhibited via abnormal methylation of DCC promoter in gastric cancer
[58]. Moreover, in nude mice, increase of DCC expression to the wild type level suppressed
the tumor growth [59] and suppressed expression of DCC in normal rat cells through
antisense oligonucleotide targeting caused anchorage-independent cell growth in vitro and
tumor formation in nude mice [60]. Besides, in a study, it has been reported that in a subset of
pancreatic and bilary cancers with homozygous deletions on 18q, SMAD4 has been shown to
be intact, where DCC is inactivated [61].
As a result, although there are some controversies and still too much questions to be
answered about the role of DCC in carcinogenesis, in support of the above-mentioned
arguments, it still remains as a putative tumor suppressor at 18q21.
DCC and Neuronal Guidance
Netrin proteins carry out their axonal guidance function through interacting with its
receptors including DCC [62] and promoting the outgrowth of commissural axons toward the
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midline in a variety of organisms [11, 34]. Unc-6 in C-elegans has been previously shown to
mediate the circumferential migration of axons to the midline in nematodes [14, 22]. Further
discovery of Unc-6 homologs, net-1 and net-2 in the chick [33], NetA and B genes in
Drosophila, where they act as guidance cues and direct proper neuronal migration [63-65],
proposed the involvement of Netrin family proteins in axonal guidance. Similarly, Netrin-1 in
mice has also demonstrated to be one of the important factors mediating migration of
commissural axons [33].
Furthermore, DCC represents homology with the unc-40 gene of C. elegans, which
encodes for a receptor protein that contributes to the axonal guidance and neuronal migration
through its interaction with the ligand UNC-6 [15, 66]. Besides, loss of function mutants of
DCC and Netrin family proteins in C. elegans [11, 66], D. melanogaster [14, 64, 65], and
mice displayed similarities in terms of axon guidance phenotypes such as axonal projection
defects [54]. These similarities and further detection of DCC expression in the axonal growth
cones of retinal ganglion cells [24, 67, 68] suggested a putative role for DCC as an axonal
guidance molecule. Dcc mutations in mice caused lack of main commissures of the central
nervous system, since many commissural axons failed to reach midline [54]. This aberrancy
lead to the failure in the development of corpus callosum, hippocampal commissure and
pontine nuclei and formation of a new ectopic commissure in the interface between the
midbrain and the hindbrain in Dcc mutant mice. In these mice anterior commissure has also
been reported to be underdeveloped [54]. In addition, DCC has been implied in the control of
spatial distribution of Net protein in D. melanogaster, which has been postulated to provide
positional information for other Net receptors in neighboring axons [69].
Interestingly, neither any abnormalities concerning intestinal growth, differentiation or
morphogenesis nor elevated risk for tumorigenesis was observed as a result of DCC loss of
function, which called the tumor suppressor function of this gene into question [2, 70]. Since
there is no in vivo evidence for DCC to promote carcinogenesis when it is lost in animal
models for cancer, the tumor suppressor potential of DCC still remains to be controversial.
However, having the capacity to induce apoptosis through acting as dependence receptor, it is
still believed that DCC can play crucial roles in tumor progression [70].
a
Almost 70% of primary colorectal cancers especially in advanced ones with hepatic
metastasis have been demonstrated to lack 18q region. Further studies showed that DCC is
involved in 90% of the 18q LOH occasions in the primary colorectal cancer, which suggests
DCC gene as a novel candidate tumor suppressor contributing colorectal carcinogenesis.
However, rare detection of point mutations within the coding region of DCC raised the
suspects about the candidacy of DCC as a tumor-suppressor gene. Besides, heterozygous
mice for Dcc displayed no tumor predisposition phenotype and other novel candidate tumor
suppressor genes have been found in the allelic loss region of chromosome 18q. Although
these findings called the tumor suppressor function of this gene into question, a number of
studies supported the notion that DCC is a tumor suppressor. For example, in nude mice, the
tumor growth is inhibited as a result of increase of DCC expression to the wild type level.
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Conclusion
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Figure 2. DCC as a chemo-attractant, chemo-repellent and dependence receptor.
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Although the tumor suppressor function of this gene has been called into question
additional roles and functions are attributed to DCC such as axon guidance. Recent studies
demonstrated that DCC is involved in axonal guidance through interacting with its ligand
Netrin-1 and promoting the outgrowth of commissural axons toward the midline in a variety
of organisms. DCC, being the main receptor for Netrin-1, may serve as a chemo-attractant
and chemo-repellent for commissural axons in the vertebral spinal cord. DCC also behaves as
a dependence receptor in the absence of its ligand, accumulates in the lipid rafts and causes
apoptosis.
In conclusion, although great progress has been made in understanding of the
mechanisms underlying the roles and functions of DCC in axon guidance and tumorigenesis,
there is still much work to be done. Further investigations are needed to provide novel
insights into the mechanisms and pathways that regulate this controversial tumor suppressor
protein and its involvement in neuronal guidance.
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ISBN: 978-1-62808-665-2
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Chapter 10
Naoki Katase1*, Tsutomu Nohno1 and Mehmet Gunduz2†
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DKK3, a Mysterious Tumor Suppressor
Gene that Possesses Multiple Functions
in Tumor Progression
Department of Molecular and Developmental Biology, Kawasaki Medical School
577 Matsushima, Kurashiki, Okayama, 701-0192 Japan
2
Departments of Medical Genetics and Otolaryngology, Head & Neck Surgery,
Faculty of Medicine, Turgut Ozal University, Ankara, 06510 Turkey
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Abstract
Introduction
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The Wnt/β-catenin pathway is one of the most important pathways in morphogenesis
and cell differentiation, and is frequently deregulated in a wide range of cancers. Wnt
pathways are regulated by secreted Wnt inhibitory molecules, which are also down
regulated by promoter methylation.
The DKK family, consisting of DKK1, 2, 3 and 4, is one of these Wnt inhibitors. The
Wnt pathway inhibitory ability differs between the DKK members. DKK3 is a very
mysterious gene in DKK family members. Although the Wnt inhibitory ability of DKK3
is still unclear, its down-regulation is reported in almost all kinds of malignancies. Hence,
DKK3 is thought to be a powerful target for cancer therapy.
In this chapter, aberrant expressions of DKK3 and its function in carcinogenesis,
tumor angiogenesis and apoptosis is reviewed.
Cancer is an aberrant cell mass that consists of abnormal cells with autonomic
proliferation ability and uncontrolled cell growth. In a recent survey, it is reported that
*
†
Corresponding Author: Naoki Katase, TEL: +81-86-4632-1111, E-mail: [email protected].
Corresponding Author: Mehmet Gunduz, TEL: +90-312-203-5103, E-mail: [email protected].
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approximately 12.7 million new cancers cases were diagnosed and 7.6 million people died of
cancer worldwide. According to the estimation of GLOBOCAN 2008, death by cancer
represents approximately 13% of all deaths each year. Now cancer is the major cause of death
both in the developed and developing world [1].
Generally, the carcinogenesis step includes multiple processes in which cellular
regulation is lost, and abnormal cells potentially possess invasive and metastatic properties.
Cancer is essentially a disease caused by an aberration in key cancer-critical genes or
oncogenic signals. However, the deregulation in the cancer-critical genes are not genetically
inherited ones, but are mostly caused by environmental factors, including exposure to
chemical mutagens such as tobacco smoking, viral or bacterial infection, obesity caused by
dietary problems or physical inactivity, physical agents and radiation [2-11]. Therefore, by
changing lifestyle habits, cancer may be preventable, unless important critical genes or
pathways are involved.
Meanwhile, the true nature of the cancer, i.e. abnormalities in cancer critical genes,
includes the loss of tumor suppressor genes (TSGs) or activation of oncogenes. Functional
aberrations in TSGs are caused by loss-of-function mutation, genetic deletion or DNA
methylation that results in reduced expression of tumor suppressive protein. On the other
hand, activation of oncogenes is the result of gain-of-function mutation, elevated protein
activity or amplification of oncogenes by increased gene copy number or chromosomal
abnormality. In normal condition, TSGs (for typical example, Rb, p53, CDKN2A etc.) are
also important genes that regulate cell cycle, DNA repair and apoptosis [12-14]. Oncogenes
exist as a proto-oncogene in the normal condition, which code functional proteins triggering
signal induction including transcription factor (c-myc), receptor tyrosine kinase (epidermal
growth factor receptor: EGFR), regulatory GTPases (Ras protein) and cytoplasmic
serine/threonine kinases (Raf kinase) [15-18]. These pivotal genes are functioning in the
orchestra of interaction among genes, thus aberrant regulation in one gene will influence the
others, resulting in abnormal regulation of the important cellular signaling that controls cell
proliferation, cellular growth, differentiation, cell survival or apoptosis.
It is well known that cancer is a heterogeneous disease, and its clinical features differ
case by case. Some show a high aggressive phenotype including high invasiveness, and is
likely to become metastatic, and some exhibit resistance to the chemotherapeutic agent or
radiation therapy. Considering this cancer heterogeneity, it is implied that cancer phenotypes
are governed not just by a single pathway, but also by complicated interactions between an
oncogenic tumor and suppressive signaling circuits [19, 20].
Intriguingly, recent research established specific mechanics to seek actual TSG/oncogene
aberrations and their involved signaling in cancers. Investigating the ―gene signature‖ or
―pathway signature‖ makes it possible to know the group of genes or pathways in the cancer
cells. This signature-centric or pathway-centric way is quite strategic for developments in
targeted molecular therapy [21, 22]. Gene signature or pathway signature analyses revealed
that deregulation in major pathways (for example: Ras, Wnt/β-catenin, Src, Myc, p53, NFκB, E2F3 and p21) are commonly observed regardless of the difference in cancer type, organ
involved, or pathological subtypes [20-22].
The Wnt signaling pathway is one of the most important pathways in which abnormality
is commonly reported in a wide range of malignancies. Wnt signaling participates in in the
physical process in adult tissue that is involved in homeostasis, and plays a critical role in the
developmental process, such as stem cell homeostasis, cell fate determination, differentiation
DKK3, a Mysterious Tumor Suppressor Gene …
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and proliferation [23]. Wnt ligands, which consist of 19 highly conserved members, act as a
multifunctional growth factor that drives cellular signaling. There are three pathways in Wnt
signaling, Wnt/β-catenin pathway [24], planar cell polarity (PCP) pathway [25] and Wnt/Ca2+
pathway [26]. The Wnt/β-catenin pathway is called the canonical pathway and the latter two
are called non-canonical pathways. In the Wnt/β-catenin pathway, cytoplasmic β-catenin is
ubiquitinated and degraded without Wnt ligand binding. When Wnt ligands bind to the
receptor complex, Frizzled and Lrp5/6, cytoplasmic β-catenin is stabilized and translocated
into the nucleus, inducing TCF/LEF mediated transcription. Distinct secreted Wnt inhibitors,
including the secreted Frizzled-related protein (sFRP) family, Wnt inhibitory factor (WIF)
and Dickkopf (DKK) family, tightly regulate Wnt signaling. The sFRP and WIF directly
sequester Wnt ligands, but the DKK family will interfere with the binding between Wnt
ligands and the receptor complex [27]. DKKs bind to its cell surface receptor complex,
LRP5/6 and Kremen, mediating internalization of Lrp (Figure 1).
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Figure 1. In Wnt/β-catenin pathway, cytoplasmic beta-catenin is ubiquitinated and degraded without
Wnt ligand binding. When Wnt ligands bind to the receptor complex, Frizzled and Lrp5/6, cytoplasmic
β-catenin is stabilized and translocated into the nucleus, inducing TCF/LEF mediated transcription.
DKK family members antagonize this pathway by binding Lrp5/6 and Kremen. Binding of DKKs with
LRP5/6 and Kremen complex resulted in endocytosis of Kremen.
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Among these extracellular Wnt antagonists, DKK3 of the Dkk Family shows unique
characteristics. So far, aberrant regulation in the DKK3 is also repeatedly reported in
malignancies. DKK3 was firstly found as an REIC (Reduce expression in cancer) or RIG
(regulated in glioma), and later identified as a member of the DKK family. DKK3 may
function not only as a tumor suppressor but also as an apoptosis inducer or tumor vessel
inducer, suggesting its multifunction in cancer condition. Moreover, even possible oncogenic
function is reported.
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In this chapter, the details of DKK3, a mysterious and absorbing molecule is discussed,
including the history, gene and protein structure and their expression profile, the function.
DKK3 Gene/Protein Structure and its function
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(1) DKK3 Gene Structure
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DKK3 gene is 46,367 bp long, containing nine exons that span over 50 kbp of genomic
DNA [28]. There are two exons in exon1, which are alternatively used in two different
transcripts. Three transcript variants are known in total. The DKK3 gene is transcribed into
three different isoforms (NM_015881, 2650bp, NM_013253, 2635bp, and NM_001018057,
2587bp). Two of them result from alternative use of the first exon (i.e. exon 1a and exon1b,
although they are both non-coding). One more variant lacks exon1. All the variants share
exons 2 to 8, and code 350 amino acid (aa) functioning protein (Figure 2).
a
Figure 2. DKK3 gene location at chromosome 11p15.2, and its annotated transcripts. Exons are
indicated as boxes, White boxes present untranscripted region (UTR), and black boxes represent
translated region. The start codon (ATG) in exon2, and stop codon (TAG) in exon 8 are indicated as
arrows.
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Human DKK3 DNA/RNA expression is widely observed in normal human tissues.
Northern blotting analyses reveal that DKK3 mRNA is expressed in the brain, heart, lung,
liver, pancreas, spleen, kidney, small intestine, colon, skeletal muscle and placenta. Amongst
them, DKK3 expression is particularly high in the heart and brain.
The pseudogene for DKK3 is not reported, and neither the germinal nor somatic mutation
is reported. 5 single nucleotide polymorphisms (SNP) are known (rs3206824, rs11022095,
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rs1472189, rs7396187, and rs2291599). The DKK3 homolog is conserved over species, in
vertebrates including zebrafish, murine, rat, chicken, dog, cow, Rhesus monkey and
chimpanzee and invertebrate, such as Dictyostelium, cnidarian, tunicate and ascidian. In
vertebrates, DKK proteins consist from 4 members (i.e. DKK1, 2, 3 and 4). Although all
these proteins possess two cysteine-rich domains, the homology among DKK1, 2 and 4 is 4150%, whereas that between DKK3 and other members is 37-40% [23, 27].
(2) DKK3 Protein
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The DKK3 protein possesses several defined regions, which may confer multiple
functions to the protein. Amino acid 1-21 is a signal peptide (SP) that characterizes this
protein as a secreted protein. Four putative N-glycosylated sites and O-glycosylated at one
site region (aa 26-46) suggest that the protein may undergo posttranslational modification
before its secretion. Two cysteine-rich domains are conserved over species. N-terminal one is
DKK_N (formerly called Cys-1) and C-terminal one is called the Colipase fold (formerly
called Cys-2). The two domains contain 10 cysteine residues and are separated by 12aa linker
region (Figure 3).
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Figure 3. All the DKK3 gene transcripts encode 350aa, 38.3kDa glycoprotein. DKK3 protein contains
N-terminal signal peptide, two cysteine rich domains (i.e. DKK-type Cys-1 and DKK-type Cys-2).
DKK-type Cys-1 is located within the DKK_N (Dickkopf N-terminal cysteine rich region) region.
DKK-type Cys-2 include prokineticin region. Two coiled-coil regions are present in the N-terminal side
and the C-terminal side. Putative N-glycosylation sites are indicated.
The Colipase fold features lipid hydrolysis and may contribute to lipid binding
(interacting with cell surface LRP5/6, for instance.) The colipase fold is solved to form an
interactive surface with finger-like structure. The presence of a coiled-coil domain suggests
possible protein-protein interaction. All these structural features facilitate Wnt/DKK
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interactions. Moreover, DKK3 possesses potential proteolytic cleavage sites by furin-type
proteases, suggesting that the protein is subject to posttranslational processing.
DKK3 protein is Extracellular secreted protein. Its intracellular localization is observed in
cytoplasm, organelle and endoplasmic reticulum.
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(3) Biological function of Dkk3
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DKK family was firstly identified in Xenopus embryogenesis [29], and named after its
role as head inducer, Dickkopf (dick=thick, kopf=head in German). Dkk binds to the Wnt coreceptor, lipoprotein receptor-related protein 5/6 class (LRP5/6), and exerts antagonistic
function for Wnt induced beta-catenin stabilization [30, 31]. Dkk plays an important role in
vertebrate antero-posterior axial patterning, limb formation, eye formation and bone
formation [23]. The Wnt signaling inhibitory ability differs between the DKK members;
DKK1 and 4 can inhibit the Wnt/β-catenin pathway, and DKK2 can both inhibit and activate
β-catenin signaling [32], and the co-receptor class of Kremen protein facilitates DKK1, 2, and
4 binding to block Wnt signaling [33]. However, DKK3 neither binds to LRP5/6 nor Kremen
[34, 35]. The receptor for DKK3 is yet to be investigated and its Wnt/β-catenin inhibitory
function is still elusive [27]. However, the Wnt modulating function of DKK3 is reported in
several kinds of malignancies including glioma [36], breast cancer [37], prostate cancer
[38,39], and lung cancer [40]. And because of its obvious tumor suppressor function, DKK3
is regarded as a tumor suppressor. Recently, intracellular function of DKK3 is noted.
Cytoplasmic DKK3 may bind to beta TrCP, and facilitate beta-catenin degradation [41]. In
cancers, DKK3 and mRNA expression is down-regulated by promoter methylation, but there
is a discrepancy between mRNA expression and protein expression in tissue samples, which
may reflect tumor heterogeneity.
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DKK3 function in tumors
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Reflecting the alias of this gene, RIG (Regulated in glioma) or REIC (Reduced
expression in cancer), DKK3 mRNA and protein expression is deregulated in a wide range of
tumors, including glioma, gastric carcinoma, colorectal carcinoma, hepatocellular carcinoma,
pancreatic cancer, leukemia, renal cell carcinoma, bladder carcinoma, prostate cancer,
testicular carcinoma, ovarian carcinoma, cervical cancer, breast cancers, non-small cell lung
cancer, mesothelioma and skin cancers. The abnormal conditions of the Wnt/β-catenin
pathway in malignancies are summarized in Table 1 and 2. This downregulation in mRNA
expression is caused by the promoter hypermethylation. Thus, Dkk3 is thought to be a
potential tumor suppressor, and is focused as a therapeutic target. However, in DKK3 protein
expression level, some reports show that the DKK3 protein expression is up-regulated,
suggesting a cancer specific expression pattern and a potential alternative role in cancer
invasion.
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Table 1.
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Table 1. (Continued)
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SCC: squamous cell carcinoma, CIS: carcinoma in-situ, CLL: chronic lympahtic leukemia, AML:Acute myeloid keukemia, ALL: Acute lymphoblastic
leukaemia.
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Table 1. (Continued)
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CIN: cervical intraepithelial neoplasia, RCC: renal cell carcinoma, RCCC: renal clear cell carcinoma, NSCLC: non-small cell lung cancer, AAH: atypical
adenomatous hyperplasia, BCC: basal cell carcinoma
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Naoki Katase, Tsutomu Nohno, Mehmet Gunduz
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The following is a detailed exposition of DKK3 aberration status in each type of
malignancy.
(1) Brain tumors
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As indicated in the name, RIG (Regulated in glioma), alias name for DKK3, DKK3
protein expression is down regulated in glioma and deregulation of DKK3 is also present in
several kinds of brain tumors.
Glioma is most common brain tumor, which accounts 25-30% of the brain tumor. Glioma
is a collective term for the neuroglia-derived tumors that includes distinct tumors: astrocytic
tumors (diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, etc.), oligodendrogliomas,
oligoastrocytoma, ependymal tumors and choroid plexus tumors.
According to the WHO classification, gliomas are divided into 4 different malignancy
grades [42]. Low expression of DKK3 protein is widely reported from grade II/III tumor
(diffuse astrocytoma) to grade IV tumor (glioblastoma), and its expression loss is reported to
correlate to tumor grade [43, 44]. The reduced DKK3 protein expression was attributed to
DNA hypermethylation, and its forced expression in the glioblastoma derived cell line
resulted in induction of JUN phosphorylation-mediated apoptosis [36]
In neuroblastoma, DKK3 mRNA expression is down regulated. DKK3 functions as a
tumor suppressor, and its expression is negatively regulated via miR92, which is up regulated
by MYCN [44, 45]. Low DKK3 expression in neuroblastoma correlates with poor prognosis.
On the other hand, DKK3 expression is high in ganglioneuroma [46].
DKK3 gene expression is observed in normal organs, and its expression is highest in the
brain and heart, thus DKK3 protein expression level or DNA methylation status may indicate
neuroblastic tumor maturation.
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(2) Head and neck cancer
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More than 90% of carcinomas in the oral, head and neck region are squamous cell
carcinoma (SCC), and SCC represents 5% of all cancers in men and 2% in women [47]. SCC
is thought to arise as a cumulative genetic or epigenetic alteration in cancer associated genes,
yet the specific genes that play a pivotal role in cancer invasion, metastasis or clinical
diverseness remain unidentified.
DNA methylation and subsequent mRNA expression reduction is a commonly observed
feature in adenocarcinomas in the digestive tract. In a similar fashion, it is also reported in
oral SCC tissue sample and cell lines [48]. Aside from this, frequent LOH in Dkk3 locus
(11p15.2) is reported, suggesting that DKK3 may play a role in carcinogenesis of the
squamous epithelium [49].
However, DKK3 protein expression status and its biological role of DKK3 in SCC are
unaccountable. DKK3 may play an oncogenic role in oral, head and neck SCC. Indeed, LOH
in DKK3 locus is frequently seen, but LOH in DKK3 locus inversely correlates with lymph
nodal metastasis and overall survival [49]. Despite frequent LOH in DKK3 locus, protein is
dominantly expressed in oral SCC tissue samples and cell lines. And DKK3 protein
expression correlates with shorter disease-free survival, and metastasis-free survival [50].
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Moreover, most recent reports demonstrated DKK3 mRNA expression is conserved in oral
SCC derived cell lines, and its knockdown decreased cellular invasion and migration [51]. In
association with these series of reports, DKK3 expression increases from epithelial dysplasia,
carcinoma in situ to invasive cancer, and is thought to be independent with Wnt/β-catenin
pathway [52].
It is suggested that DKK3 may be involved in SCC carcinogenesis in the oral, head and
neck region. However, its detailed function has yet to be investigated.
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(3) Gastrointestinal cancers
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Aberration of the Wnt/β-catenin pathway is a well-known key event in carcinogenesis
steps in gastrointestinal adenocarcinomas. Deregulation of the signaling is traditionally
attributed to mutations in Axin, adenomatous polyposis coli (APC), and β-catenin, which
cause pathway hyper-activation. Moreover, Wnt/β-catenin signaling is also modulated
through various other mechanisms, down-regulation of the Wnt inhibitor, cross talk with
other altered signaling pathways are examples of them [53]. Therefore, down-regulation of
the Wnt inhibitor, including DKK3, is a hot topic in this field.
In the esophagus, most of the cancers are squamous cell carcinoma. Although there are
only a few reports on DKK3 expression in esophageal SCC, some reports demonstrated that
DKK3 DNA is hyper-methylated in esophageal cancer patient samples and cell lines and
concluded that methylation of DKK3 predicts risk of recurrence [54, 55]. However, another
report indicates that the DKK3 protein is overexpressed and that the DKK3 protein
expression correlates with invasive depth, lymph nodal metastasis and an advanced TNM
stage [56].
In gastric adenocarcinoma cell lines, DKK3 mRNA expression is down regulated and
methylation of DKK3 is a prognostic predictor for shorter survival [55, 57, 58]. However, in
tissue samples, DKK3 protein expression is also observed in the tumor endothelium adjacent
to the cancer tissue, and DKK3 protein expression in cancer cells is associated with pT-stage
and UICC stage and correlates with a favorable prognosis [59]. In conclusion, reduced DKK3
mRNA expression by CpG methylation is thought to be involved in gastric cancer
development, and might be a potential clinical target.
In colorectal adenocarcinoma cell lines, DKK3 expression is down-regulated both in the
mRNA and protein level. Forced overexpression of DKK3 mRNA results in G0/G1 cell cycle
arrest, induction of apoptosis and reduced cell proliferation. Increased cytoplasmic β-catenin
is also noted [60]. In clinical tissue samples, DKK3 protein expression is decreased compared
to corresponding normal tissues, and DKK3 expression correlates with invasion depth, TNM
stage and dedifferentiation [61]. From these reports, DKK3 might be involved in
carcinogenesis of colorectal cancer via the Wnt/β-catenin pathway.
(4) Liver cancer
The most representative liver cancer is hepatocellular carcinoma (HCC). In HCC and
cirrhosis-related HCC tissue samples, DKK3 mRNA expression is low because the promoter
hypermethylation and hypermethylation of DKK3 may correlate to shorter progression-free
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survival in cirrhosis-related HCC. Hypermethylation is more frequent in high-grade tumors
[62, 63]. However, as for protein expression, one report describes that DKK3 protein
expression is up-regulated in HCC and hepatoblastoma tissue samples [64]. DKK3 may be
involved in tumorigenesis of HCC and associated with dedifferentiated nature.
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(5) Pancreatic cancers
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Pancreatic cancer is one of the most aggressive human cancers, with an exceedingly poor
prognosis because of its late disclosure of symptoms, rapid progression, frequent metastasis
and insensitivity to chemotherapy and radiotherapy [65]. Therefore, identification of genes
and pathways that contribute to pancreatic cancer progression is eagerly anticipated. DKK3 is
a possible target for pancreatic cancer therapy.
DKK3 expression is low in pancreatic cancer cell lines (MIA PaCa-2 and AsPC-1), due
to DNA methylation. DKK3 expression in transfection of expressing plasmids decreases cell
proliferation and β-catenin expression [66]. However, another report indicates that DKK3
expression is overexpressed in PANC-1 cell line (derived from human pancreatic ductal
carcinoma), and that its down-regulation results in a reduction in cellular proliferation [67].
DKK3 may be involved in carcinogenesis in pancreatic carcinoma via Wnt/β-catenin
signaling.
(6) Hematopoietic neoplasm and leukemia
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The possible function of DKK3 as an immune modulator and involvement in
hematopoietic neoplasms are reported. As for chronic lymphatic leukemia (CLL), CLLderived cell lines demonstrated DKK3 methylation ranging from 23-37%. DKK3 methylation
is also observed in CLL patients, ranging from 18.7-61% [68]. A small population of acute
myeloid leukemia (AML) patients shows DKK3 methylation [69, 70]. DKK3 methylation is
also reported in acute lymphatic leukemia (ALL) derived cell lines and patients, and DKK3
methylation is a prognostic predictor of disease free survival [71]. As for the function of the
immune modulator function, it is reported that recombinant DKK3 may alter CD14+
monocyte into novel phenotypes, which demonstrate dendritic cell like appearances and IL-4,
GM-CSF. Administration of recombinant DKK3 results in tumor regression with CD11c+,
CD8+ T-cell infiltration [72]. From these reports, immunological aspects of DKK3 are noted.
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(7) Gynecological cancers
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Gynecologic malignancies include cancer of the uterus, ovaries, cervix, and endometrial
cancer. The estimated incidence of cases are more than 80,000 cases per year, which accounts
for more than 11% of all malignancies in women [73]. Particularly, ovarian cancer exhibits
the most aggressive nature of all gynecologic cancers. Overall cure rates for ovarian cancer
are limited to 30% [74]. Therefore, a new therapeutic strategy is urgently needed. Although
the number is a few, possible involvement of Wnt/β-catenin signaling and its inhibitors is also
discussed.
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In cervical squamous cell carcinoma (SCC) tissue samples and cell lines, DNA
methylation of DKK3 is reported [75]. Overexpression in the cervical SCC cell line results in
reduction of the cellular β-catenin level [41]. DKK3 methylation is also reported in cervical
adenocarcinoma, and DKK3 DNA methylation status may correlate with a larger tumor size
and shorter disease-free survival [76]. Taken together, DKK3 methylation and aberrant
Wnt/β-catenin signaling may be involved in cervical SCC.
As for ovarian cancer, DKK3 mRNA expression is decreased in ovarian cancer tissue
[77], and low DKK3 mRNA levels correlate with high-stage and a high incidence of lymph
nodal metastasis. Another report suggested that serum DKK3 protein level is low in ovarian
cancer patients compared to non-cancerous subjects, and low serum DKK3 levels correlate
with a high frequency of lymph nodal metastasis [78].
In endometrial cancer tissue samples, DKK3 mRNA expression is down regulated and
overexpression in endometrial cancer cell lines results in reduced cell proliferation and betacatenin mediated TCF activity [79].
As shown above, DKK3 may be involved in a wide range of gynecological malignancies.
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(8) Breast cancer
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Breast cancer remains the most commonly diagnosed malignancy among females [80].
Positive associations between environmental and individual factors and increased risk of
breast cancer were pointed out. Thus, prevention is a highly feasible approach to breast cancer
control [81]. Indeed, breast cancer sometimes becomes difficult to control. The involvement
of DKK3 in breast cancer is discussed in the context of aberration in Wnt/β-catenin signaling.
DNA hypermethylation of DKK3 is reported both in breast cancer tissue samples and cell
lines, and DKK3 DNA methylation status may be a prognostic factor for disease free survival
and overall survival [82, 83, 84]. Forced expression in breast cancer cell lines result in
induction of JNK-mediated apoptosis and reduction of anticancer drug resistance [85]. The
knockdown of DKK3 by shRNA transfection revealed the possible function of DKK3 as a
modulator of the Wnt/beta-catenin signaling modulator in breast cancer [37].
In summary, DKK3 may be involved in carcinogenesis of breast cancer, and may
modulate Wnt/β-catenin signaling.
(9) Urologic cancers
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The involvement of DKK3 and its possibility for use in cancer treatment is most actively
reported in the field of urology. As for bladder cancer, DKK3 is thought to be a candidate
therapeutic target. The details will be discussed below, in the section, ―DKK3 as a candidate
cancer treatment target‖.
In renal cell carcinoma (RCC), DKK3 mRNA expression is down regulated because of
promoter CpG island methylation. Stable transfection of DKK3 in RCC cell lines does not
affect the Wnt/β-catenin pathway, but induces apoptosis via the JNK pathway [86].
Methylation of DKK3 is also observed in renal clear cell carcinoma (RCCC) [87].
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Prostate cancer and testicular cancer
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So far, mutation in the DKK3 gene is not reported in any kind of malignancy, but SNP in
the DKK3 gene is reported in RCC, and rs1472189 SNP correlates with distant metastasis
[88].
DKK3 methylation is observed in bladder cancer, and forced expression in cancer cell
lines induces JNK mediated apoptosis [89, 90, 91].
In conclusion, DKK3 methylation may be involved in carcinogenesis in RCC and bladder
carcinoma.
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Similar to urologic cancers, DKK3 is focused as a therapeutic target for prostate and
testicular cancer.
In prostate cancer, mRNA and protein expression are down regulated and DKK3 protein
expression in prostate cancer decreases gradually in prostate carcinogenesis. DKK3 protein
expression loss may correlate to tumor grade. Overexpression of DKK3 in the prostate cancer
model may ameliorate tumor progression [39, 92]. And high DKK3 protein levels are
reported in the seminal plasma of prostate cancer patients [93]. Overexpression in prostate
cancer cell lines induces JNK-mediated apoptosis [38] and decreases lymph nodal metastasis
in the prostate cancer mice model [94, 95].
In testicular cancer, DKK3 expression is down regulated and forced expression in cancer
cell lines induce JNK-mediated apoptosis [96].
As shown above, DKK3 methylation may be involved in carcinogenesis in prostate and
testicular cancers.
Lung cancers
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Reduced DKK3 mRNA level is first reported in human non-small cell lung cancer
(NSCLC) tissue samples [97]. Decreased expression ofDKK3 mRNA is due to DNA
methylation, and DKK3 may regulate cancer cell growth via the Wnt/β-catenin pathway [40].
DKK3 methylation is also observed in precarcinomatous lesions, atypical adenomatous
hyperplasia [98].
In addition to lung NSCLC cases, DKK3 expression is also down regulated in the
mesothelioma cell line, and overexpression of DKK3 induces JNK-mediated apoptosis [99].
These reports suggest that DKK3 may be involved in NSCLC via Wnt/β-catenin signaling
regulation.
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Skin and bone cancer
DKK3 protein expression is down regulated in skin cancers [100]. In malignant
melanoma tissue sample and cell lines, DKK3 mRNA expression is strongly reduced, and the
stable expression of DKK3 in malignant melanoma cells reduces cellular migration [101]. As
for bone tumors, the osteosarcoma-derived cell line Saos2, shows decreased expression of
DKK3, which may modulate Wnt/β-catenin signaling [102].
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DKK3 and tumor vessels
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As mentioned above, aberrant expression of DKK3 is reported in a wide range of cancers
in almost all organs. However, the function of DKK3 is not fully clarified, and is sometimes
conflicting report by report. Indeed DKK3 is a potential therapeutic target, however, further
investigation is necessary.
In the following articles, the multifunctional aspect of DKK3, particularly in tumor vessel
formation, and possible clinical use are discussed.
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Recent reports revealed the existence of a new function of DKK3 in tumor angiogenesis.
It is well known that tumor nests require abundant blood vessels in order to supply nutrition
and oxygen. Thus, tumor cells stimulated tumor stroma, facilitating production of
neoangiogenesis [103]. Nowadays, the differences between tumor vessels and normal vessels
are discussed, and it is revealed that tumor endothelial cells possess a distinct nature at the
molecular level, compared with normal endothelial cells [104]. This concept indicates tumor
vessels to be a new therapeutic target.
DKK3 protein expression in tumor vessels is noted in glioma, melanoma [105], oral
squamous cell carcinoma and its precursor lesion [50,52], gastric cancer [59], prostate cancer
[92], pancreatic cancer [106] and colorectal cancers [107]. In colorectal cancers,
immunohistochemical analysis revealed that vessels in/adjacent to the cancer tissue shows
DKK3 protein expression, whereas normal vessels do not. This implies pro-angiogenic
function of the DKK3 protein [105,107]. In pancreatic cancer, DKK3 protein expression in
tissue samples revealed that DKK3 protein expression is observed both in cancer cells and
tumor endothelium. Moreover, DKK3 expressing endothelium is sensitive to anticancer
drugs. Low DKK3 protein expression in tumor endothelium correlates with a worse clinical
outcome [106]. Supporting this research with clinical samples, stable overexpression of
DKK3 increased microvessel density in melanoma mice model [105].
The detail about DKK3 endothelial expression is still unclear. However, one possible
explanation for this is regulation among receptor tyrosine kinase Axl, angiopoietin-2 (ANG2) and DKK3 in endothelial cells [27]. Generally, Axl expresison is up-regulated in cancer
cells and tumor endothelium. Knockdown of Axl expression in human umbilical vein
endothelial cells (HUVEC) results in up-regulation of Axl-2 and down-regulation of DKK3.
And DKK3 knockdown results in reduced tube formation [108].
Although further investigations are required, DKK3 may be a potent anti-cancer
therapeutic target.
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DKK3 as a candidate cancer treatment target
As the reduced expression ofDKK3 mRNA or protein is observed in all kinds of
malignancies, it is no wonder that we hypothesize that overexpression of DKK3 might
ameliorate hopeless cancers. Indeed, pre-clinical evidence includes the adenovirus-mediated
DKK3 transfection model, which is known as Ad-REIC/DKK3 [28, 85, 90, 91, 94, 96, 109-
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114]. Investigations using Ad-REIC are especially focused on the urology field (prostate
cancer, testicular cancer)
As for prostate carcinoma, Ad-REIC treated PC3 cells showed significant tumor
reduction and growth inhibition in the BALB/c mice tumor injection model [109, 110].
Similarly, when RM-9 cells after Ad-REIC treatment was injected into prostate tissue of
C57/BL6 mice, tumor growth and lymph nodal metastasis was significantly decreased [94]
In testicular cancer, NCCIT cells treated with Ad-REIC was injected into BALB/c mice,
then 40% of the tumor disappeared and the rest of the tumors showed reduced tumor growth
[96]
In the mice gastric scirrhous carcinoma model, intraperitoneal administration of
adenovirus vector carrying DKK3 significantly decreases tumor dissemination and increases
recruitment of killer T cells [113]. Ad-REIC treatment was also reported in breast cancer.
Intratumoral injection of Ad-REIC in mice resulted in delayed tumor growth and upregulation of apoptosis [85].
The mechanism that Ad-REIC decreases tumor growth and ameliorates disease in animal
models is mainly due to JNK-mediated apoptosis induction, which is independent of Wnt/βcatenin canonical pathway. However, it bears on a wide variety of aspects, such as apoptosis
induction, and inhibition in lymph nodal metastasis. All the effects contribute to better
survival in animal models.
Ad-REIC might be a powerful tool for cancer treatment. But as indicated previously,
DKK3 possesses multiple functions including a possible oncogenic role. Further investigation
will substantialize clinical use of Ad-REIC.
Conclusion
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Wnt/β-catenin signaling and its regulators are one of the important pathways often
deregulated in various kinds of cancers. The DKK family is one of the Wnt regulators that
antagonizes Wnt ligands, and is commonly down regulated in cancers by CpG methylation.
DKK3, a member of DKK family, of which Wnt inhibitory activity is still questionable, but
regarded as a putative Wnt signaling inhibitor and tumor suppressor.
DKK3 behaves as a multifunctional gene, including tumor suppression via inhibition of
Wnt/β-catenin signaling, participation in tumor vessel formation and apoptosis induction.
Currently, DKK3 research is an engrossing theme that has potential for therapeutic
applications.
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ISBN: 978-1-62808-665-2
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Chapter 11
The Functions and Roles of the Unique
Tumor Suppressor Gene PTEN
is
Omer Faruk Karatas,1,2 Esra Guzel1 and Mustafa Ozen1,3,
1
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2
Molecular Biology and Genetics Department,
Erzurum Technical University, Erzurum, Turkey
3
Houston, TX, US
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Phosphatase and tensin homolog (PTEN), is a well-documented tumor suppressor
gene, which was discovered in 1997 as a result of identification of a frequently lost
region on chromosome 10q23. PTEN primarily acts at the plasma membrane and
negatively regulates PI3K/AKT signaling pathway through converting PIP3 to PIP2.
PTEN signaling pathway conveys the signals from cell surface receptors to the effector
proteins in association with other tumor suppressor and oncogenic signaling pathways. It
is one of the most frequently mutated or deleted tumor suppressors in human sporadic
cancers worldwide. Since it is known as not belonging to a protein family, it is a unique
protein, which has been shown to participate in carcinogenesis and whose aberrant
expression has been detected in several primary tissues or established tumor cell lines. In
this chapter we have reviewed and summarized the most current evidences that implicate
the importance of PTEN in cellular functioning in association with its tumor suppressive
role through unraveling the recent insights into its expression, function and activity in
normal and pathological states. We believe that this chapter will help investigators not
only working in the cancer research field, but also studying PTEN in other diseases, to
understand the functions of PTEN.
Keywords: Tumor suppressor, cancer, PTEN, PI3K/AKT

E-mail: [email protected].
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Omer Faruk Karatas, EsraGuzel and Mustafa Ozen
Introduction
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Phosphatase and tensin homolog (PTEN), also known as MMAC1 (mutated in multiple
advanced cancers-1) or TEP1 (tensin-like Phosphatase-1), was originally discovered in 1997,
as a result of identification of a frequently lost region on chromosome 10q23 [1, 2] and
named as so due to encoding a phosphatase and exhibiting high homology to tensin and
auxilin. Being the second most frequently mutated or deleted tumor suppressor [3] in human
sporadic cancers including brain, bladder, breast, melanoma, lung, prostate, renal and
endometrial cancers [4], the relevance of PTEN in development and cancer was further
revealed with the utilization of germline knockout Pten mice mutants. Homozygous loss of
Pten caused embryonic lethality, while heterozygous mice for Pten, developed dysplasia of
several tissues, cancers of multiple origins, and a lethal haploinsufficient lymphoproliferative
disease [5-7]. Altered PTEN expression through promoter methylation has also been reported
in several tumors [8, 9]. In addition to cancer, germline loss and mutations of PTEN are
associated with several hereditary autosomal dominant disorders such as Cowden disease,
Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Ducros disease (LDD) and Proteus
syndrome, which share common symptoms such as neurological disorders, multiple
hamartomas and cancer susceptibility [10, 11].
This chapter summarizes the importance of PTEN in cellular functioning in association
with its tumor suppressive role through unraveling the recent insights into its expression,
function and activity in normal and pathological states.
Structure of PTEN
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PTEN gene spans 105 kb on chromosome 10q23 and is made up of 9 exons (Figure 1A).
It is a relatively small, multi-domain polypeptide (55 kDa) that is made up of 403 amino
acids. As it is demonstrated in Figure 1C, PTEN contains five domains consisting of 1) an
amino terminal phosphatase domain homologous to chicken tensin, 2) a PIP2 binding
domain, 3) two consecutive PEST (rich in proline, glutamate, serine, and threonine residues)
homology domains, which regulates protein stability via the ubiquitin-proteasome pathway,
and 4) a PDZ-binding domain, and 5) the C2 regulatory domain, which is implicated in
recruitment of PTEN to plasma membrane [12] and may be related to protein stability [13].
As to the phosphatase domain of PTEN, it has shown to contain a central five-stranded sheet and 6 α-helices wrapping the core [14]. The targets of dephosphorylation activity of
PTEN are demonstrated to be Tyr-, Ser- and Thr-phosphorylated residues of highly acidic
substrates in vitro [15]. Although the tumor suppressor potential is mostly associated with the
N-terminal lipid phosphatase activity, at least 40% of the mutations are localized in the Cterminal domain [16] (Figure 1B).
Therefore, it has been suggested that PTEN may have additional functions through its C
terminal domain, which is involved in tumor suppressor capability of PTEN [17]. Moreover,
crystal structure analysis of PTEN carried out by Lee et al. indicated that C2 regulatory
domain has a -sandwich structure, which is interpreted as a basis for interaction of PTEN
with DNA and proteins [18].
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The Functions and Roles of the Unique Tumor Suppressor Gene PTEN
Figure 1. Phosphatase and tensin homolog (PTEN), is a unique tumor suppressor that is localized on
chromosome 10q23. A simplistic schematic representation of PTEN gene (A), mutation hot-spots
regions throughout its exons (B) and the functional domains of PTEN (C).
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Furthermore, PTEN contains a catalytic signature motif, which is a common active site
signature of protein tyrosine phosphatases [19]. This catalytic signature motif constitutes the
phosphate-binding domain and includes the hot-spot bases for cancer associated PTEN
mutations [20, 21]. Residing in the amino terminal region, the catalytic motif shows
homology to the actin-binding protein tensin 1 (TNS1) and auxilin, the cofactor of ATPase
heat shock cognate 70 (HSC70), which are not actually related to catalytic activity of PTEN
[1, 2].
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Regulation of PTEN Expression
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Expressional regulation of PTEN involves a series of mechanisms involving
transcriptional activation and repression, epigenetic silencing, microRNA regulation and posttranslational regulation. Further interactions with PTEN-interacting proteins also play roles in
the activation and repression of PTEN function.
PTEN expression is strictly controlled, and although it is assumed to be constitutively
expressed in normal tissues, it is, later showed to be both transcriptionally activated and
repressed by several regulators [22]. Transcription factors such as early growth regulated
transcription factor 1 (EGR1) [23], peroxisome proliferator-activated receptor  (PPAR)
[24], p53 [25] and human sprouty homolog 2 (SPRY2) [26] activates transcription of PTEN
through binding its promoter. On the other hand, regulators such as Mitogen-activated protein
kinase kinase-4 (MKK4) [27], Transforming growth factor (TGF) [28], JUN [29], JNK–
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nuclear factor-κB (NF-κB, [27] has shown to suppress PTEN transcription through direct or
indirect mechanisms.
In addition, epigenetic mechanisms are also involved in transcriptional repression of
PTEN by, for instance, a zinc-finger transcription factor called sal-like protein 4 (SALL4),
which recruits an epigenetic repressor complex containing a chromatin-remodelling ATPase
and a histone deacetylase to the PTEN promoter [30]. In several types of cancer, dramatic
reduction of PTEN gene expression is detected because of promoter silencing by DNA
methylation [31-33].
In the post-transcriptional level, microRNAs (miRNAs), which are small, approximately
18-24 nucleotide-long, non-coding and endogenously synthesized ribonucleic acids (RNAs),
are involved in the regulation of gene expression [34]. They bind to 3‘-untranslated regions
(3‘UTR) of target mRNAs and cause either mRNA degradation or translational blockade
[35]. It is thought that miRNAs modulate the expression of at least 60% of all protein coding
human genes through working in combination with each other and fine-tuning of the target
mRNA levels [36, 37]. They are shown to be involved in several crucial biological processes
such as development, proliferation, and apoptosis through their spatial and temporal
expression [38, 39].
Expressional deregulation of miRNAs is associated with pathogenesis of several diseases,
including human cancers and they can act as potent tumor oncogenes or tumor suppressor
genes [40, 41]. MiRNAs have recently been demonstrated to be targeting PTEN and resulting
in tumorigenesis and formation of PTEN associated diseases through inhibiting its expression.
For example, deregulation of miR-17~92 cluster is associated with lymphoproliferative
disease and autoimmunity [42], whereas altered expression of miR-19 is related to leukemia
and Cowden disease [43]. In addition, deregulation of miR-21 has been demonstrated to alter
the expression of PTEN in multiple cancers [44, 45].
PTEN Expression and Function
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Since PTEN is known as not belonging to a protein family, it is a unique protein, which
means in times of PTEN loss, there are no other family members to substitute for it.
Therefore, the loss of PTEN is catastrophic for cells [46]. Its expression has been shown to be
detected in embryonic cells starting from the time when blastocysts aged 3.5 days [47] and
ubiquitous expression of PTEN is maintained until late stages of the embryonic development
(E15-19) [6].
In addition to its expression in extra-embryonic tissues, which has been demonstrated by
both in situ hybridization and immunohistochemistry analyses, PTEN is predominantly
expressed in embryonic tissues/organs including central nervous system components, bone,
liver, heart, skin and gastrointestinal tract. Similar expression pattern is maintained
throughout the lifetime and its roles in the development and functioning of these organs have
also been reported [6, 48, 49]. PTEN expression is associated with several biological
functions, differing from embryogenesis to sexual organ development in adults.
Controlling the organ size through modulating the checkpoints for cell proliferation and
soma growth is another key role of PTEN through development [50]. Furthermore, induction
of apoptosis and cell cycle arrest along with maintaining chromosomal integrity are among
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the well-defined functions of PTEN in the cellular level [18, 51, 52]. Cell migration is another
cellular mechanism that is postulated to be negatively regulated by PTEN through directly
dephosphorylation of p125FAK and alteration of MAP kinase activity [52, 53]. Besides, its
expression is also important for the regulation of stem cell self-renewal and proliferation
[54, 55].
PTEN, a multifunctional phosphatase, has both potential to dephosphorylate lipids and
proteins. It carries out its lipid dephosphorylation activity, which is associated with tumor
suppression capability of PTEN [1, 56] mostly at the plasma membrane. Recruitment of
PTEN to the membrane, via interaction with other proteins such as MAGI1b, MAGI2,
MAGI3, MAST3 and SAST, through its PDZ-binding domain (Figure 2), is required because
of the localization of its target, PIP3, at the plasma membrane [22]. PTEN expression,
however, is also observed in both cytosol and nucleus within the cell [57, 58]. Detection of
PIP3 and PIP2 in the nucleus ascribed putative roles for PTEN in the nucleus, although it is
not as well-described as the role of PTEN at the plasma membrane [59]. Further studies
revealed that loss of nuclear PTEN resulted in formation of neoplasias and tumorigenesis,
attributing a tumor suppressor role for nuclear PTEN [60].
In addition, nuclear PTEN has shown to reduce the levels of cyclin D1 and phosphoMAPK, playing important roles in the cell cycle arrest. Meanwhile, cytoplasmic PTEN
intervenes apoptosis through downregulating phospho-PKB levels and upregulating p27kip1
levels [61, 62].
Figure 2. PIP3 represents the major substrate of the lipid phosphatase activity of PTEN. It is responsible
for the activation of the serine-threonine kinase, AKT. Phosphorylation helps the stabilization of PTEN
and the ubiquitin ligase NEDD4-1 has shown to be responsible for both mono- and poly-ubiquitylation
of PTEN. Recruitment of PTEN to the membraneis performed via interaction with other proteins such
as MAGI1b, MAGI2, MAGI3, MAST3 and SAST.
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As a lipid phosphatase, PTEN demonstrates phosphatase activity against the 3‘ position
of phosphatidylinositol 3,4,5-triphosphate (PIP3), the second messenger that is produced by a
potent proto-oncogenic lipid kinase, phosphoinositide 3-Kinase (PI3K), and therefore,
negatively regulates the PI3K/AKT signaling pathway and suppresses tumorigenesis [63].
PIP3, representing the major substrate of the lipid phosphatase activity of PTEN [9, 21], is
responsible for the activation of the serine-threonine kinase, Akt, and known to be playing
crucial roles in antiapoptosis, proliferation and oncogenesis [9] (Figure 2). Therefore, loss of
PTEN lipid phosphatase function causes elevated levels of PIP3 and subsequent derepression
of PI3K/AKT pathway, which stimulates proliferation and tumorigenesis [20, 47].
PTEN is also known to demonstrate phosphatase activity against protein substrates. The
phosphatase activity results in subsequent inactivation of related proteins. Focal adhesion
kinase FAK (PTK2), a non-receptor protein tyrosine kinase [64], p130Cas, a FAK
downstream effector [52], Shc, PDGFR and PTEN itself [52, 65, 66] are among the identified
direct protein targets of PTEN. Additionally, receptor tyrosine kinases are also found to be
targeted by PTEN. For example, the receptor of platelet-derived growth factor physically
interacts with PTEN and it is dephosphorylated as a result of protein phosphatase activity of
PTEN [17]. PTEN also targets and inactivates upstream molecules of MAP kinase such as
RAS and IRS-1 [67, 68], and downstream effectors of the same pathway including ETS-2, a
transcription factor whose DNA-binding ability is controlled by phosphorylation [69] and
another transcription factor Sp1, through its protein phosphatase activity [70].
On the other hand, it has been reported that dephosphorylation activity of PTEN is
restricted to the intracellular signaling molecules and extracellular signal-regulated kinases
cannot serve as direct targets of PTEN for its phosphatase activity [71].
PTEN has also ascribed functions independent of its phosphatase activity [22]. JNK has
demonstrated to be a functional target of PTEN, in which no direct involvement of
phosphatase activity detected [71].
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Post-translational Regulation of PTEN
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PTEN post-translational regulations involve phosphorylation, acetylation, oxidation and
ubiquitination. Phosphatase activity of PTEN is regulated through phosphorylation of its
C-terminal tail at Ser380, Thr382, Thr383 or Ser385 positions, which results in inhibition of
its lipid phosphatase activity. In the meanwhile phosphorylation helps the stabilization of
PTEN through promoting a closed PTEN conformation, which blocks its membrane
localization [72, 73] (Figure 2). Moreover, PTEN is susceptible to acetylation at Lys125–
Lys128 residues by p300/CREB-binding protein (CBP)-associated factor (PCAF) and at
Lys402 residue by CBP, which has shown to be linked to inhibition of its catalytic activity.
Acetylation also improves protein-protein interactions with PDZ domain-containing proteins
[74]. As to the oxidation of PTEN, a catalytic Cys nucleophile is oxidized, which is important
in the modulation of PTEN‘s catalytic activity. Furthermore, having two canonical PEST
domains, which is a signature of involvement of the ubiquitin pathway for ubiquitin-mediated
proteasomal degradation [75, 76], regulation of PTEN is also controlled by ubiquitylation.
The ubiquitin ligase NEDD4-1 (neural precursor cell expressed, developmentally downregulated gene 4-1) has shown to be responsible for both mono- and poly-ubiquitylation of
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PTEN [77], whilst only poly-Ubiquitylation is recognized as a target for proteasomal
degradation through the C-terminal PEST domain of PTEN (Figure 2). Mono-ubiquitylation
at the Lys13 and Lys289 residues, which are reported to be mutated in somatic cancers and
Cowden disease, respectively, is necessary for the PTEN nuclear–cytoplasmic shuttling [78].
Interestingly, in certain types of cancer samples, wild-type PTEN gene has been observed
with low and even in undetectable levels, which is most probably associated with abnormal
post-translational regulation of PTEN [79, 80], pointing the importance of post-translational
regulation.
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PTEN-Controlled Signaling Pathways
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PTEN signaling pathway conveys the signals from cell surface receptors to the
transcription factors in association with other tumor suppressor and oncogenic signaling
pathways. PTEN primarily acts at the plasma membrane and negatively regulates PI3K/AKT
signaling pathway through converting PIP3 to PIP2. As ligand-bound growth factor receptor
tyrosine kinases or Ras activates PI3K, which is a heterodimeric kinase, PIP3 is produced via
phosphorylation of PIP2 by PI3K [81]. PIP3 binds and recruits AKT, the major downstream
target of PI3Ks [82], to the plasma membrane. Binding of PIP3 to AKT results in colocalization of AKT and phosphoinositide-dependent kinase 1 (PDK-1) through interaction of
their pleckstrin homology (PH) domains. Thereafter, PDK-1 phosphorylates AKT at Thr 308
residue in its kinase domain [83]. Additional phosphorylation of AKT at Ser 473 residue
within the carboxyl-terminal regulatory domain by phosphoinositide-dependent kinase 2
(PDK2) is needed for the complete activation of AKT signaling [84]. Upon activation of
AKT, it is transported to the nucleus, where it exhibits its functions. Activated AKT
modulates the downstream targets to be involved in a variety of cellular functions such as cell
survival, cell cycling, metabolism and angiogenesis [85].
Moreover, PTEN also regulates mTOR/S6K signaling pathway, and by this way it
controls translation mechanisms and thus influence the cell size [86, 87]. Mammalian target
of rapamycin (mTOR, also known as mechanistic target of rapamycin or FK506 binding
protein 12-rapamycin associated protein 1) is serine/threonine protein kinase that is involved
in the regulation of several cellular processes such as, cell growth, proliferation, cell motility,
cell survival, protein synthesis, and transcription [88, 89]. Germline mutations of PTEN, as a
negative regulator of mTOR/S6K signaling have been shown to dysregulate the mTOR
pathway and cause development of familial cancer syndromes [88, 90].
In addition to PI3K/AKT and mTOR signaling pathways, PTEN has also been reported to
interact with TGF-/SMAD pathway andWnt/-catenin pathway, and contribute to
expressional regulation of homeobox genes, such as NKX3.1 [91] and hepatic nuclear factors
[92], pointing the role of PTEN in multiple levels through the development.
PTEN and Cancer
Analysis of either primary tissues or established tumor cell lines showed that PTEN
mutations and deletions have been observed in a variety of human [1, 93-97], especially with
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a high frequency in glioblastoma, endometrial and prostate cancers [17]. These mutations
include homozygous deletion, frame-shift, inframe deletion, truncation and point mutations
[1, 98]. Missense mutations, as well as nonsense and frame-shift mutations of PTEN, has
been documented to reside frequently in the phosphatase domain [97, 99]. Most of the
missense mutations have shown to result in significant reduction of phosphatase activity
through induction of early termination of translation [21, 100-102]. Common missense point
mutations of PTEN include H123Y (endometrial cancer), L57W (glioblastoma), G165R
(glioblastoma), T167P (breast cancer) and C124S [102].
On the other hand, even though identified mutations seems to disperse throughout the
PTEN gene and the tumor suppressor potential is mostly associated with the N-terminal lipid
phosphatase activity, almost 40% of all germline mutations occur in the 5th exon, which only
encodes for 20% of the whole protein. This represents the biological significance of the
phospholipid domain of PTEN that is encoded from the 5th exon [103]. Studies has also
revealed that truncated PTEN products lacking the C2 domain and the PDZ-biding motif,
because of nonsense and frame-shift mutations occurred within the C2 domain–C-tail
junction, lost their stability and functionality [102].
Moreover, a loss of PTEN has been frequently detected especially in late-stage brain,
prostate and endometrium tumors. This implies that PTEN‘s involvement in carcinogenesis
occurs mostly during tumor progression instead of initiation [1, 2].
PTEN and Other Diseases
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In addition to cancer, PTEN is also implicated in a variety of familial cancer
predisposition syndromes such as Cowden disease (CD), Bannayan-Riley-Ruvalcaba
syndrome (BRRS), Lhermitte-Ducros disease (LDD) and Proteus syndrome (PS), which have
been collectively called as PTEN hamartoma tumor syndromes (PHTSs) [17, 104]. PHTSs
have common symptoms like neurological disorders, multiple hamartomas and predisposition
to cancer [17, 105]. 85% of Cowden disease patients have shown to be carriers for germline
mutations of PTEN [106]. Besides, 65% of Bannayan-Riley-Ruvalcaba syndrome cases [107]
and 20% of Proteus syndrome and Proteus-like syndrome cases have also shown to be
positive for germline mutations of PTEN [108]. CD, being discovered in 1963 by Lloyd and
Dennis, is a rare autosomal dominant hereditary genodermatosis disorder [109], which is
characterized by mucocutaneous skin findings in more than 95% of the cases and multiple
hamartomatous polyps of ectodermal, mesodermal, and endodermal origins [110]. There is
also increased susceptibility to cancer like breast, thyroid, endometrial, colon and renal cell
carcinomas [111, 112]. For example, predisposition to breast cancer in women with CD, has
shown to be elevated, in which the lifetime risks has been estimated to increase from 11%,
which was documented as the risk for normal population, to 25-50% [113, 114]. Besides,
Lhermitte–Duclos disease, a dysplastic gangliocytoma of the cerebellum, is another
hamartoma syndrome, which is often associated with CD and accepted as pathognomonic for
it. LDD is diagnosed with diffuse hypertrophy of the stratum granulosum of the cerebellum
(106). Furthermore, Bannayan-Riley-Ruvalcaba Syndrome is also a rare autosomal dominant
PTEN hamartoma tumor syndrome, which is characterized by macrocephaly, multiple
lipomas, intestinal hamartomatous polyps, haemangiomas and pigmented macules of the
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glans penis [115-117]. Additionally, having clinical features like hamartoses, lipomas, and
overgrowth, a Proteus-like syndrome patient has been reported to be positive for a germline
PTEN R335X mutation and shown to be a carrier for a second ‗‗hit‘‘ mosaic R130X mutation
in his affected tissues [118]. The most frequently detected PTEN mutations in PHTSs and
their germline mutation frequency in total are summarized in Table 1. Deregulation of PTEN
function is also implicated in diseases other than PHTS and cancer, such as diabetes and
autism [22]. Experiments carried out in conditional PTEN-knockout mice demonstrated that,
specific deletion of PTEN from adipocyte or pancreatic cells, didn‘t result in development of
carcinomas, instead, in addition to being healthy and fertile, and they were protected from
streptozotocin-induced diabetes [119, 120], although the mice lacking PTEN specifically in
pancreatic -cells were shown to be significantly smaller than control animals [121].
Table 1. The most frequently detected PTEN mutations in PHTSs and their germline
mutation frequency in total for each disease
G129E
R233TER
H123R
C124R
R130TER
IVS6, T-G, +2
1-BP DEL, 696A
M35R
L70P
R130Q
R335TER
1-BP INS, A
C124S
1-BP DEL, 802G
5-BP DEL, NT347
-764A-G
-861G-T
R233TER
S170R
R130TER
1-BP DEL, 1390C
Y178TER
Q214TER
E256TER
R335TER
DEL
E157TER
L112P
R335TER
1-BP DEL, 507C
[10]
[10]
[126]
[126]
[126]
[127]
[127]
[127]
[128]
[129]
[130]
[131]
[128]
[132]
[132]
[133]
[133]
[117, 128]
[128]
[126]
[116]
[116]
[116]
[116]
[130]
[133]
[104]
[135]
[133]
[108]
Frequency of Germline
Mutation of PTEN
Ref.
85%
[125]
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Cowden
Disease
Mutations
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PHTSs
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BannayanRileyRuvalcaba
Syndrome
LhermitteDucrosDisease
Proteus-like
Syndrome
65%
[107]
50%
[134]
20%
[136]
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Conclusion
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Besides, conditional PTEN-knockout mice, in which PTEN is deleted in the cerebral
cortex neurons and hippocampus demonstrated several aberrant social behaviors, augmented
response to sensory stimuli and learning disabilities [122], which resembles the symptoms of
autism spectrum disorders. In another study, which includes 18 patients with autism spectrum
disorders, germline missense PTEN mutations in 3 patients have been found [123].
All the above-mentioned diseases, which are associated with several PTEN mutations,
indicate the wide-range of clinical reflections of PTEN alterations, and attributes novel
functions independent of its tumor suppressor role.
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More than ten years of investigation has showed the importance of PTEN in cellular
functioning in association with its tumor suppressive role. Although it has shown to be
primarily responsible for conveying the signals from cell surface receptors to the transcription
factors through acting at the plasma membrane and negatively regulating the PI3K/AKT
signaling pathway, functions independent of its phosphatase activity, which is mostly carried
out within the nucleus, has also been ascribed.
In addition, since its discovery, it has demonstrated to be involved in regulation of several
cellular processes such as cell cycle transition, chromosomal integrity, cell migration, stem
cell self-renewal and proliferation.
Most importantly, PTEN, being the first designated lipid phosphatase, is one of the most
frequently affected genes in cancers [124]. In addition to carcinogenesis, its aberrant
expression and mutations are implicated in many familial cancer predisposition syndromes,
such as Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Ducros disease
and Proteus syndrome, which have been collectively called as PTEN hamartoma tumor
syndromes.
Knowing the fact that its expression is controlled through a series of mechanisms
involving transcriptional activation and repression, epigenetic silencing, microRNA
regulation and post-translational regulation, novel therapeutic and diagnostic approaches can
be developed via targeting these mechanisms.
For example, it is interesting to note that miRNAs have been recently demonstrated to be
targeting PTEN and resulting in tumorigenesis and formation of PTEN associated diseases
through inhibiting its expression. Thus, profiling the changes in miRNA expression in cancer
tissues originated from PTEN aberrancy is very important for enlightening possible
mechanisms of pathogenesis. This might also help to develop miRNA biomarkers for the
early detection, diagnosis of these cancers, since miRNAs are promising candidates for novel
therapeutic applications against cancer.
As a conclusion, although great progress has been made in the understanding of the
mechanisms underlying the PTEN regulation and along with its roles and functions in cell
growth and tumorigenesis, there is still much work to be done.
Further investigations are needed to provide novel insights into the mechanisms and
pathways that regulate this unique tumor suppressor protein and develop novel therapeutic
approaches and tools for cancer prevention and therapy.
243
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syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3kinase/Akt pathway. Am. J. Hum. Genet. 2003 Aug;73(2):404-11.
[134] Eng C. PTEN: one gene, many syndromes. Hum. Mutat. 2003 Sep;22(3):183-98.
[135] Sutphen R, Diamond TM, Minton SE, Peacocke M, Tsou HC, Root AW. Severe
Lhermitte-Duclos disease with unique germline mutation of PTEN. Am. J. Med. Genet.
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[136] Breathnach CS. The stability of the fetal oxygen environment. Ir. J. Med. Sci. 1991
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Chapter 12
Functions of the Tumor Suppressor
Gene APC
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Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa
and Yoshio Naomoto
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Department of General Surgery, Kawasaki Medical School
Keywords: APC: adenomatous polyposis coli; FAP: familial adenomatous polyposis;
colorectal cancer
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Introduction
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Adenomatous polyposis coli (APC) is a multifunctional protein commonly mutated in
colon cancer. Mutations in adenomatous polyposis coli are associated with most colon
cancers however the precise mechanisms how these mutations induce neoplasms are not
cleared [1].
Over twenty years ago, mutations in the APC gene were identified as the causative lesion
in autosomal dominant colorectal cancer or familial adenomatous polyposis coli (FAP)[2]. In
addition, over the eighty percent of sporadic colon cancers have been reported to possess
truncating mutations of APC gene [3]. Following these great discoveries, APC is recognized
as one of the most important tumor suppressor genes, and the mutations in the APC gene may
directly result in colorectal cancer [4].
In this chapter, we will start describing the history of the APC, cloning, location and
structure of the gene. Later we will discuss the protein function of APC, oncogenic
mechanisms of APC mutations, interaction for many cellular molecules, the specific
relationship to the Wnt signaling pathway, and a newly discovered candidate for gene
targeted therapy.

Phone +81-86-225-2111 fax +81-86-232-8343, E-mail [email protected].
254
Tomoki Yamatsuji, Munenori Takaoka, Takuya Fukazawa et al.
Cloning of APC Gene
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In terms of a discovery of APC gene, in 1987 its location was firstly identifiedon
Chromosome 5, and in 1991 cloned simultaneously and independently by two groups [5-12].
According to their findings, the APC gene has an 8,538 bp open reading frame, and consists
of 15 transcribed exons [8–10], on the long (q) arm of chromosome 5 between positions 21
and 22, from base pair 112,118,468 to base pair 112,209,532. The APC gene has been shown
to contain an internal ribosome entry site. This gene is expressed in a variety of fetal and adult
tissues, including mammary and colorectal epithelium [8]. It encodes for a 312-kDa protein,
2,843 amino acids long [8]. Inactivation of the APC gene product constitutes the initial step in
the development of colorectal cancer in Familial adenomatous polyposis (FAP).
The protein sequence does not contain transmembrane regions or nuclear targeting
signals, suggesting cytoplasmic localization.
In researchers‘ search for APC gene, Kinzler et al. identified several genes such as FER
(176942), MCC (159350), SRP19 (182175), and TB2 (REEP5; 125265), in addition to the
APC gene itself within a 5.5-Mb region of DNA linked to FAP. All genes were expressed in
normal colonic mucosa [6]. The APC gene product was predicted to contain coiled-coil
regions and was expressed in a wide variety of tissues.
Joslyn et al. identified 3 genes within small deleted regions on chromosome 5q12 found
in 2 unrelated patients with FAP [12]. One of these, termed DP2.5, was found to be the APC
gene [7], and the other 2 genes were SRP19 and DP1 (REEP5) [12]. Northern blot analysis
identified a 10-kb APC mRNA [7]. Independently, Hampton et al. isolated 2 overlapping
YACs containing the MCC gene; one of the YACs also included the complete APC gene
[13]. In 1993, cDNA clones representing transcripts expressed in human fetal brain and
coding for the 5-prime end of the APC gene were isolated and sequence analyses revealed an
alternative 5-prime untranslated region comprising at least 103 bp. This finding suggested
that 2 APC-specific promoter elements exist, giving rise to 2 different untranslated regions
(UTRs) [14].
Within the alternative UTR, 3 additional AUG codons was identified in the location of 5prime to the intrinsic APC initiation site, suggesting that these codons may be relevant for the
translational regulation of APC gene expression [14]. Horii et al. noted that transcriptional
initiation of APC occurs at 3 sites in 2 distinct nontranslated exons at the 5-prime end of the
gene [15].
Studies of transcripts from human colorectal tumor cell lines suggested the presence of
mutations in the transcriptional control region. At least 5 different forms of 5-prime
noncoding sequences, which were generated by alternative splicing, were detected [15].
Interestingly, the splicing mechanism appeared to be regulated in a tissue-specific fashion,
and 1 transcript, expressed exclusively in brain, contained an extra exon.
Gene Structure of APC
As mentioned above, the APC gene contains 15 exons [7], including an oligomerization
domain, an armadillo repeat-domain, a 15- or 20-residue repeat domain for binding to betacatenin, serine alanine methionine proline (SAMP) repeats for axin binding, a basic domain
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for microtubule binding and a C-terminal domain binding to EB1 and DLG proteins [16].
Exon 15 is large and comprises more than three quarters of the coding region of the gene.
Figure 1 reveals the schematic representation of APC protein and binding domains.
The most important functional domains of the APC gene appear to be the first
SAMP(axin binding) repeat at codon 1580 [17] and the first, second and third 20-amino acid
repeats (20AARs) involved in beta-catenin binding and degradation.
Sulekova and Ballhausen identified a novel coding exon of the APC gene, which was
located 1.6-kb downstream from exon 10 [18]. To that point, this 54-bp exon was the smallest
coding exon in the gene and termed exon 10A.. It is alternatively spliced and inserted inframe into mature transcripts; it gives an APC protein with an additional 18 amino acids.
APC exon 10A flanking sequences were presented so that this exon could be included in
mutation screening procedures [18].
Xia et al. described an alternatively spliced APC transcript which had not been reported
previously [19]. Within this transcript, they found an evolutionarily conserved but previously
unidentified exon between the known exons 10 and 11. The exon contains a heptad repeat
motif [18].
Karagianni et al. identified an alternatively spliced APC transcript in mouse embryonic
stem cells and colon tissue [20]. The transcript contains an untranslated exon, which was
designated as exon N. Transcripts bearing exon N, which were spliced to either exon 1 or
exon 2, were detected in all mouse tissues examined. A promoter region within exon N has
features of a housekeeping gene, including high average GC content and lack of CAAT and
TATA boxes. The researchers mapped the promoter about 40 kb upstream of the initiating
methionine, and transient transfection experiments showed strong promoter activity [20].
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Figure 1. Schematic presentation of APC protein. The 2,843–amino acid APC protein and its functional
sites are indicated. The N-terminal region has a domain that regulates APC oligomerization. Repeated
sequences with similarity to the Drosophila armadillo protein and its vertebrate homolog β-catenin
(armadillo repeats) are localized in the N-terminal side of APC. Multiple independent 20–amino acid
repeats that mediate binding to β-catenin and several binding sites for the Axin protein (SAMP repeats)
are localized in the center of APC. The C-terminal side of APC has a basic region that is involved in
microtubule (MT) binding and interactions with the proteins EB1 and hDlg. Modified from
Reference 41.
Most domains of this protein are solved structurally and exhibit high intrinsic disorder
and flexibility as a monomer, and a low content of stable secondary structure. Thus it is a
member of the intrinsically unstructured proteins.
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Function of APC
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APC is expressed constitutively within the normal colonic epithelium. The APC gene
product is a 310-kDa-homodimeric protein, which is localized in the cytoplasm and the
nucleus. APC is a multifunctional protein involved in several cellular life processes [6, 21].
Wild-type APC are critical to cytoskeletal integrity, cellular adhesion and Wnt signaling.
In in vitro and in vivo studies it has been shown that APC inhibits the canonical Wnt signal
pathway, which is essential for tumorigenesis, cell multiplication and differentiation,
development and homeostasis of a variety of tissues. Through intracellular mechanism, APC
has a master role in the process of suppressing the classic Wnt signal pathway [22].
It is discovered that APC also has an important role in other basic vital activities,
including the adhesion and migration of cells, organization of actin and microtubule skeleton
network, spindle formation and chromosome segregation. Deactivation of APC make cell lose
adhesion and cause the tumorigenesis. APC affects the cell adhesion through accommodating
the distributions of β-catenin and E-cadherin between the cytoplasm and the cell membrane
[23]. Wild-type APC interact with a tumor suppressor protein, DLG, which regulates cell
cycle progression from the Go/G1 to the S phase of the cell cycle [24]. Recently it has been
reported that APC–human disc large complex is responsible for the above-mentioned effect in
cell cycle in a manner without relying on the effect of β-catenin.
In addition, APC may act as a negative regulator of β-catenin signaling. APC combines
with GSK3-β and actin to form a complex that promotes β-catenin phosphorylated and
consequently ubiquitin-dependent degradation of β-catenin. The β-catenin/Tcf4 complex
regulates the proto-oncogene and cell cycle regulator c-myc, the G1/S-regulating cyclin D1,
the gene encoding the matrix-degrading metalloproteinase, matrilysin, the AP-1 transcription
factors c-jun and fra-1 and the urokinase-type plasminogen activator receptor gene [25, 26].
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Diseases Caused by APC Mutation
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It is clear that APC has a key role in number of vital life processes. Mutation of APC
gene results in occurrences of various kinds of diseases and produces incomplete APC
protein. The mutation is not only related to the familial adenomatous polyposis (FAP) but
also to sporadic colorectal cancer [27].
Familial adenomatous polyposis (FAP) is one of the autosomal-dominant inherited
diseases characterized by the development of small adenomatous polyps widely throughout
the large intestine, which is associated with a very high risk of colorectal cancer. The number
of polyps increases with the growing age accompanied by many characteristics of different
extracolonic manifestations. Among them, benign tumors usually progress to carcinomas if
they are not removed in time. FAP was first reported in 1925 and scientists had described in
detail the pathology characteristics. FAP is traditionally diagnosed based on the presence of
more than 100 adenomatous colorectal polyps. Genetic testing is now standard for the
diagnosis of FAP. FAP accounts for less than 1% of all colorectal cancer cases seen in
practice. Polyps often appear in teenagers or young people in their third decade of life. The
risk of cancer usually depends on the number of adenomatous polyps. This leads to an almost
100% chance of malignant transformation in at least one of these polyps by the fifth decade.
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At present, FAP is diagnosed by colonoscopy and the commonly used treatment method
is surgery that involves total colectomy and proctomucosectomy with ileal pouch-anal
anastomosis. The majority of FAP patients (over 70%) develop extracolonic manifestations
[28]. Most extracolonic manifestations have little clinical significance, but some lesions can
cause serious complications and even lead to death.
The most common extracolonic manifestation is congenital hypertrophy of the retinal
pigment epithelium (CHRPE) found in 60–90% of FAP patients. Desmoids are benign tumors
of the connective tissues that can lead to life-threatening complications through their sheer
size and impingement on vital structures. They occur in 5–10% of FAP. It has been
discovered that the CHRPE and desmoids are related to the mutation at definite region of the
APC gene [29]. Moreover, in the mutation sites of APC database, more than 300 mutations in
the APC gene have been identified in families with classic and attenuated types of familial
adenomatous polyposis.
Most are insertion, deletions and nonsense mutations that lead to frame shifts or
premature stop codons resulting in truncation of the APC gene product. The most common
mutation in familial adenomatous polyposis is a deletion of five bases in the gene [30]. The
majority of the serious clinical symptom representations are caused by minor defect in codon
1309.
Mutation at this site may cause the early development of colorectal polyps. The genetic
basis of FAP relies on the genetic inheritance mutation of APC gene [31, 32]. Colon cancer is
one of the most common malignancies in the USA and Western Europe, and is also one of the
main factors of disease incidence and fatality caused by cancers throughout the world [33].
The great majority (80%) of patients with colorectal cancer have sporadic disease with no
evidence of having inherited the disorder.
In the remaining 20% a potentially definable genetic component exists. In the past decade
germ line genetic mutations conferring high lifetime risk of colorectal cancer in carries have
been found, accounting for 5–6% of all colorectal cases [34]. The most common mutation in
colon cancer is inactivation of APC. Genetics studies using mouse model showed that
mutation of APC gene has a close relationship with intestinal tumorigenesis. Somatic
mutations in APC gene has been detected in the majority of colorectal cancers.
At least four genetic changes appear sequentially to ensure colorectal cancer evolution,
including an oncogene (KRAS) and three anti-oncogenes (APC, SMAD and p53) [35]. The
functional defect of APC is precisely the triggering factor of these cascade changes, which
ultimately lead to the malignant transformation of large intestine. The mutation of APC gene
is one of the early events in the process of sporadic colorectal cancer. In all, 60–80 in 100 of
sporadic colorectal cancers carry APC gene mutation and a similar frequency was reported in
colorectal adenomas [36].
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Relationship between Wnt Signaling and the APC Tumor-Suppressor Gene
in Activating the β-Catenin
APC is a classical tumor suppressor protein that plays a dominant role in Wnt signaling,
in part by regulating the degradation of β-catenin. Wnt signals influence the stability of a
protein complex containing β-catenin, conductin and GSK3 (glycogen synthase kinase 3).
258
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Gene Therapy and Targeted Therapy
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In the absence of Wnt or the presence of wild-type APC protein, β-catenin is degraded. In
the presence of Wnt, or the absence of APC, β-catenin target genes-including c-myc are
expressed.
Consequently, Myc expression induced the expression of the polyamine ornithine
decarboxylase (ODC) which is a proto-oncogene. The APC gene product indirectly regulates
transcription of several critical cell-proliferation genes, through its interaction with the
transcription factor β-catenin [37]. APC binding to β-catenin leads to ubiquitin-mediated βcatenin destruction; loss of APC function increases transcription of β-catenin targets. When
APC does not have an inactivating mutation, β-catenin does. These mutations can be
inherited, or arise sporadically, often as the result of mutations in other genes that produce
chromosomal instability.
A mutation on APC or β-catenin must be followed by other mutations to lead to
tumorigenesis; however, in carriers of an APC inactivating mutations, the risk of colorectal
cancer by age 40 is almost 100% [25].
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Hargest et al. have lipofected APC gene into the mouse colon and showed that
transfection of APC gave prolonged high-level expression of the transgene, an important
basis for gene therapy [38]. More, it has been reported that activation of PPAR inhibits APCdependent suppression of colon carcinogenesis and suppression of β-catenin inhibits the
neoplastic growth of APC-mutant colon cancer [39, 40]. APC remains an attractive target for
therapeutic intervention because its mutation is a common and early event in the continuum
of colorectal tumor progression. Therefore, APC and its related genes are attractive targets for
the design of therapeutic and chemopreventive strategies for colorectal cancer patients.
Further investigation into the biology, biochemistry, and genetics of APC will no doubt result
in the realization of these therapies
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APC gene was found about twenty years ago classified as a tumor suppressor gene, those
mutations are associated with most colon cancers although how these mutations affect the
development of cancer was not fully understood.
In this chapter, we have learned the history of the APC gene, cloning, location and
structure of the gene. After that we described the protein function of APC, oncogenic
mechanisms of its mutations, involvement in the Wnt signaling pathway, and we have
discussed a novel target of gene therapy. APC is a traditional tumor suppressor gene. It would
be involved in oncogenic signaling of the most colorectal cancers in the world. Therefore it
should be a critical therapeutic target in the near future.
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Chapter 13
Structure and Function of the Tumor
Suppressor Gene p16
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Zeynep Tarcan1, Catherine Moroski Erkul1, Bunyamin Isik2,
Esra Gunduz1 and Mehmet Gunduz1,
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Departments of 1Medical Genetics and 3Family Medicine, Faculty of Medicine,
Turgut Ozal University, Istanbul, Turkey
Abstract
1. Introduction
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Since its identification in 1993, the P16INK4a gene has been one of the most
extensively studied tumor suppressors. As one of the three genes located in the unique
INK4b-ARF-INK4a locus, its dysregulation is a common event in the initiation and
progression of many different cancers. In some cancer types, such as pancreatic
carcinoma, it is downregulated in up to 98% of cases. Thus it takes a place alongside P53
as an essential diagnostic tool and thereapeutic target in disease. The role of P16INK4a in
cell cycle regulation, primarly through its inhibition of CDK4/6-pRb, is the most studied
and best understood function of this mult-tasking molecule. It also has well documented
roles in mechansisms of senescence, apoptosis, anoikis and the DNA damage response.
Its role in the inhibition of inflammatory processes has also revealed a potential role in
the suppression of rheumatoid arthritis. The aim of this review is to give a general
overview of the various functions that P16INK4a plays in oncogenic transformation and
cancer progression and a brief look at its potential as a therapeutic target.
Cancer may be thought of as the culmination of a multitude of genetic and biochemical
aberrations that result in unrestricted cell growth. Cellular proliferation is a complex and
tightly controlled process that relies on the proper expression, function, and co-ordination of

Corresponding author: Mehmet Gunduz, MD, PhD, Department of Medical Genetics, Faculty of Medicine, Turgut
Ozal University, Turkey. Anadolu Bulvari 16A Gimat Ankara, Turkey. e-mail: [email protected].
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many genes and proteins throughout the entire cell cycle. There are a number of cell cycle
checkpoints that allow cells to ―pause‖, make sure that the current phase will be completed
successfully and to ensure that the raw materials required to complete the next phase are
available. This also allows replicating cells to repair any DNA damage that may have
occurred or, in cases when damage is too ovewhelming, to undergo programmed cell death.
These checkpoints are integral to maintaining proper cellular function and genomic integrity
and for guarding against the proliferation of cells with serious genomic defects. The ability to
escape these checks and balances and continue proliferating even in the presence of
significant damamge is one of the hallmark features of cancer cells [1].
Cyclins are a group of proteins whose expression and activity are triggered by mitogenic
stimuli and are necessary for entry into and progression through the cell cycle. They are
regulated by both changes in gene expression and rates of degradation. There are several
different cyclin proteins (Cyclin A, B, D and E) and each has unique characteristics and
patterns of expression throughout the cell cycle. Another group of proteins integral to the cell
cycle are cyclin dependent kinases (CDKs), a family of serine/threonine protein kinases
whose concentrations remain constant but whose activity is regulated by cell cycle phasespecific changes in cyclin concentrations. Cyclins bind to specific CDK partners to form
protein complexes. These cyclin-CDK complexes, which are activated by CDK-activating
kinases (CAKs) and inhibited by cyclin dependent kinase inhibitors (CDKIs), together form
functional holoenzymes that drive cells through cell cycle transitions [2]. In normal cells,
these complexes contain two additional components, PCNA and P21 [3]. Among the cyclin
family of proteins, Cyclin D1 has emerged as a gene with significant oncogenic potential.
Depending on cell type, Cyclin D1 forms complexes with CDK4 and/or CDK6 in G1 of the
cell cycle. These complexes, as well as the cyclin E-CDK2 holoenzyme, are invovled in the
transition from G1 to S phase, a critical regulatory point during the cell cycle [4]. Cyclin E
and CDK2 are expressed ubiquitously and their expression increases at the later stages of G1
[5]. The G1 to S phase transition is activated in part by the phosphorylation of retinoblastoma
protein (pRb) by the cyclin D-CDK4/6 and cyclin E-CDK2 complexes. The phosphorylation
of pRb results in the release of pRb-bound E2F transcription factors. These transcription
factors are then free to activate (via E2F-1, 2 and 3) or repress (via E2F-4 and 5) the
expression of genes whose products are required for entry into S phase and commitment to
mitosis [2, 6].
Also essential to cell cycle control, in this case for the negative regulation of growth, are
CDK inhibitors. One CDK inhibitor in particular, P16INK4a (inhibitor of CDK4 variant A) is
the focus of this chapter. P16INK4a and cyclin D competitively bind to CDK4/6. Thus when
P16INK4a is bound to CDK4/6, cyclin D cannot bind CDK4/6. In the absence of the cyclin DCDK4/6 complex, E2F transcription factors remain bound to hypophosphorylated pRb and
together they are translocated to the cytoplasm. Cell cycle progression is thereby prevented.
However, in the absence of pRb, cyclin E expression is increased and the inhibition of cyclin
D-CDK4/6 by P16INK4a does not prevent progression to S phase [5].
Since these cyclins, CDKs and their regulators are central to cellular proliferation, it is
not surprising that they play an important role in the process of tumor development. This also
suggests they may have potential for use as clinical diagnostic markers and therapeutic targets
in cancer [7].
The P16INK4a gene is located on human chromosome 9p21 region [8-10]. In addition to its
function in cell cycle regulation, P16INK4a (also referred to as CDKN2A, MTS1, and INK4a)
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has a critical role in cellular senescence, anoikis, apoptosis, and aging. It is a member of the
INK4 family of genes which also includes P15INK4b, P18INK4c, and P19INK4d. All INK4 family
members have structural similarity and can inhibit cyclin D kinase activity, but they also have
unique biological functions [5, 11]. P16INK4a has been identified as a tumor suppressor based
on its germline and somatic inactivation and abberant expression (both downregulation and
overexpression) in many tumor types [12]. Several types of P16INK4a mutations have been
identified in tumors including homozygous deletions, promoter hypermethylation, loss of
heterozygosity, and point mutations; however it is mainly via homozygous deletion of the
gene or promoter methylation that results in P16INK4a inactivation [12-14]. Consequently, the
clinical assessment of P16INK4a expression is an important aspect of cancer diagnosis,
prognosis and therapy [15, 16].
2. CDK Inhibitors
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There are a number of positive and negative regulators involved in cell cycle control. In
addition to P16INK4a, some of the important negative regulators in G1 include the other INK4
family proteins P15INK4b, P18INK4c, and P19INK4d as well as the Cip/Kip family proteins P21
(also known as WAF1, CAP20 and CIP1), P27 (KIP1), and P57 (KIP2). P21 and P27 can
inhibit progression through G1 in at least two points of the pathway; one is through steric
inhibition of CAK-activation via binding to cyclin-CDK holoenzymes and another is by
inhibiting the phosphorylation of pRb by CAK-activated cyclin-CDK holoenzymes [2]. It
should be noted that P21 is involved in both positive and negative regulation of cell cycle
progression. Along with cyclin, CDK and PCNA, P21 is an integral component of G1 to S
phase transition-promoting complexes. When all of these components exist in the complex at
a 1:1 ratio, the effect of P21 on growth is positive. If the ratio of P21 increases in relation to
the other components in the complex, it then acts as a negative regulator [2]. In addition to
cell cycle transition points, these two families of proteins have functions in transcription, the
DNA damage response, apoptosis and other processes [17]. Among the positive regulators of
the cell cycle are, of course, the cyclins and CDKs [16, 18].
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3. The INK4 Gene Family
The INK4 family is made up of four proteins, P16INK4a, P15INK4b, P18INK4c, and P19INK4d.
P16
and P15INK4b are encoded by the INK4b-ARF-INK4a locus located on chromosome
9p21 [19] (Figure 1). This locus codes for three different proteins, P16INK4a, P15INK4b and
P19ARF (not to be confused with P19INK4d). P19ARF/P14ARF, or simply ARF, is also a tumor
suppressor acting in the p53 pathway and is likely regulated by similar (or even the same)
mechanisms as P16INK4a and P15INK4b (Figure 2). However, this protein is completely
unrelated to the INK4 family of CDK inhibitors [20]. Also, while the INK4a nd INK4b
proteins are highly conserved across many species this is not the case for ARF, despite the
fact that they share the same genetic locus and have all been implicated in tumorigenesis to
one degree or another [20]. The genetic locus of P18INK4c is found at chromosome 1p32, and
that of P19INK4d is located at 19p13. The P15INK4b protein contains 138 amino acids and has a
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INK4a
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MW of 14.7-kDa. P18INK4c is composed of 168 amino acids and has a MW of 18.1-kDa and
P19INK4d contains 166 amino acids and as a MW of 17.6-kDa (19) (Figure 3).
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Figure 1. The INK4b-ARF-INK4a locus. The same gene locus encodes three proteins, P15INK4b,
P16INK4a, and P14ARF. P15INK4b has only two exons, whereas P16INK4a and P14ARF have three exons; the
latter two genes share exons 2 and 3, but P16INK4a is coded by exon 1α and P14ARF is coded by exon 1β.
Figure 2. Function of INK/ARF moleculer pathways in normal and tumor cells. P16INK4a binds and
inactivates CDK4/6 complexes. pRb cannot be phosphorylated and this results in cell cycle arrest in
normal cells. In tumor cells, P16INK4a binds and activates CDK4/6-cyclin D. Thus, pRb is
phosphorylated and the E2F transcritption factor is released and cell cycle progression proceeds.
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Figure 3. INK4 Family members.
Figure 4. Function of INK/ARF moleculer pathways in normal and tumor cells. P16INK4a binds and
inactivates CDK4/6 complexes. pRb cannot be phosphorylated and this results in cell cycle arrest in
normal cells. In tumor cells, P16INK4a binds and activates CDK4/6-cyclin D. Thus, pRb is
phosphorylated and the E2F transcritption factor is released and cell cycle progression proceeds.
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Despite similar structure and inhibitory activity of CDK4 and CDK6, only P16INK4a has
clearly demonstrated a tumor suppressor capacity, primarily through its role in cell cycle
control (Figure 4). Somatic mutations and altered protein expression (downregulation and
overexpression) have been detected in a multitude of different cancers. In fact, after P53,
P16INK4a is one of the most frequently altered genes in human cancer. Thus, it is curious that
the other INK4 family members have not been identified as having a more important role in
the pathogenesis of cancer. While it is true that mutations in the other family members have
been identified in scattered cases of cancer, they do not even begin to compare with the extent
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of P16INK4a aberrations in cancer. This suggests that one or more of P16INK4a‘s other functions
may be integral to its status as a tumor suppressor.
4. Discovery of P16INK4a
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In 1993, Xiong et al. reported a 16-kDa protein associated with CDK4 in SV40 (simian
virus 40) transformed human diploid fibroblasts [3]. It was given the name P16INK4A by this
group due to its inhibitory effect on CDK4. Around the same time, researchers were
investigating the existence of a melanoma susceptibility gene at the 9p21 locus. Two different
groups that isolated and studied P16INK4A named it MTS1 (multiple tumor suppressor 1) and
CDK4I (CDK4 inhibitor). It was given yet another name, CDKN2, for the human genome
project [19]. Today, the gene is typically referred to either as P16INK4A (or simply INK4A)
or CDKN2A. Since its initial discovery, P16INK4a has become a widely studied gene in human
cancer. P16INK4a is one of the most frequently mutated genes in human cancers [21]. It is
estimated that P16INK4a is mutated in about 20% of breast cancer, 65% of non-small cell lung
carcinoma, 30% of colorectal cancer, 60% of bladder cancer, melanoma, leukemia and
multiple myleoma, 50-70% of head and neck squamous cell carcinoma, 70% of esophageal
cancer and 85% of pancreatic carcinoma [22-24].
5. Structure of P16INK4a Gene and Protein
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The coding of the three genes located in the INK4b-ARF-INK4a locus is somewhat
complex. P15INK4a is coded by exons 1 and 2 and P16INK4a is coded by exons 1 thorugh 3.
However, between exons 2 and 3, there lies an alternate form of exon 1 that has its own
promoter, and is referred to as exon 1β. The third gene in this locus, ARF, is coded by this
alternate form of exon 1 along with exons 2 and 3 [6, 20]. Thus P16INK4a and ARFshare a
common exon 2 and exon 3. However, despite sharing these two exons, these proteins do not
share any amino acid homology. This is because exon 2 is translated in a different reading
frame in each protein (hence the name ‗ARF‘, alternative reading frame, for P19/ARFP14ARF)
[20].
Approximately 100 kB downstream of this three-gene locus there is a highly conserved
gene, MTAP. Looking at the organiziaton of these four genes in several organisms gives clues
as to how this unique locus may have evolved. In the puffer fish, Fugu rubripes, a homolog
of INK4b lies immediately next to MTAP. It is thus thought that P16INK4a may have arisen
from a gene duplication event. However, the presence of exon 1β physically separating these
two genes complicates this simple explanation. It has been suggested that this phenomenon
may be due to a need for common regulation of the genes within the INK4b-ARF-INK4a
locus [20].
P16INK4a is a 156 amino acid protein with a MW of approximately 16-kDa. The solution
structure of full-length P16INK4a was solved in 1998 by Byeon et al. [25]. Central to the
P16INK4a protein are its four ankyrin repeats [26, 27] which provide its structural scaffold and
mediates protein-protein interactions [20]. Ankyrin repeats are 33 amino acids in length and
are found in many different types of proteins. It is estimated that approximately 6% of
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proteins coded by human genes contain these motifs [28]. Different ankyrin repeats found in
the same proteins can differ from one another in terms of amino acid sequence, allowing for
structural and functional diversity. Ankyrin repeat-containing proteins are rather distinct from
globular proteins in that they do not have a hydrophobic core and their tertiary structure does
not include stabilizing contact between residues distant from one another on the polypeptide
chain [27, 28]. Each of the four repeats found in P16INK4a has a helix-turn-helix motif except
for the first half of the second ankyrin repeat which only has one helical turn [25, 27]. The
helices stack into bundles in a linear fashion facilitated by internal hydrophbic residues. The
helix-turn-helix motifs are linked by loops that are positioned perpendicular to the helix. Each
of the three loops have little amino acid sequence homology suggesting that each plays a
unique role in different P16INK4a functions [27]. Most of the hydrogen bonds that allow INK4
proteins to bind CDK6 are not found in other ankyrin repeat-containing proteins, again
suggesting that sequence diversity is a mechanism that allows functional specificity [28].
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6. P16INK4a as a Tumor Suppressor
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As a negative regulator of cellular proliferation, it has an important role in the prevention
of tumor formation. Approximately 50% of all human cancers demonstrate P16INK4a
inactivation, with a range from about 25% to 70% [15]. Inactivation has been detected in the
following types of cancer: head and neck, esophageal, biliary tract, liver, lung, bladder, colon
and breast carcinomas; leukemia; lymphomas; and glioblastomas [4, 24, 29, 30]. Biological
and biochemical analysis of P16INK4a germ line mutations in some cancer types show
abnormalities of P16INK4a protein function [12, 31, 32] further evidence that P16INK4a plays a
role as a tumor suppressor. The P16INK4a gene is inactivated in head and neck squamous cell
carcinoma. Genetic and epigenetic analyses show that expression of P16INK4a gene is lost in
74% of head and neck squamous cell carcinoma and promoter hypermethylation occurs in
27% [33]. P16INK4a is inactivated most frequently in pancreatic carcinomas with 98% of cases
demonstrating a loss of P16INK4a function. Many mechanisms of P16INK4a gene inactivation
have been identified in these tumors, including homozygotic deletions, loss of heterozygosity,
point mutations and promoter methylation [34, 35].
Other research has demonstrated germline mutations in P16INK4a in familial melanoma
[36]. For instance, in familial melanoma, the most prevalent changes (41% of cases) are
associated with missense mutations or deletions in the INK4/ARF locus [15, 37].
7. Specific Functions of P16INK4a
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P16INK4a is a tumor supprossor gene whose function in cell cycle regulation is extensively
documented. P16INK4a is involved in other cellular processes including (but not limited to)
senscence, apoptosis/anoikis and DNA repair. In addition to its apparent role in the control of
the G1 to S transition, P16INK4a may also regulate the cell cycle via its inhibition of cyclin
dependent kinase 7 (CDK7)-carboxyl-terminal domain (CTD) kinase activity [38, 39].
Phosphorylation of this essential CTD, which is located in the largest subunit of RNA
polymerase II, is involved in the regulation of transcription both in vitro and in vivo. In vitro
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7.1. Other Fuctions of P16INK4a
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experiments have indicated that the CDK7 subunit of the general transcription factor TFIIH
can carry out this phosphorylation. P16INK4a has been found to associate with TFIIH as well as
the CTD of RNA pol II. Furthermore, this association was observed to inhibit the
phosphorylation of the CTD and contribute to cell cycle arrest [38, 39]. This function
suggests a P16INK4a-mediated connection between basal transcription mechanisms and the
regulation of cell cycle progression. Experiments with chimeric INK4 family proteins indicate
that this activity is specific to P16INK4a, apparently carried out by residues on its aminoterminal end [39].
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One of the first lines of research that led to the identification of P16INK4a was the attempt
to find a melanoma susceptibility gene near the 9p21 locus. P16INK4a mutations are present in
almost all human melanoma cell lines [40] and p16INK4a knock-out mice are susceptible to
tumor formation, including skin melanomas [41]. In families with germline mutations in
P16INK4a sunburn is a very high risk factor. One of the three major signalling cascades in
higher organisms, collectively referred to as mitogen activated protein kinases (MAPKs), is
the c-Jun N-terminal kinases (JNKs). These signal transduction molecules are activated in
response to extracellular stimuli, inflammatory cytokines, and environmental stress (including
UV radiation) [42, 43]. They are involved in activation of the c-Jun/activator protein-1 (AP-1)
transcription factor complex. AP-1 transcription factors regulate gene expression in response
to stress, growth factors and cytokines and thus is involved in controlling processes like
proliferation, differentiation and apoptosis [44]. They also can cause neoplastic
transformation and skin carcinogenesis in mice [45, 46]. Furthermore, tumor formation is
inhibited in c-Jun knock-out mice [47]. Choi et al. reported in 2005 that P16INK4a, but not
other INK4 proteins, binds JNK1 and JNK3 in vitro and in vivo with or without exposure to
UVC. Upon exposure to 60J/m2 UVC, this association increased 10-fold. After UV-exposure,
its interaction with JNK proteins inhibits their ability to bind to c-Jun, which in turn inhibits
c-Jun phosphorylation [42]. This function of P16INK4a thereby contributes to the prevention of
neoplastic transformation.
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7.1.1. P16INK4a in Apoptosis
Apoptosis is a controlled form of cell death that is essential to growth and development
of organisms and functions in actively proliferating cell types in adults such as skin and the
gastrointestinal tract. Apoptosis is also essential in preventing the replication of cells that
contain overwhelming amounts of DNA damage, thus ensuring that cells that harbor
potentially deleterious mutations do not proliferate. Along with unrestrained proliferation,
defects in apoptosis are another hallmark of cancer. The absence of proper apoptotic
mechanisms aids proliferation beyond a cell‘s normal replicative lifespan. It also facilitates
other mechanisms associated with cancer such as angiogenesis. Defective apoptosis presents a
challenge for many chemotherapeutic agents that rely on apoptosis for their effect [48].
Evidence of P16INK4a mediated P53-dependent apoptosis has been provided by studies in
numerous cell lines and mouse models. Ectopic overexpression of P16INK4a and P53 in tumor
cell lines induces apoptosis. When delivered together to a mouse xenograft tumor model, their
combined expression is capable of inhibiting tumor growth [49]. In several non-small cell
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lung cancer cell lines ectopic expression of P16INK4a and P53 led to G1 arrest and several days
later an induction of apoptosis. This effect was caused by direct downregulation and
hypophosphorylation of Rb and indirect downregulation of the anti-apoptotic factor, bcl-2.
The mechanism of apoptosis was later shown to involve caspase-3 activation followed by
cleavage of PARP, MDM2 (a negative regulator of P53) and Rb [50]. A pan-caspase inhibitor
significantly reduced the observed phenotype and in all experiments wild-type P53 was
requisite for apoptosis to occur [50, 51].
P16INK4a has also been reported to modulate apoptosis after DNA damage. After
treatment of P16INK4a-deficient U2OS cells (osteosarcoma cell line) and p16 null mouse
embryonic fibroblasts (MEFs) with UV, apoptosis was triggered. However, in isogenic cell
lines EH1 and EH2 which express P16INK4a, very little apoptosis was observed after UV
exposure. The same was true of p16 wild-type MEFs [48]. Conversely, after treatment of the
same cell types with cisplatin, a chemotherapeutic agent that causes DNA damage and
induces both p53-dependent and independent apoptosis, U2OS cells underwent minimal
apoptosis while P16INK4a proficient cells exhibited significant levels of apoptosis.
Interestingly, while the MEFs exhibited an immediate response to cisplatin, the EH1 and EH2
cell lines underwent apoptosis only after being blocked at the G2/M checkpoint after 48h of
treatment. The extent of the G2/M arrest was cisplatin dose-dependent. Cell synchronization
experiments as well as experiments carried out in quiescent cells, suggested that cisplatin only
causes apoptosis in proliferating cells [48].
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7.1.2. P16INK4a in Tumor Cell Invasion
As a tumor grows and acquires more aggressive proliferative capacity, changes take place
both within the tumor and in surrounding stromal tissue that allows invasion and metastasis to
take place. Invasion occurs when tumor cells acquire the ability to begin ―invading‖ adjacent
normal tissue. This is primarily caused by aberrant activation of cell motility and changes in
adhesion properties. The latter is associated with matrix degradation and remodeling. Tumor
cells must also harbor an ability to adapt to the new local tissue environment into which it
invades. Metastasis requires yet more changes which allow cells that have managed to reach a
conduit for travel to distant locations to survive in the blood/lymph circulation without a
connection to supporting tissue [52]. One characteristic that invasion and metastasis appear to
share is the activation of motility since metastasis has been observed to occur in tumors
without any apparent invasive properties. P16INK4a has been associated with the acquisition of
the ability to invade surrounding tissue in serveral cancer types via its effect on factors
directly involved in the processes of cellular motility. By comparing stromal fibroblasts in the
tumor microenvironment with fibroblasts in cancer-free, histologically normal tissue isolated
from the same patient, Al-Ansari et al. demonstrated that 83% of the cancer associated
fibroblasts (CAFs) in the breast cancer cases they studied have lower P16INK4a expression at
both the mRNA and protein level [53]. In a later study, they show, both in cell lines and in a
mouse xenograft breast cancer model,that this downregulation results in enhanced invasion
and VEGF-A-dependent angiogenesis via activation of Akt protein kinase. P16INK4a normally
represses the expression and secretion of VEGF-A via its suppression of the Akt/mTOR
signalling pathway. Suppression of this pathway prevents HIF1-α, a downtstream effector of
Akt/mTOR, from transactivating VEGF-A. Inhibition with a VEGF-A-specific inhibitor
molecule suppressed the pro-angiogenic effect caused by this P16INK4a deficiency [54].
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Several studies over the past decade have found that P16INK4a plays a role in inhibiting
angiogenesis and cell migration via inhibition of the expression of cell surface receptor
alpha(v)beta(3) integrins [55]. One study conducted in the malignant glioma cell line SNB19
found that adenoviral expression of P16INK4a combined with knock-down of uPAR, a gene coregulated with alpha(v)beta(3), inhibited integrin-mediated cell adhesion, migration, and
proliferation [56]. This combination was also found to be capable of inhibiting angiogenesis
and activating cell death in the U251 glioma cell line [57]. Another study conducted in
HUVEC cells came to similar conclusions, where P16INK4a was involved in a pathway that
controls angiogenesis of endothelial cells via alpha(v)beta(3)-mediated migration [58]. The
alpha(v)beta(3) integrins have also been implicated in invasion in melanoma [16] and
pancreatic cancer [7].
The gamma2 chain of laminin 5 is associated with invasion in some types of carcinoma
(Figure 5). P16INK4a and gamma2 chain of laminin 5 were co-expressed in regions of
microinvasion and margins of squamous cell carcinoma of skin and oral cavity. P16INK4a
expression was, on the other hand, undetectable in benign hyperplastic lesions. Keratinocytes
at the edges of wounds show a similar co-expression of P16INK4a and gamma2, thus
suggesting a possible role for P16INK4a in wound healing [59]. This capacity may be
commandeered by cancer cells to facilitate invasion.
Beta-catenin is a well-established oncogene in colorectal cancer. P16INK4a expression is
regulated by beta-catenin (Figure 5) and is associated with low survival in colon cancers with
an infiltrative front of invasion [60]. In a study investigating the expression and association
between beta-catenin, P16INK4a and c-Myc in colorectal cancer at various stages of
tumorigenesis and progression, there was an increase in nuclear expression of all three
proteins correlated with severity of disease. Nuclear overexpression of beta-catenin and
P16INK4a were associated with tumors having lymph node metastasis but not distant metastasis
[61].
Figure 5. The gamma2 chain of laminin 5 is associated with invasion in some types of carcinoma and
p16 expression is regulated by beta catenin.
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In a study investigating the expression of cell cycle regulatory proteins in endometrial
carcinomas, Horree et al. show, via immunohistochemical staining of tissue, that the invasive
front of these tumors, as compared to central regions of the tumors, have higher rates of
proliferation and lower rates of cell cycle regulatory proteins. P16INK4a displayed higher
expression in about 25% of the 39 cases included in their experiments [62].
In work published by Zhang et al. in 2012, the use of a P16INK4a-specific artificial
transcription factor (P16ATF) was able to re-activate P16INK4a by demethylation of CpG
islands that had inactivated P16INK4a (and other tumor suppressor gene) expression. CpG
islands are found in about 60% of human gene promoter regions and consist of many CpG
dinucleotide repeats. Their methylation usually results in irreversible inhibition of gene
expression [63]. Thus, the ability to re-activate gene expression via artificial transcription
factors is a potentially powerful tool. Both the transient and stable expression of P16ATF not
only led to re-activaiton of P16INK4a but also successful inhibiton of cell migration and
invasion. This targeted approach may be preferable to the global DNA methylation inhibitors
currently used in cancer treatment as it may reduce or completely do away with the toxic side
effects associated with such drugs [64].
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7.1.3. P16INK4a in Anoikis
Cells possess surface receptors on their membranes to which adhesion molecules such as
integrins and cadherins can bind to. This allows them to maintain connections to the
extracellular matrix and/or to neighboring cells. When these connections are lost, cells
normally undergo a form of apoptosis referred to as ―anoikis‖. Cells that have the capacity to
avoid anoikis are able to survive in suspension and thus can travel to distant sites. If these
cells are cancerous, they can form new sites of tumor growth. Thus proper function of the
biomolecular pathways involved in anoikis signalling is essential for the prevention of
metastasis. In 2004, Douma et al. showed that repression of anoikis in vivo can induce
metastasis. Using a retrovirally transfected cDNA library screen, non-transformed rat
intestinal epithelial cells, known to be highly sensitive to anoikis, were transferred from
standard adhesive culture plates to ultra-low cluster plates containing a gel layer that prevents
cell attachment. Most cells rapidly underwent apoptosis but some clones survived. The TrkB
clone, in particular, was able to form large spheroid aggregates growing in suspension. When
stimulated with BDNF, TrkB‘s primary ligand, these cells became even more resistant to
apoptosis. To carry out experiments with these cells in mice, TrkB-expressing and
TrkB/BDNF co-expressiong cells were engineered to also express luciferase, which would
facilitate non-invasive in vivo imaging. Luciferase activity disappeared after 6 days in nude
mice injected with control cells. However, TrkB-expressing cells were able to colonize the
lungs and heart and form rapidly proliferating tumors. Even more striking was the ability of
TrkB/BDNF co-expressing cells to cause metastasis throughout the body. This effect was
determined to be caused by the activation of PI3K/Akt signaling pathways by TrkB [65].
TrkB is overexpressed in several cancer types including pancreatic, ovarian and
neuoblastoma. And loss of P16INK4a expression is known to activate Akt signalling [54].
More direct evidence of P16INK4a involvement in anoikis comes from work performed in
a number of different cancer cell lines. In 2000, P16INK4a was reported to induce anoikis via
transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin
receptor in the pancreatic cell line Capan-1, the hepatocellular carcinoma cell line SKHep1,
and the melanoma cell line NKI4 [66]. When these P16INK4a-deficient cells were transfected
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with P16INK4a expression vector, the cells‘ ability to carry out anoikis was restored. siRNA
mediated knock-down of alpha(5) integrin chain in P16INK4a-expressing cells did not undergo
anoikis. A follow-up study carried out in pancreatic cell lines investigated the involvement of
K-Ras in suppression of anoikis. Two common features of pancreatic cancer are activation of
K-Ras and inactivation of P16INK4a, P53 and SMAD4. Since K-Ras is known to regulate
P16INK4a, the authors wished to determine whether the converse was also true [67]. Capan-1
cells exhibit loss of P16INK4a and an activating mutation in K-Ras. When P16INK4a was reintroduced to Capan-1 cells, K-Ras protein was destabilized, its activity was suppressed, and
anoikis mechanisms were restored [68].
Subsequent investigation into the role of alpha(5)beta(1) fibronectin receptor have
confirmed previous findings of the role it plays in anoikis [69]. Andre et al. were able to
restore epithelial tumor cell sensitivity to anoikis after stable transfection with a P16INK4a
expression vector. They found P16INK4a to be implicated in the process of protein
glycosylation. Glycosylation is simply the addition of a glycan (an oligosaccharide or
polysaccharide) to a protein. These glycans have effects not only on protein structure but also
are involved in cell adhesion and signalling mechanisms that can trigger apoptosis or
proliferation. Abberant glycosylation is a characteristic observed in malignant transformation.
The authors found that expression of P16INK4a was associated with an increase in expression
of fibronectin cell-surface receptors and that this was responsible for restoring sensitivity to
cell death by anoikis [70].
P16INK4a loss is a common occurrence in HCC and, like pancreatic cancer, HCC is very
lethal. Restitution of P16INK4a in HCC cell lines was reported to induce anoikis via decreased
phosporylation of Akt and Survivin. After levels of survivin decreased, there was a limited
supply of this protein for CDK4 to import into the nucleus. This caused cell cycle arrest and
anoikis. The authors also demonstrated the ability of P16INK4a to induce apoptosis via
Akt/Survivin downregulation in vivo in a mouse xenograft model. P16INK4a is thus a
potentially useful therapeutic target in these difficult to treat cancers [71].
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7.1.4. P16INK4a in Senescence
Senescence is typically defined as irreversible cell cycle arrest. There are two types of
senescence which are defined by the type of stimulus they are triggered by. Replicative
senescence is caused by telomere shortening and dysfunction which is a normal part of the
cellular and organismal aging process. Premature senescence is a stress-induced phenotype
typically triggered by overwhelming DNA damage, replicative stress, oncogene activation
and reactive oxygen species generation. The pathways involved in the cellular response to this
stress are the DNA damage response (DDR), P16INK4a/pRb and ARF/P53/P21 [72]. While
senescence pathways are active in pre-malignant tissue, they are bypassed and/or inhibited in
cancerous cells and tissues. This is another mechanism whereby cancer cells attain their
endless replicative capacity.
Genes involved in the suppression of senescence are upregulated in cancer cells and
genes involved in the induction of senscense are downregulated. Thus, reactivation of
senescence pathways may be possible by activation of these senescence-inducing genes or
inhibition of senescence suppressing genes [72]. Cells that have undergone senescence exhibit
changes in organelle structure and function, many of which adversely affect mitochondria and
mtDNA [73]. P16INK4a appears to be involved in both the initiation and maintenance of
cellular senescence.
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One of the side-effects of doxorubicin which limits its use in the clinic is cardiomyopathy
and heart failure. This is caused by extensive apoptosis of mature cardiomyocytes and
progenitor cells. This includes cardiac progenitor cells as well as endothelial progenitor cells
(EPCs), that latter which are recruited from bone marrow. However, at lower doses of
doxorubicin, cells experience telomeric dysfunction and enter a state of stress induced
premature senescence (SIPS). P16INK4a and JNKs are both implicated in this process.
There is evidence that cellular senescence and aging are also induced by P16INK4a
expression in different progenitor cells such as neural progenitor cells [74], pancreas islet
progenitor cells [75], and hematopoietic stem cells [76].
8. Regulation of P16INK4a Expression
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P16INK4a gene expression is regulated by many different mechanisms at transcriptional,
post-transcriptional and post-translational levels. Its physical location within the unique
INK4b-ARF-INK4a locus means that its transcriptional regulation is, at least in some
instances, intimately connected to the regulation of the ARF tumor suppressor. It also means
that parsing the precise regulatory mechanisms associated with this gene has been a
complicated endeavor. Finally, as is the case for many genes, the specific regulatory
mechanisms involved are cell-type and context dependent.
Several transcriptional activators of P16INK4a have been identified. There is an ETS
binding site in the P16INK4a promoter that can be activated by Ets-1 and Ets-2 transcription
factors (TFs). Conversely, other ETS family TFs, Pea3, Sap1 and Elk1, are capable of causing
a reduction in promoter activity. PPAR gamma TF also binds the promoter and inhibits P16
upregulation. De-phosphorylation of PPAR gamma leads to it release from the promoter and
upregulation of P16INK4a [77].
Approximately 2 kb upstream of the P16INK4a promoter regions lies an area that canbe
bound by H2A.Z protein. H2A.Z protein along with the transcription factor CTCF can modify
the P16INK4a locus in a manner that inhibits epigenetic silencing via chromatin remodelling of
the P16 locus [78].
RAS-RAF-MEK signalling induces P16INK4a expression (Figure 6). The precise
mechanism is not clear but it may occur via binding of the P16INK4a promoter by ETS2 TF or,
alternatively, via secondary signalling through P38. MYC also appears capable of
upregulating P16INK4a expression possibly through binding of the promoter. However in
both of these cases, the kinetics of expression do not match up well with the proposed
causative mechanism and thus more research is needed to clarify these ambiguities. And in
the case of MYC, it can activate BMI-1 which is a Polycomb Group (PcG) gene and inhibitor
of INK4a, further complicating the picture [20].
P16INK4a is also regulated by the AP-1 family of transcription factors. The overexpression
of JUNB causes upregulation of p16INK4a while another AP-1 family member, c-Jun, represses
P16INK4a [20].
P16INK4a expression can also be activated by interactions at the P16INK4a locus between
P300 and Sp1 which result in hypermethylation of histone H4 [79]. It has also been reported
to be upregulated by inhibitor of growth 1 (ING1) [80]. Repressors of the locus include
TAL1, ATM, EGR1, ZBT7B, and AML1 (RUNX1) [20].
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Figure 6. RAS-RAF-MEK signalling in p16 expression.
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9. Overexpression of P16INK4a in Tumors
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Thus far, we have seen that P16INK4a loss or downregulation is a common occurrence in
many different cancer types. It plays an integral role in a host of cellular processes that guard
against oncogenic transformation and its absence or aberrant expression allows a variety of
pathological changes to occur. These changes contribute not only to oncogenic transformation
but also to the invasive and angiogenic capacity of a tumor and the ability to metastasize.
However, in some cancers, after malignant transformation takes place, the expression of
P16INK4a is reactivated [15]. Detection of the overexpression of P16INK4a can be used as a
diagnostic tool [29, 81] in some tumor types, such as cervical cancer and perianal lesions.
Milde-Langosch et al. reported overexpression of P16INK4a, as detected by Western blot
(WB) and immunohistochemistry (IHC), in about 20% of 60 breast cancer tumor samples
tested. Furthermore, this expression was associated with other markers indicating a poor
prognosis [82]. In another study of gynecological cancer, Armes et al. found that invasive
serous papillary ovarian cancer had strong P16 expression throughout the tumors. It was
particularly common in grade 3 carcinomas [83]. A similar observation was reported by
Chiesa-Vottero et al. in an examination of uterine serous carcinoma and ovarian high-grade
serous carcinoma [84]. Overexpression has also been observed in high-grade ovarian serous
carcinomas and uterine leiomyosarcomas [85, 86]. P16INK4a overexpression can also be used
as a marker of dysplasia and neoplasia in cervical epithelial biopsy samples. Using IHC,
Klaes et al. found significant overexpression of P16INK4a in cervical intraepithelial neoplasm
(CIN) I lesions (n=47) that were associated with high-risk HPV types. Overexpression was
also found in all CIN II lesions (n=32), CIN III lesions (n=60) and 58 of 60 invasive cervical
cancers. This was in contrast to a lack of observable P16 staining in normal cervical
epithelium (n=42), inflammatory lesions (n=48), and CIN I lesions associated with low-risk
HPV types (n=7) [87].
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Both P16INK4a downregulation and overexpression have been observed in colon cancer
[88-90]. In colorectal cancer, P16INK4a overexpression is associated with poor prognosis, and
varies by case according to sex, distal location, and tumor grade and stage [15]. Zhao et al.
found overexpression of P16INK4a in 71.7% of Chinese patients with Hodgkin lymphoma. In
some studies of pre-cancerous and cancerous lesions of the oral cavity, increasing P16INK4a
expression assayed by IHC has been associated with increasing level of malignancy [91].
However, in some studies of oral squamous cell carcinomas P16INK4a overexpression was
correlated with favorable prognosis [92]. This apparent contradiction may be due to
differences between the cancer sub-types that were studied or age-dependence. The latter
study was conducted in young patients (age 18-39) and thus may reflect different mechanisms
or germline versus somatic mutations.
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10. Aberrant Subcellular Localization
of P16INK4a in Cancer
In addition to downregulation and overexpression, aberrant subcellular localization of
P16
has been reported in several cancer types. Other cell cycle regulatory proteins that
have aberrant cytoplasmic expression in tumors are PTEN and P27. Zhao et al. used P16INK4a
and CDK4 IHC to examine changes in subcellular localization of these proteins in a series of
colon tissue samples that ranged from normal histopathlogy to adenoma and carcinoma.
While 95% of normal epithelia (n=42) had P16INK4a positive staining localized to the nucleus,
25% of adenomas (n=43) had cytoplasm-only positive staining of P16INK4a and 75% had
cytoplasmic/nuclear staining. Out of 73 carcinomas, the proportion of samples exhibiting P16
localization in the cytoplasm increased to 62% and cytoplasmic/nuclear staining was
observed in 37% of carcinomas. Furthermore, in cancer cells, the staining intensity of
P16INK4a was weak and sporadic [63]. Cytoplasmic P16INK4a expression has been described
in astrocytomas and is associated with poor prognosis [29]. It has also been observed in terine
leiomyosarcomas and in these it is typically associated with malignant tumors. In
gastrointestinal stromal tumors (GISTs), Haller et al. describe P16INK4a cytoplasmic
expression to be associated with poor prognosis but the precise mechanism involved in
unclear. They also describe a set of GISTs in which low nuclear expression, also corrleated
with poor prognosis, is associated with E2F1 transcription factor upregulation which causes
increased proliferation [93]. The authors suggest that the proteins detected in cytoplasm and
nucleus are actually two dinstict splice variants of P16INK4a, as had been previously reported
by Lin et al. in 2007 [94].
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INK4a
11. P16INK4a as a Therapeutic Target
Re-activation of P53 and P16INK4A can reduce tumor burden via the induction of apoptosis
and senscense, at least in murine models [95]. Promoter hypermethylation of P16INK4a or
cytoplasmic P16Ink4a sequestration by anion exchanger 1 (AE1) has been identified as an
alteration that contributes to malignant transformation. Hence, both p16INK4A can be restored
and premature senescence can be induced in cancer cells by demethylating therapies or
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ISBN: 978-1-62808-665-2
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Chapter 14
The Functions and Roles of RB1
in Cancer
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Erkan Koparir,1 Asuman Koparir1 and Mustafa Ozen1,2,
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Houston, TX, US
Abstract
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The retinoblastoma tumor-suppressor gene (RB1) was cloned in 1986 and called as
the prototype of tumor- suppressor genes. Up to now, we know there have been hundreds
of reviews published and announced RB1 is most studied gene. Loss of RB1 gene
function predispose childhood retinoblastoma tumor affecting approximately 1 in 15.000
children, also many other human cancers. RB1 associate multiple pathways and has
critical roles in any steps of cell cycle. It is also clear that RB1 regulates cell
differentiation, apoptosis and genomic stability. RB1 acts in these processes through most
especially regulating the function of E2F transcription factors by affecting G1 signaling
pathway. pRb is the product of RB1 and associates with p107 and p130 cellular proteins.
All of these related proteins named as pocket protein family which have similar structures
and functions. Three viral oncoprotein induce transformation by binding pocket domain
of these proteins including pRb, p107, p130. Therapeutic applications have improved for
restore pRb functions such as p53 inducer Topotecan, Mdm2 inhibitor Nutlin-3 and
HDAC inhibitors.
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Keywords: RB1, retinoblastoma, tumor-suppressor genes, E2F, pocket proteins

E-mail: [email protected], [email protected].
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Introduction
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The retinoblastoma tumor-suppressor gene (RB1) was the first identified tumor
suppressor, which has demonstrated to have a central role in cancer research [1, 2]. Since it
regulates multiple cellular processes such as cell proliferation, cell differentiation, apoptosis
and genomic stability, whose aberrancies result in oncogenesis, it has become one of the most
studied proteins.
Inactivation of RB1 as a result of deleterious mutations causes retinoblastoma, which
develops during childhood period, however, deregulation of the pathways, where the protein
product of RB1 (pRB) functions as a primer regulator of E2F transcription factors, has shown
to be involved in pathogenesis of several human cancers [3, 4]. This chapter summarizes the
significance of RB1 in cellular functioning in relation to its tumor suppressive role through
unraveling the recent insights into its expression, function and activity in normal and
pathological states.
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1. The RB1 Gene
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RB1, was cloned in 1986 [5] and found to be located at chromosome 13q14.2, consisting
of 27 exons. This tumor suppressor gene has shown to be transcribed into a 4.7-kb messenger
RNA (mRNA) [6, 7].
It is a member of a large gene family, which includes two other RB-related genes called
as retinoblastoma-like 1 (RBL1) and retinoblastoma-like 2 (RBL2). RB1, RBL1 and RBL2
genes encode for structurally related proteins; pRb, p107 and p130, respectively [2].
2. RB1 and Retinoblastoma
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RB1 mutations, which cause retinoblastoma, result from bi-allelic inactivation or loss of
the retinoblastoma 1 gene (RB1) [8]. According to Knudson‘s ―two-hit hypothesis‖ (9), which
is originated from retinoblastoma gene, two mutational events or two hits are required for
tumor onset.
A patient may have inherited a germline mutation from a parent and that would constitute
the first "hit" leading to the cancer, then a second mutational event, or second ―hit‖ results in
the development of the disease [10-12]. RB1 gene has shown to be heterozygous mutant in
patients who are diagnosed with sporadic bilateral or familial retinoblastoma. These first
―hits‖ were either inherited or developed de novo in parental germline cells or during
embryonic development. In both cases, all cells of the affected individual turns to be carrier
for RB1 mutation and a second mutation in the retina cells during the early childhood period
results in the development of bilateral retinoblastoma. These heritable cases constitute
approximately 40% of all cases, which are predisposed to retinoblastoma during childhood.
Non-heritable RB cases made up of 60% of all cases and most of them develop unilaterally,
with mutations occurring locally within the affected retina only [2, 13, 14].
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3. The Pocket Protein Family
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Figure 1. Functional domains of the pRb protein.
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pRb and its related cellular proteins p107 and p130 comprise the family of ―pocket‖
proteins that have structural and functional similarities (Figure 1). All three proteins contain a
large pocket domain, which is required to mediate the binding site for many of viral and
cellular proteins [15, 16].
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The pocket proteins carry out overlapping functions such as binding E2Fs, restraining
cell cycle progression, and serving as substrates for CDKs. It has been demonstrated in
different studies that loss of p107 or p130 represents cell cycle defects similar to those
lacking pRb [17, 18], whereas in contrary to pRb loss, mutational inactivation of p107 or
p130 was rarely observed in human cancers [19].
The pocket proteins can bind DNA tumor virus oncoproteins as well as endogenous
nuclear proteins, which promote cellular proliferation. These mitogenic oncoproteins,
including adenovirus E1A protein, simian virus 40 (SV40) large T antigen and human
papillomavirus E6 and E7 protein, inactivate the function of Rb and result in tumor formation
[20-24].
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4. Functional Domains of the pRb Protein
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Rb, p107, p130, as pocket proteins, share a structural element called the A/B pocket [25,
26]. The A/B pocket, which contains the LXCXE motif, is needed for binding of E2Fs,
HDACs, and viral oncoproteins [27-30]. Cyclin E–CDK2 complex inactivates pRb through
binding its phosphorylation site of the N terminus. Cyclin A–CDK2 and cyclin D–CDK4
binds phosphorylation site of the C terminus and inhibit function of pRb. C-Abl protooncogene and the p53 inhibitor Mdm2 also have binding sites in the C terminus (Figure 1)
[26, 30, 31]. pRb protein have 16 CDK phosphorylation sites that regulate the pRb activity
throughout the cell cycle. Most of the RB1 gene mutations localize in the A/B pocket domain
[33].
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5. Rb in Cell Cycle Regulation
5.1. E2F and E2F Regulated Genes
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The ‗pocket protein‘ family of cell cycle regulators is made up of pRB and and its related
cellular proteins p107 and p130. These proteins are primarily responsible for preventing the
G1–S transition via regulation of E2F-responsive genes. In quiescent cells, RB proteins
repress the transcriptional activity of E2Fs. When the cell growth is induced and the cell cycle
entry is promoted, cyclin dependent kinases are activated. This leads to the phosphorylation
and as a result deactivation of pRB, which cause activation of E2F transcription factors and
G1-S transition [1, 2, 16, 33].
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E2F transcription factors serve as targets for the Rb protein‘s growth-inhibitory action
[22, 23, 34]. It has become clear that E2F1 is a member of sequence-specific DNA-binding
transcription factors family and pRb composes a complex with several members of this
family, especially with transcriptional activators E2F1-3 and transcriptional repressors E2F45 [35, 39]. pRb interacts with transcriptional activators E2F1-3 and contributes to active gene
silencing through restraining the binding of transcriptional co-activators, and through
recruitment of histone deacetylases (HDAC), ATPases, and DNA methyltransferases to the
promoters of target genes (Table 1) [40, 41].
E2F transcription factors have reported to regulate multiple genes, which are responsible
for cell cycle regulation, nucleotide synthesis and DNA replication. DNA polymerase-I and
thymidine kinase, thymidylate synthase and dihyrofolate reductase, apoptosis regulators;
caspases and apaf-1 and several proto-oncogenes are also shown to be regulated by these
transcription factors. [42-47].
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5.2. RB Phosphorylation and CDK’s/CDKI’s
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Phosphorylation and cyclin-dependent kinase (CDK) activation regulate pRb-E2F
interaction [48-50]. CDK enzymes consist of two components, which are catalytic CDK
subunit and regulatory cyclin subunit.(kitap referans verilmeli ve başka yerlerde)
Phosphorylation of pRb was shown to be synchronized and it is hypophosphorylated during
the cell cycle‘s G1 phase. Unphosphorylated form of pRb, the active form, can directly bind
to and inhibit activity of E2F activators. During the G1/S transition and G2/M phases of cell
cycle, pRb is phosphorylated. By the end of mitosis, pRb is dephosphorylated and returns its
active form (Figure 2) [16, 43, 45, 49, 51, 52].
When pRb is hyperphosphorylated by CDKs induced by growth stimulation, E2F
accumulates and thus causes S-phase entry [53-56]. After phosphorylation of pRb, E2F gets
free from it and elevates the expression of genes that are necessary for cell division [43].
Cyclin D and cdk4 functions are induced by cyclin E and cdk2 and promote inactivation of
Rb. Cyclin D and cdk4 have been shown to phosphorylate pRb in early G1, and whereas
cyclin E and cdk2 phosphorylates pRb in the late G1. Cyclin E and cdk2 are also regulated by
E2F, having primary role for activation of replication [40, 54, 56]. Consequently, pRb/E2F
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pathway controls both activation of DNA replication and the regulation of the G1-S
transition. All these data proposed pRb as a general cell cycle regulator (Figure 2).
Regulation of pRb by phosphorylation is a convoluted process in which, multiple CDKs
are involved. They phosphorylate pRb through 16 potential phosphorylation sites on pRb
[57].
CDK activity is regulated primarily by two parameters: the level of proper cyclins and
cyclin-dependent kinase inhibitors (CDKI‘s) [58]. Two families of CDK inhibitors are used to
control the G1–S transition and antagonize pRb phosphorylation. The first family consists of
INK4 proteins (p15, p16, p18, p19), which specifically inhibit kinases activated by D-type
cyclins and the second one is Cip/Kip (p21, p27, p57) family, which inhibit D-, E-, and Atype cyclin–CDK complexes [4, 59-63].
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Figure 2. Regulation of pRb in coordination with the cell cycle. Regulation of E2F-dependent
promoters by Rb.
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Table 1. E2F transcription factors have reported to regulate multiple genes, which are
responsible for cell cycle regulation, nucleotide synthesis and DNA replication [33]
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Cell cycle
Regulation
Cyclin A
Cyclin E
CDK1
E2F1
E2F2
E2f3
Skp2
HP1
DNA replication and
Nucleotide synthesis
DNA polymerase 1
Thymidylate synthase
Thymidine kinase
Dihyrofolate reductase
Apoptosis
Proto- oncogenes
Caspase- 3
Caspase- 7
Caspase- 8
Apaf1
b- myb
c-Myb
c- Myc
Mutations or decreased expression of CDK inhibitors in many types of tumors has been
attributed to genetic and epigenetic changes. Mutations of p16INK4a cyclin kinase inhibitor
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are the second most frequent mutations after RB1 in human cancers, disturbing the regulation
of the pRb/E2F pathway. The p16 protein controls the Cyclin D/cdk4 kinase activity. The
absence of p16 activity is functionally equivalent to loss of RB1, since in absence of p16
Cyclin D/cdk4 kinase activity increases and leads to pRb phosphorylation and subsequent
E2F accumulation. Furthermore, loss of p16 function is relevant in a variety of sporadic
cancers, but inherited p16INK4a mutation is observed in melanoma [64].
5.3. RB1 and HDAC1
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pRb binds to E2F transcription activators and repress expression of their target genes
through preventing the binding of transcriptional co-activators, and then leading to
recruitment of HDACs (Figure 2). pRb should have intact pocket domain in order to directly
interact with HDAC1. This interaction has shown to be decreased as a result of RB1
mutations [40, 41, 65].
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5.4. RB1 and Skp2
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Independent from E2F, pRb protein can also modulate cell cycle through other
mechanisms. One of these mechanisms involves interaction of pRb with Skp2 that prevents
degradation of the CDK inhibitor p27 and subsequently results in p27 accumulation.
Inhibition of cyclin E-CDK activity by p27 is essential for normal progression of cell cycle.
Besides, phosphorylation of pRb by the cyclin E-CDK complex inhibits activity of pRb itself
and its downstream effectors, E2F transcription factors. Existence of mitotic signals results in
binding of Skp2 to p27, which causes its subsequent degradation.
When p27 is degraded, the inhibition of cyclin E-CDK activity decreases and
phosphorylation of pRb is allowed. This results in inhibition of pRb and promotes G1 to S
phase progression. In the absence of growth-promoting signals, pRb binds to Skp2, and
prevents it from binding to p27. In this case, free p27 inhibits the cyclin E-CDK activity and
results in G1 cell cycle arrest [66, 67].
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5.5. RB1 and Viral oncoproteins
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The pocket proteins can bind DNA tumor viral oncoproteins in addition to endogenous
nuclear proteins, which induce cell proliferation. These oncoproteins, such as adenovirus
E1A, SV40 large T antigen and human papillomavirus E6 and E7 proteins, interact with pRb
and prevents pRb/E2F binding and as a consequence cause activation of genes that are
transcriptionally regulated by E2F [68].
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6. Other Roles for RB1
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6.1. RB1 and Apoptosis
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6.2. RB1 and Differentiation
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In addition to controlling cell proliferation and progression, RB1/E2F pathway is also
involved in the control of apoptosis induction by regulating the function of E2F1 [69]. When
E2F1 expression is elevated, it can activate apoptosis in certain cell types, and in the absence
of RB1, E2F-1 inappropriately induces apoptotic signals. E2F1‘s function of regulating
apoptosis exists in multiple ways. First mechanism is controlling the accumulation of p53
through upregulation of ARF activity. The ARF protein is able to bind the Mdm2, which
functions as a p53 ubiquitin ligase and destroys p53. Thus, loss of Rb function results in E2F1
activation that induce the ARF/Mdm2/p53 pathway, causing cell cycle exit and apoptosis [7075]. Second mechanism involves the p53 homologue, p73. E2F1 stimulates p73 transcription
that causes p53-independent apoptosis [76, 77]. Additionally, E2F1 triggers apoptosis by
regulation of genes, which are important for apoptosis, such as apoptosis protease activating
factor-1 gene (Apaf1) and pro-caspases [78, 79].
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pRb has a role in the differentiation process of muscle cells, which is controlled by a set
of four myogenic transcription factors: MyoD, myogenin, MRF4 and Myf-5. All of MyoD,
myogenin, MRF4 and Myf-5 have similar functions, but their expression are detected at
different stages of skeletal muscle development. MyoD and Myf-5 are expressed in the early
stages of embryogenesis and are associated with muscle lineages. MRF4 acts in later stages
and it is responsible for myotube maturation. pRb affects this process in early stage by
regulating transcription factor MyoD in two ways. Firstly, pRb induces MyoD to interact with
the coactivator myocyte enhancer factor 2 (MEF2). Secondly, pRb blocks deacetylation of
MyoD by directly binding histone deacetylase 1 (HDAC1), thus pRb prevents MyoDHDAC1 interaction and allows expression of MyoD transcriptional targets. p21 is a probable
MyoD target and its expression is promoted by MyoD. It is included in differentiation
program to cell cycle arrest by inhibiting CDK-mediated pRb phosphorylation [33, 80-83].
7. RB1 and Other Cancers
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The RB1/E2F pathway has critical role in controlling cell growth and pRb affects some
other pathways controlling replication, apoptosis as has been mentioned before. Thus, it is
clearly known that the normal function of the pathway is destroyed by oncogenic mutations.
In addition to retinoblastoma, RB1 gene mutations results in loss of Rb function and occurs in
various types of human cancers, which are called as second primary malignancies including
osteosarcomas, sarcomas, small cell lung carcinomas, breast, bladder, and prostate
carcinomas, myelomas, and leukemias [84-87].
294
8. Therapeutic Applications
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RB1 pathway‘s damages are common feature of tumorigenesis, so restoration of Rb
function initially seemed to be a useful way for correcting the disorders. Here are some
challenges waiting to be solved. First, for not to let even rare cells with unrestored Rb
function since any such cell can potentially give rise to tumors, a highly efficient dose of
targeted drug delivery should be aimed. Second, as it is known that multiple mutations are
associated with RB1 gene-related cancers, therefore, restoration of just one mutated function
may not result in a fully functional restoration of the normal cell cycle [88, 89].
A novel therapeutic agent, Nutlin-3, is a Mdm2 inhibitor, which prevents association of
Mdm2 with p53 [90]. Nutlin-3 treatment is currently in phase I clinical trial, and has
demonstrated to restore the p53 pathway in RB cells, and thus induce p53-mediated
apoptosis. Topotecan, another therapeutic agent, can inhibit topoisomerase-I and trigger DNA
double-strand breaks, which promote apoptosis, through p53-dependent and -independent
pathways. When p53 inducer Topotecan and Mdm2 inhibitor Nutlin-3 are used together, they
function synergistically and 82-fold reduction in tumor burden was observed in mouse
models after subconjunctival injection [33]. Unlike to systemic chemotherapeutic agents, no
side effects were noted in animal models following Nutlin-3 and Topotecan combination
therapy [91].
The HDAC inhibitors (HDACi) are another class of targeted therapies and currently they
are in phase I clinical trial for treatment of RB. HDACi therapy has been found to be effective
in cells with increased E2F1 activity by inducing expression of apoptotic factors [92]. Due to
the loss of pRb, E2F1 activity is known to elevate, thus RB-derived cell lines are sensitive to
HDACi therapy and go apoptosis.
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Conclusion
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Since the discovery of RB1, remarkable progress has been made in the understanding of
RB1 function. Although the critical roles of pRb in regulating cell proliferation,
differentiation, genome integrity and apoptosis have been clearly identified, there are several
outstanding questions, where it acts in processes such as differentiation. As the pRb and E2F
deregulation leads to multiple cancer development, it is possible that the pRb can have
alternative tumor-promoting activities. There should be additional research projects to
understand all mechanisms and to develop effective and applicable targeted treatment
approaches.
[1]
[2]
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ISBN: 978-1-62808-665-2
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Chapter 15
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Endoplasmic Reticulum
Protein 29 (ERp29) and Cancer:
Molecular Functions, Mechanisms
and Clinical Implication
Daohai Zhang
Discipline of Pathology, School of Medical Sciences,
University of Sydney, Sydney, Australia
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Abstract
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The Endoplasmic reticulum (ER) is the primary subcellular organelle of synthesis
and folding of secretory and membrane bound proteins.
Disturbance of ER homeostasis results in accumulation of unfolded and/or misfolded
proteins, leading to the ER stress that has been associated with disease progression
including cancer.
In the ER system, ERp29 belongs to the non-classical molecular chaperones and
lacks its redox-active function due to the absence of an active motif consisting of double
cysteines. While ERp29 plays a critical role in protein secretion as an escort carrier,
accumulating evidence has demonstrated a potential role of ERp29 in cancer cell survival
and carcinogenesis.
However, its molecular function and pathological role in cancer cells have not been
fully investigated. Recent studies demonstrated that ERp29 is an emerging tumor
suppressive molecule by inducing cell growth arrest in breast cancer.
This review will provide an overview of current understanding of ERp29 in
modulating cell cycle arrest, resistance against genotoxic stress, epithelial-mesenthymal
reverse transition and epithelial cell morphogenesis in breast cancer cells. The clinical
importance of ERp29 in disease recurrence, as well as being a potential therapeutic target
for preventing metastasis, is also discussed.

Email: [email protected].
302
Daohai Zhang
Introduction
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The endoplasmic reticulum (ER), an intracellular organelle of all eukaryotic cells, is
complex membrane system constituted of an extensively interlinked network of membranous
tubules, sacs and cisternae. It is the main subcellular organelle that transports different
molecules to their subcellular destinations or to the cell surface [1, 2]. The ER membrane
spans from the nuclear envelope to the secretory vesicles and this structure is closely related
to its multifacet cellular functions such as synthesis and sorting of secretory and membrane
proteins, biosynthesis of lipids, degradation of glycogen, detoxification reactions and
maintenance of intracellular calcium homeostasis and storage [2, 3]. In the ER system, protein
folding is executed by a complex process that involves folding, assembly, modification,
quality control, and recycling. Correctly folded and assembled proteins are packaged into
membrane carriers and are transported from the ER to various cellular or extracellular
destinations through the Golgi complex. These carriers are formed by the activity of cytosolic
protein complexes involved COP II (coat complex II) machinery [4]. However, under certain
conditions that affect proper protein folding, the inappropriately folded or assembled proteins
are exported to proteasome and are subsequently degraded in the cytosol, a process known as
ER-associated degradation [5]. When the misfolded or unfolded proteins are unable to be
degraded and thereof accumulated in the ER, the unfolded protein response (UPR) [6, 7] is
activated to relieve the ER stress. In response to UPR, X-box-binding protein 1 (XBP-1), a
gene of ER stress sensor, is alternatively spliced by the activated endonuclease domain of
inositol-requiring enzyme 1 (IRE1) [8]. Under these conditions, ER chaperones, including
endoplasmic reticulum protein 29 (ERp29)/protein disulfide isomerase (PDI)-like
proteins/glucose-response protein 78 (GRP78 or BiP), are up-regulated to bind to denatured
or aggregated cellular proteins thereby alleviating their refolding, which facilitates cell
survival and attenuates apoptotic stimuli.
The ER contains a number of molecular chaperones physiologically involved in protein
synthesis and maturation. Of the ER chaperones, PDI-like proteins are characterized by the
presence of a thioredoxin domain and function as oxido-reductases, isomerases and
chaperones [9]. Oxido-reductase activity is present in the chaperones with an active-site
double-cysteine (CxxC) motif, such as PDI, ERp72 and ERp57. The CxxC motif allows the
protein to cycle between a reduced and an oxidized state [10]. As such, PDI-related proteins
have various functions, including noncovalent chaperone activity and disulfide bond
formation and reduction [9, 11]. Furthermore, redox-inactive PDI-like proteins including
ERp29, ERp27 and thioredoxin-related transmembrane protein 2 (TMX2) do not have the
CxxC motif [12]. ERp29 is recognized as a characterized resident of the cellular ER, and it is
expressed ubiquitously and abundantly in mammalian tissues [13]. ERp29 was proposed to be
involved in the UPR as a factor facilitating transport of newly synthesized secretory proteins
[14]. The increased expression of ERp29 was demonstrated in certain cell types both under
the pharmacologically induced UPR and under the physiological conditions (e.g., lactation,
differentiation of thyroid cells) [15, 16]. In most cases, ERp29 interacts with BiP/GRP78 to
exert its function under ER stress [17].
Recently, it was reported that ERp29 was up-regulated under conditions that
homocysteine or dopamine invokes ER stress [18, 19], or when cells were exposed to
radiation [20]. Furthermore, ERp29 could potentiate resistance to doxorubicin (DOX) and
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Endoplasmic Reticulum Protein 29 (ERp29) and Cancer
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radiation by up-regulating Hsp27 in cancer cells through down-regulating the expression of
eIF2α [21, 22]. Hence, ERp29 is associated with resistance to oxidative and radiation stress
and may play a potential protective role against stress. In cancer cells, the function of ERp29
has been actively addressed [23, 24], and several important roles have been reported. Recent
studies have demonstrated significant involvements of ERp29 in: 1) cancer cell survival
against genotoxic stress induced by DOX and radiation [21, 22, 25]; 2) cell growth arrest
through modulating ER stress [26]; 3) mesenchymal-epithelial transition (MET), cell polarity
and epithelial morphogenesis [24, 27]. The current review will focus on the functions of
ERp29 as a novel tumor suppressive molecule and the key signaling pathways affected by
ERp29 in cancer cells.
ERp29: Structure and Biochemical Function
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ERp29 localizes in the luminal compartment of the ER and contains a PDI-like structure
that consists of an N-terminal domain homologous to the thioredoxin-like domains in PDI,
and a C-terminal domain with similarity to the P5 subfamily of PDI [12, 13, 28]. Despite the
structural resemblance to PDI as manifested by the thioredoxin-like N-terminal domain,
ERp29 has no characteristic disulfide isomerase or chaperone activity [12], but exhibits
chaperone-like properties at both the biophysical and cellular levels [29, 30]. There is a single
cysteine residue at the N-terminal domain (Cys157) indicating that ERp29 may present in
either the reduced state or a mixed-disulfide bonded states. These two states may enable
ERp29 to bind to substrates with different affinities. It was found that ERp29 can form dimers
or higher oligomers with PDI [17]. In addition, ERp29 could change its conformation through
noncovalent interactions with other ER factors to drive the substrate binding and release cycle
[31]. Its N-terminal domain is critical for dimerization and this serves as a general mechanism
to regulate its ER activities [32]. Moreover, both ERp29 N-terminal and C-terminal domains
are essential for inducing the local unfolding of polyomavirus to initiate the ER membrane
penetration process [32-34].
In addition to the ER chaperone functionality, ERp29 can act to escort secretory proteins
through post-ER compartments [12, 14, 35]. The existences of ERp29 in conditioned cell
media [14] and in milk [36] are consistent with an escort role from the ER to the cell surface.
It was found that, in rat spermatozoa, ERp29 is increased significantly on the sperm surface
as well as in the cytoplasm of epididymal epithelia from caput to cauda during sperm
epididymal maturation [37]. The present of ERp29 on mouse sperm membrane and upregulation as the sperm undergoes epididymal maturation may implicate other potential
function in mammalian fertilization and sperm-oocyte fusion [38]. Generally, the ubiquitous
expression of ERp29 in the secretory cells [14] and involvement in the secretion of
thyroglobulin and other secretory proteins support ERp29‘s function as a secretion
factor/escort chaperone [14]. In fact, ERp29, as a bona fide molecular chaperone or cochaperone [30, 35], binds to thyroglobulin and mediates its secretion [39].
ER29 also plays a critical role in secretion and transport of membrane proteins. This is
supported by a recent finding that ERp29 facilitates the appropriate processing and assembly
of connexin43 hemichannels [35]. ERp29 regulates folding of newly synthesized integral
membrane proteins like cystic fibrosis transmembrane conductance regulator CFTR [40],
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indicting a key role of ERp29 in the regulation of CFTR biogenesis. Interestingly, the
classical ER chaperones BiP/GRP78 and endoplasmin/GRP94 do not interact with CFTR
stably [41-43], and neither BiP nor calnexin has central roles in ER-associated degradation of
CFTR [44-46]. Instead, ERp29 showed a robust interaction with ΔF508-CFTR in the absence
of cross-linking. Hence, ERp29 could favor hydrophobic substrates such as integral
membrane proteins [47].
Recent evidence also implicated the importance of ERp29 for polyomavirus infection
through its C-terminal domain [34]. During this process, ERp29 can alter the conformation of
polyomavirus' coat protein VP1 and internal protein VP2 through disruption of Py's disulfide
bonds. Cooperation of ERp57 and PDI with ERp29 facilitates unfolding of the VP1 Cterminal arm [48]. This stimulates polyomavirus to bind to the surface of the ER membrane
and then perforates ER membrane [31, 33]. Induction of a conformational change in the
polyomavirus VP1 protein in turn facilitates passage of the virus across the ER membrane and
successful infection [31]. This biophysical characterization suggests that ERp29 is
functionally distinct from the classical ER chaperones [29, 49].
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ERp29, ER Stress and Cell Growth Arrest
1. ER Stress-Related PERK/p-eIF2α Pro-Apoptotic Pathway
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The ER is a major signal transducing organelle that senses and responds to changes in
cellular homeostasis. ER stress can be induced by UPR or by viral infection. The first
response of ER stress involves up-regulation of genes encoding ER chaperones, which
increase protein-folding/assembly activity and prevent protein aggregation in the ER. These
ER resident molecular chaperones include chaperones of the heat shock protein family,
including BiP/GRP78 and its co-chaperone partners, chaperone lectins and the foldase family
of PDI and ERp29.
Under normal conditions, Bip binds to stress transducers, such as double-stranded RNA
(PKR)-like ER kinase (PERK) [50], activating transcription factor 6 (ATF6) [51], and IRE1
[52], to form complexes. However, at abnormal conditions, Bip dissociates from these
transducers and binds to ER luminal un/misfolded proteins and activates the ER stress
response [53] which mediates multiple molecular biological processes via ER stress sensors
[54, 55] (Figure 1).
Phosphorylation of eukaryotic translation-initiation factor 2α (eIF2α) by PERK is a
characteristic of PERK pathway activation. To reduce the risk of further accumulation of
misfolded proteins in the ER, this pathway is activated to block the initiation of protein
translation and attenuate global protein synthesis (Figure 1). Moreover, to overcome the ER
stress, a subset of mRNAs is able to bypass translational blockage and synthesize proteins
that participate in the UPR/ER stress response. One of the critical downstream targets is
activating transcription factor 4 (ATF4) which is upregulated and subsequently induces
transcription and expression of a set of genes to cope with ER stress [50]. For instance, if
misfolded protein levels are consistantly elevated, ATF4 stimulates transcription of CHOP
gene (C/EBP homologous protein, also called GADD153) to support the ER stress-induced
apoptotic program [56] (Figure 1).
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Figure 1. Depiction of ER stress and the regulatory role of ERp29 in ER stress signaling. Under ER
stress, the ER-associated transmembrane sensors PERK, IRE-1α and ATF6 are activated by
homodimerization and autophosphorylation (for PERK and IRE1α) and proteolytic cleavage at the
Golgi (for ATF6). Activation of PERK leads to phosphorylation of eIF2α, resulting in termination of
global translation and selectively translation of ER stress-associated proteins, such as ATF4. Prolonged
ER stress consistently activates p-PERK/p-eIF2α pathway to induce cell apoptosis. The CHOPregulated GADD34 inhibits eIF2α phosphorylation via PP1 to restore translational function of eIF2α
and ER homeostatsis. Active ATF6 translocates into nucleus to promote transcription of XBP-1 and ER
chaperones. XBP-1 mRNA is spliced by activated IRE-1 to generate a shorter spliced variant (XBP-1s)
which encodes an active XBP-1s transcription factor. p58IPK is one of the targets regulated by XBP-1s,
and prevents eIF2α phosphorylation by binding to PERK. ERp29 enhances XBP-1s/p58IPK to
antagonize cell apoptosis regulated by p-PERK/p-eIF2α pathway.
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On the other hand, CHOP also stimulates the expression of growth arrest and DNA
damage inducible protein 34 (GADD34), a feedback inhibitor on eIF2α phosphorylation by
protein phosphatase type 1 (PP1) [57]. This feedback regulation on translation ensures
general translational function of eIF2α and ER homeostasis.
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2. ER Stress-Related XBP-1 Cell Survival Signaling
IRE1 is a bifunctional molecule with serine/threonine protein kinase and
endoribonuclease (RNase) activity in its cytosolic domain [58]. Dissociation from GRP78
triggers its dimerization and autophosphorylation to activate its downstream process [55].
Activation of IRE1 involves its oligomerization and trans-autophosphorylation of the
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kinase domains. Activated IRE1 removes an intron from unspliced XBP-1 (XBP-1u)
mRNA to generate spliced and activated XBP-1 (XBP1s) [8] (Figure 1). XBP-1s is a
highly active transcription factor and is one of the key regulators of ER folding capacity
[59, 60]. XBP-1s activates genes that enhance ER protein-folding capacity and degradation
of misfolded ER proteins [61] and phospholipid synthesis for the expansion of ER
membranes under ER stress [52, 59].
Recent studies showed that prolongation of IRE1 signaling during ER stress can
promote cell survival [62, 63]. It was also shown that heat shock protein 72 (HSP72)
physically interacts with the cytoplasmic part of IRE1 to protect cells from ER stressinduced apoptosis by prolonging XBP-1 splicing [64]. Thus, IRE1-activated XBP-1s
serves as an important adaptive mechanism under stress. Interestingly, the unspliced form
of Xbp-1u mRNA encodes a rapid-turnover protein that can function as a dominant
negative factor to inhibit XBP-1s activities [65, 66] and causes increased apoptosis of
tumor cells [67]. In contrast, high expression level of XBP-1s was shown to associate with
increased tumor survival [67].
ATF6 is a transcription factor with basic leucine zipper (bZIP) motif and localizes on ER
membrane. GRP78 binds to ATF6 and prevents its translocation to the Golgi apparatus.
Under ER stress, ATF6 dissociates from GRP78 and is transported to the Golgi apparatus
where it is cleaved by Golgi-resident site-1 and site-2 proteases (S1P and S2P) (Figure 1). In
general, two isoforms of ATF6, ATF6α and ATF6β, are activated during ER stress by the
same mechanism [68].
Upon migrates to the nucleus, activated ATF6α stimulates transcription of unspliced
XBP-1 gene [55] and the genes that participate in ER-associated degradation [69] (Figure 1).
XBP-1s is essential for the regulation of several UPR target genes including p58IPK [61]. Of
interest, p58IPK can directly bind to PERK to inhibit PERK phosphorylation, resulting in
cope with ER stress.
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inhibition of phosphorylation of eIF2α [70, 71]. As such, ATF6 activation helps the cell to
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3. ERp29 Inhibits eIF2α /CyclinD1/2 to Induce G0/G1 Arrest via Activating
p38 Phosphorylation
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Tumor cell growth arrest and survival have also been mechanistically linked to ER stress
signaling [72]. In particular, PERK autophosphorylation phosphorylates eIF2α at Ser-51 upon
stress and consequently attenuates global protein translation and induces G0/G1 arrest [73].
The role of p38 activation in inducing cell growth arrest by regulating the PERK/p-eIF2α
pathway has been established in human squamous carcinoma cells [72, 74]. The mechanistic
link between ER stress and tumor cell growth arrest through p38 activation suggests an
important role of p38-regulated networks in modulating tumor cell quiescence, survival and
apoptosis [72, 74, 75]. Given that ERp29 induces G0/G1 arrest in breast cancer cells [24, 26],
activation of p38 may play a critical role in this aspect. Indeed, studies from the gene
expression microarray revealed a significant reduction of urokinase plasminogen activator
receptor (uPAR), β1-integrin and epidermal growth factor receptor (EGFR) [24], an important
upstream regulatory complex modulating extracellular signal-regulated kinase (ERK) and p38
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activity [76] (Figure 2). Activation of ERK facilitates cell proliferation and tumorigenesis,
whereas activation of p38 promotes G0/G1 arrest [76].
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Figure 2. ERp29 induces cancer cell growth arrest via p38 activation. Over-expression of ERp29 in
MDA-MB-231 cells decreases expression of the uPAR–β1-integrin–EGFR ternary complex, leading to
activation of p38 and suppression of phosphorylated extracellular signal-regulated kinase (p-ERK).
Phosphorylation of p38 inhibits the expression of basal eIF2α and cyclin D2 and increases the
expression of cyclin-dependent kinase inhibitors (p15, p16 and p21), thus causing the G 0/G1 arrest.
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When ERp29 was over-expressed in MDA-MB-231 cells, the basal level of p38 was
reduced, while its phosphorylation was remarkably enhanced [77]. Meanwhile, ERK
activation was concomitantly inhibited by ERp29 in these cells [24] (Figure 2). Interestingly,
the enhanced p38 phosphorylation by ERp29 in MDA-MB-231 cells inhibited the expression
of basal eIF2α, while the relative phosphorylation of eIF2α was not significantly changed.
This may suggest a novel regulatory mechanism of ERp29 in modulating eIF2α activity and
protein translation.
In general, inactivation of eIF2α by phosphorylation induces G0/G1 arrest and cell
survival by blocking cyclin D1/D2 translation/stability [73]. The ERp29-reduced expression of
basal eIF2α causes repression of cyclin D1/D2 expression, in particular of cyclin D2 level [77].
Knockdown of ERp29 by shRNA in MCF-7 cells resulted in up-regulation of cyclin D2, with
no significant effect on cyclin D1 expression. These results further suggest ERp29 induces
G0/G1 arrest, to a greater extent, via suppressing cyclin D2 expression. On the other hand,
ERp29 expression increases the levels of cyclin-dependent kinase inhibitors (e.g., p15, p21
and p27) at both mRNA and protein levels [24, 77], thereby leading to G0/G1 arrest (Figure
2). The role of p38 activation in regulating cyclin D2 and p15 was experimentally verified by
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4. ERp29 Activates XBP-1s/P58IPK Cell Survival Pathway
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gene knockdown or pharmacological inhibition of p38 activity [77]. Therefore, increased
expression of ERp29 can induce growth arrest through modulating p38 phosphorylation and
eIF2α expression.
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XBP-1s is the transcription factor that controls p58IPK expression during the UPR [61].
The protein p58IPK can directly bind to PERK to inhibit PERK phosphorylation, resulting in
inhibition of eIF2α phosphorylation [70]. Consequently, up-regulation of p58IPK upon ER
stress may relieve eIF2α phosphorylation and restore general protein translation.
ERp29 over-expression in MDA-MB-231 cells led to activation of XBP-1 by stimulating
splicing [78] and a significant up-regulation of p58IPK [77]. Activation of XBP-1/p58IPK by
ERp29 may play a key role in suppressing eIF2α phosphorylation induced by p38phosphorylation (Figure 1). Indeed, in our cell models, silencing of p58IPK stimulated eIF2α
phosphorylation and activated expression of ATF4/CHOP and cleavage of caspase-3 [26].
Enhanced phosphorylation of eIF2α is a mechanism leading to attenuation of general protein
synthesis and activation of ATF4/CHOP under ER stress [79]. Hence, up-regulation of p58IPK
may facilitate cell survival under stress by repressing eIF2α phosphorylation (Figure 1). The
interplay between p38 phosphorylation and p58IPK up-regulation has key roles in modulating
ERp29-induced cell growth arrest and survival. This new knowledge could lead to novel and
effective therapies against drug-resistant cancer cells, e.g., by targeting p58IPK.
ERp29 and Mesenchymal-Epithelial Transition
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1. Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial
Transition (MET)
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The epithelial-mesenchymal transition (EMT) is an essential process during
embryogenesis [80] and its pathological activation during tumor development increases
primary tumor cells to metastasize [81, 82]. The pathological conditions such as
inflammation, organ fibrosis and cancer progression facilitate EMT [83]. The EMT is
characterized as: (1) loss of their junctions, for example, adherens junctions (AJs) and tight
junctions (TJs); (2) loss of apical–basal polarity; (3) reorganization of their cytoskeletal
components and distribution; and (4) gain of migration and invasion abilities [84]. The
involvement of EMT in cancer progression has been considered to be an important process
for the tumor cells to ‗escape‘ from the primary site and to metastasize [85].
E-cadherin is considered to be a key molecule that provides the physical structure for
both cell–cell attachment and the recruitment of signaling complexes [86]. Loss of E-cadherin
is a hallmark of EMT and the EMT-inducing factors can impair the expression or function of
E-cadherin to initiate epithelial reorganization [87]. Therefore, characterizing transcriptional
regulators of E-cadherin expression during EMT has provided important insights into the
molecular mechanisms underlying the loss of cell–cell adhesion and the acquisition of
migratory properties during carcinoma progression [88].
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Clinically, loss of E-cadherin is widely observed in invasive and metastatic carcinoma
and correlates with the aggressiveness of tumors and poor survival rates in cancer patients
[89]. This observation is consistent with that the enhanced expression of the transcriptional
repressors of E-cadherin is associated with tumor progression and lymph node metastasis
[90].
While the epithelial cancer cells can be transited to mesenchymal-like cells to initiate
metastasis, mesenchymal cells can also regain a fully differentiated epithelial phenotype via
the mesenchymal–epithelial transition (MET) [80, 91]. The MET may also have a role in
carcinogenesis by mediating the establishment of distant metastatic tumors at secondary sites
[92]. Indeed, recent studies demonstrated that distant metastases in breast cancer expressed an
equal or stronger E-cadherin signal than the respective primary tumors and the re-expression
of E-cadherin was independent of the E-cadherin status of the primary tumors [93]. Similar
findings were also reported in which E-cadherin is re-expressed in bone metastasis or distant
metastatic tumors arising from E-cadherin-negative poorly differentiated primary breast
carcinoma [94], or from E-cadherin-low primary tumors [95]. The gain of E-cadherin
expression during metastasis has been well studied in MDA-MB-468 breast cancer cell line
[96]. Analysis of MDA-MB-468 xenografts revealed that some tumor cells exist a metastable
phenotype [97, 98], characterized by the expression of both vimentin and E-cadherin [96],
The cells at the invasive front showed a positive expression for vimentin and negative
expression for E-cadherin, consistent with an EMT. On the other hand, the lymphovascularinvaded tumor cells showed a gradual transition of invaded tumor cells from mesenchymal to
metastable and then to the epithelial phenotype, indicating that a MET process occurs as an
early event in the metastatic process [97, 98]. The interaction of nonmetastatic mesenchymallike cells and metastatic epithelial-like cells accelerates their metastatic colonization in
prostate and bladder cancer cells [99]. Therefore, the EMT and MET process may co-exist
and work co-operatively in driving metastasis.
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2. Molecular Regulation of EMT
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Regulation of E-cadherin expression is mediated via transcriptional repressors including
zinc-finger proteins (e.g., Snai1 and Slug), zinc-finger E-box-binding proteins (e.g., ZEB1/2),
the basic helix-loop-helix proteins (e.g., Twist) and Ets-1 [100-102] (Figure 3). Importantly,
knockdown of these repressors induces the re-expression of E-cadherin [103] and restores the
epithelial phenotype [104]. In addition, several known signaling pathways, such as those
involving transforming growth factor-β (TGF-β), Notch, fibroblast growth factor and Wnt
signaling pathways, have been shown to trigger epithelial dedifferentiation and EMT [105,
106]. These signals exert their action through modulating transcription factors that repress
transcription of epithelial genes, such as those encoding E-cadherin and cytokeratins, and that
activate transcription programs that facilitate fibroblast-like motility and an invasive
phenotype [88, 106].
Recently, the involvement of microRNAs (miRNAs) in controlling EMT has been
emphasized [107-109]. miRNAs are small non-coding RNAs (~23 nt) that silence gene
expression by pairing to the 3′UTR of target mRNAs to cause their posttranscriptional
repression [110]. To date, the miR-200 family has been shown to be major regulators of EMT
through silencing the EMT-transcriptional inducers ZEB1 and ZEB2, which in turn repress
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miR-200f in a double-negative feedback loop [111, 112]. MiR-200f opposes EMT by directly
targeting genes involved in motility and invasion [113, 114]. Therefore, expression of miR200f in normal and cancer cells promotes the maintenance of an epithelial phenotype [115].
miR-200 and miR-205 RNAs are negative regulators of EMT and their expression is
decreased in cells that have undergone a full EMT under different stimuli [116, 117].
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Figure 3. ERp29 regulates mesenchymal-epithelial transition. Exogenous expression of ERp29 in
mesenchymal MDA-MB-231 breast cancer cells suppresses transcription and protein expression of Ecadherin transcription repressors (e.g., ZEB2, SNAI1 and Twist), resulting in re-expression of Ecadherin and re-establishment of epithelial cell phenotype. ERp29 over-expression also inhibits
expression of mesenchymal cell markers (e.g., vimetin, N-cadherin and fibronectin) and increases
expression of differentiation markers (e.g., cytokeratin 19) in this cell model.
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Recent studies have shown that miR-200f members including miR-200h, miR-200a, miR200c and miR-141 are epigenetically regulated in cancer and normal tissue [118, 119].
Indeed, epigenetic regulation is a critical mechanism affecting miR-200f expression to
modulate EMT-MET [120].
Recent studies also indicated a significant role of miRNAs as a prodisposed factors for
cancer cell metastasis. For instance, the elevated levels of the epithelial miR-200 family in
primary breast tumors associate with poorer outcomes and metastasis [121]. The enhanced Ecadherin re-expression by the miR-200 family members was driven via the repression of ZEB
family genes [122, 123]. These findings support a potential role of ‗epithelial‘ miRs in MET
to promote metastatic colonization [124].
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3. ERp29 Promotes MET in Breast Cancer
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The role of ERp29 in regulating MET has been established in breast cancer cells (Figure
3). When ERp29 was over-expressed in mesenchymal MDA-MB-231 cells, the spindle-like
fibroblastic morphology was remarkably changed to a cobble-stone-like phenotype, which is
identical to that observed in epithelial MCF-7 cells [24] (Figure 3). Accompanying the
phenotypic change, over-expression of ERp29 resulted in cytoskeletal reorganization with
loss of filamentous stress fibers and cortical actin formation. Reorganization of the actin
cytoskeleton is tightly linked to myosin-driven contraction initiated by MLC phosphorylation
[125, 126]. ERp29 expression markedly reduced the level of MLC phosphorylation,
suggesting its critical role in ERp29-regulated cortical actin formation and epithelial
phenotype [127]. The extracellular matrix (ECM) component fibronectin, which is involved
in cell transition from quiescence to proliferation [128], was concomitantly decreased by
ERp29 over-expression. Importantly, ERp29 expression reactivated both transcription and
protein expression of epithelial cell marker E-cadherin and regulated its membranous
localization. Meanwhile, the mesenchymal cell marker vimentin was highly reduced and the
epithelial differentiation marker cytokeratin 19 was increased [127] (Figure 3). The ERp29‘s
role in inducing MET was further substantiated in epithelial MCF-7 cells where knockdown
of ERp29 led to a fibroblast-like cellular phenotype, enhanced cell spreading, decreased
expression of E-cadherin and increased expression of vimentin [24, 127]. Given that Ecadherin expression inhibits the EMT process [129], reactivation of E-cadherin by ERp29
supports its role in modulating MET in breast cancer cells.
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4. ERp29 Targets E-Cadherin Transcription Repressor
The transcription repressors such as Snai1, Slug, ZEB1/2 and Twist [100-102] have been
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considered to be the main regulators for E-cadherin expression. Mechanistic studies revealed
that ERp29 expression significantly down-regulated transcription of these repressors, leading
to their reduced nuclear expression in MDA-MB-231 cells [24, 127] (Figure 3). Consistent
with this, the extracellular signal-regulated kinase (ERK) pathway which is an important upstream regulator of Slug and Ets1 was highly inhibited [24]. Apparently, ERp29 up-regulates
the expressions of E-cadherin transcription repressors through repressing ERK pathway.
Interestingly, ERp29 over-expression in basal-like BT549 cells resulted in incomplete MET
and did not significantly affect the mRNA or protein expression of Snai1, ZEB2 and Twist,
but increased the protein expression of Slug [127]. The differential regulation of these
transcriptional repressors of E-cadherin by ERp29 in these two cell-types may occur in a cellcontext-dependent manner.
5. ERp29 Antagonizes Wnt/ β-Catenin Signaling
Wnt proteins are a family of highly conserved secreted cysteine-rich glycoproteins. The
Wnt pathway is activated via a binding of a family member, such as Wnt1, Wnt3, Wnt3a,
Wnt7A, or Wnt10B, to a frizzled receptor (Fzd) and the LDL-Receptor-related protein co-
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receptor (LRP5/6). There are three different cascades that are activated by Wnt proteins:
namely canonical/β-catenin-dependent pathway and two non-canonical/β-catenin-independent
pathways that include Wnt/Ca2+ and planar cell polarity [130]. Of note, the Wnt/β-catenin
pathway has been extensively studied, due to its important role in cancer initiation and
progression [131]. β-catenin binds to E-cadherin as a part of adherens junctions (AJs) in the
absence of Wnt. The presence of Wnt promotes formation of a Wnt–Fzd–LRP complex,
recruitment of the cytoplasmic protein Disheveled (Dvl) to Fzd and the LRP phosphorylationdependent recruitment of Axin to the membrane, thereby leading to release of β-catenin from
membrane and accumulation in cytoplasm and nuclei.
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Figure 4. ERp29 over-expression ―turns-off‖ activated Wnt/β-catenin signaling. In mesenchymal MDAMB-231 cells, high expression of nuclear β-catenin activates its downstream signaling involved in cell
cycles and cancer stem cell self-renewal. When ERp29 is over-expressed in this cell model, nuclear βcatenin is relocated at the membrane where it binds to E-cadherin, and Wnt/β-catenin signaling is
switched off. Meanwhile, over-expression of ERp29 results in up-regulation of TCF3 and increases
expression of genes involved in differentiation. N: Nucleus.
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In the cytoplasm, the unbound β-catenin is constantly degraded by a complex composed
of the scaffold proteins Axin and adenomatous polyposis coli (APC), the casein kinase 1
(CK1) and glycogen synthase kinase 3 beta (GSK3β) [132]. β-catenin is sequentially
phosphorylated by CK1 and GSK3β, ubiquitinated by β-Trcp ubiquitin ligase and degraded
by proteasome [133]. However, mutations of APC and Axin, or β-catenin cause constitutive
Wnt activation [134]. When β-catenin is absent in nuclear, the transcription factors T-cell
factor/lymphoid enhancer factors (TCF/LEF) recruits co-repressors of the TLE/Groucho
family and function as transcriptional repressors. However, nuclear β-catenin replaces
TLE/Groucho co-repressors and recruits co-activators to activate expression of Wnt target
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genes. The most important genes regulated are those related to proliferation, such as Cyclin
D1 and c-Myc [135, 136] (Figure 4), which are over-expressed in most β-catenin-dependent
tumors.
Wnt pathway is required for driving the stem cell/progenitor compartment. This pathway
is altered together with other stem cell-regulating pathways such as Hedgehog and Notch
signaling, which supports the cancer stem cells (CSC) model [137]. In fact, nuclear
expression of β-catenin and/or mutation in this gene or in other genes of the pathway such as
Axin1 are frequently found in poorly and undifferentiated carcinomas, and the Wnt/β-catenin
pathway is necessary for the maintenance of CSCs. In breast cancer, the Wnt pathway is upregulated in CSCs by Wnt ligands secreted by the tumor microenviroment [138]. Thus,
repressing this pathway by increasing the stability of β-catenin-degrading complex [139] is an
alternative therapeutic strategy for the treatment of β-catenin-dependent tumors.
As a novel tumor suppressive molecule, ERp29 significantly decreased the expression of
cyclin D1/2 [77], one of the downstream targets of activated Wnt/ β-catenin signaling [136],
indicating an inhibitory effect of ERp29 on this pathway. Indeed, when ERp29 was overexpressed in mesenchymal MDA-MB-231 breast cancer cells, nuclear β-catenin was
translocated from nucleus to membrane where it forms complex with E-cadherin [27] (Figure
4). This causes a disruption of β-catenin/TCF/LEF complex and abolishes its transcription
activity.
Meanwhile, expression of ERp29 in this cell type increased the nuclear expression of
TCF3, a transcription factor regulating cancer cell differentiation while inhibiting selfrenewal of cancer stem cells [140, 141]. Hence, ERp29 may play dual functions in
mesenchymal MDA-MB-231 breast cancer cells by: 1) suppressing activated Wnt/β-catenin
signaling via β-catenin translocation; and 2) promoting cell differentiation via activating
TCF3 (Figure 4). Because β-catenin serves as a signaling hub for the Wnt pathway, it is
particularly important to focus on β-catenin as the target of choice in Wnt-driven cancers.
Though the mechanism by which ERp29 expression promotes the disassociation of βcatenin/TCF/LEF complex in MDA-MB-231 cells remains elusive, activating ERp29
expression may be a promising therapeutic intervention for the poorly differentiated and Wntdriven tumors.
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ERp29 and Epithelial Cell Integrity
1. Cell Adherens and Tight Junctions
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Adherens junctions (AJs) and tight junctions (TJs) are composed of transmembrane
proteins that adhere to similar proteins in the adjacent cell [142]. The transmembrane region
of the TJs is composed mainly of claudins, tetraspan proteins with two extracellular loops
[143]. AJs are mediated by Ca2+-dependent homophilic interactions of cadherins [144] which
interact with cytoplasmic catenins that link the cadherin/catenin complex to the actin
cytoskeleton [145].
The cytoplasmic domain of claudins in TJs interacts with occludin and several zona
occludens (ZO) proteins (ZO1-3) to form the plaque that associates with the cytoskeleton
[146]. The AJs form and maintain intercellular adhesion, whereas the TJs serve as a diffusion
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2. Apical–Basal Cell Polarity
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barrier for solutes and define the boundary between apical and basolateral membrane domains
[147]. The AJs and TJs are required for integrity of the epithelial phenotype, as well as for
epithelial cells to function as a tissue [86].
Especially, the TJs are closely linked to the proper polarization of cells for the
establishment of epithelial architecture [148]. During EMT, the expression of proteins that are
responsible for the formation of AJs, TJs and apical–basal polarity is affected, resulting in
loss of cell polarity in epithelial cells [149]. In fact, cancer development is frequently
associated with the failure of epithelial cells to form TJs and to establish correct apico–basal
polarity [150]. For instance, alterations of TJs protein expression and distribution cause the
loss of contact inhibition of cell growth [151]. In addition, reduction of ZO-1 and occludin
were found to be correlated with poorly defined differentiation, higher metastatic frequency
and lower survival rates [152, 153]. Hence, TJs proteins have a tumor suppressive function in
cancer formation and progression.
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Epithelial cells maintain two types of cell polarity, planar and apical–basal polarity [154].
The apical–basal polarity of epithelial cells in an epithelium is characterized by the presence
of two specialized plasma membrane domains: namely, the apical surface and basolateral
surface [154]. The asymmetrical distribution of lipids and proteins between both apical and
basal domains is caused by polarized trafficking and the presence of a physical frontier
established by the apical junctional complex. In this complex, TJs provide a tight seal while
AJs maintain the adhesion between neighboring cells [142, 155].
In general, apical–basal cell polarity of epithelial cells is determined by three core
interacting protein complexes that influence the assembly and localization of the junctional
complexes. These protein complexes include: (1) the partitioning-defective (PAR) complex;
(2) the Crumbs (CRB) complex; and (3) the Scribble complex [154, 156, 157]. In mammals,
PAR complex is composed of two scaffold proteins (PAR6 and PAR3) and an atypical
protein kinase C (aPKC), and is localized to the apical junction domain for the assembly of
TJs [158, 159].
The Crumbs complex is formed by the transmembrane protein Crumbs (Crb) and the
cytoplasmic scaffolding proteins such as the homologue of Drosophila Stardust (Pals1) and
Pals-associated tight junction protein (Patj) and localizes to the apical [160]. The Scribble
complex is comprised of three proteins, Scribble (Scrib), Disc large (Dlg) and Lethal giant
larvae (Lgl) and is localized in the basolateral domain of epithelial cells [161]. Decreased
expression or mis-localization of these core polarity proteins may have a causal link to
suppression of mammalian tumorigenesis [162-164].
Accumulating evidence supports that apical–basal cell polarity is established as the result
of mutually antagonistic interactions between the PAR, Crumbs and Scribble complexes,
thereby leading to the distribution of proteins in a polarized manner [165]. The PAR and
Crumbs complexes cooperate to establish the apical domain and the assembly of TJs, whereas
the Scribble complex has a key role in the definition of the basolateral plasma membrane
domain [148, 160]. The Par6/Par3/aPKC in PAR complex has a pivotal function in
polarization and this process is triggered by protein kinases such as Rac1/Cdc42 GTPases
[166, 167].
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Indeed, activated Cdc42 is recruited by Par6 to the PAR complex where it causes the
activation of aPKC and phosphorylation of Par3. Phosphorylated Par3, in turn, promotes the
formation of an active PAR complex at the apical domain and the assembly of the junctional
structure. At this stage, Crumbs complex is critical to stabilize active PAR complexes. On the
other hand, Lgl proteins of the Scribble complex compete with Par3 for binding to the PAR
complex, thus sequestering the active PAR complex away from the apical junction domain
[168].
Conversely, Lgl phosphorylation by aPKC inactivates the Scribble complex [156].
Therefore, the basolaterally located Scribble complex functions as an antagonist of the apical
localization of the active PAR complex.
3. Molecular Regulation of AJs, TJs and Cell Polarity
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Accumulating evidence indicates that EMT inducers concomitantly regulate the
expression of genes involved in the formation of AJs and TJs. Apart from E-cadherin, Snail
and ZEB factors down-regulated the components of the TJs, including occludin and several
members of the claudin family [169, 170]. Mechanistically, Snai1/Snai2 can directly bind to
the conserved E-box elements in the corresponding promoters of occludin, claudin-1 and
claudin-7 [170, 171]. Gene profiling studies showed that expression of Snai1, Snai2, E47 or
other EMT regulators promotes EMT through down-regulating claudin-4, the junctional
adhesion molecule-1 (JAM-1/JAM-A) and Dlg3 in carcinoma cells and in Madin-Darby
canine kidney (MDCK) cells [172, 173]
All these E-cadherin repressors are recognized as key inducers of EMT. Indeed,
expression of these EMT inducers results in genetic EMT programs including genes
regulating epithelial and mesenchymal phenotypes and genes involved in cytoskeletal
reorganization, cell movements and cell survival [88]. Recently, it has been reported that
EMT inducers can directly target members of the Crumbs and the Scribble complexes to
regulate their expression in different cell systems. For instance, ZEB1 binds at specific
proximal E-box sequences of CRB3 and PATJ gene promoter to repress their transcription as
demonstrated by promoter analysis and chromatin immunoprecipitation assays [174].
Similarly, ZEB1 silencing also targets polarity gene LGL2 promoter to upregulate its
expression in colorectal carcinoma cells [174].
By the similar mechanism, ZEB1 silencing leads to enhanced transcription of CRB3,
PATJ and the human homolog of lethal giant larvae 2 (HUGl2/LGL2), as well as genes for
TJs components (JAM-1; occludin, claudin-7) [174]. In breast and colorectal carcinoma cells,
silencing of ZEB1 leads to partial reversion of the epithelial phenotype and the relocalization
of CRB3 and/or LGL2 to the membrane to restore apical–basal polarity [175]. Snai1 shows a
similar function by acting through distal E-box sequences of the CRB3 promoter in breast
carcinoma cells [174]. Apparently, EMT transcriptional inducers act cooperatively to repress
polarity proteins to reinforce EMT.
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4. ERp29 Restores AJs and TJs
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ERp29 involves the establishment of the apical–junctional complex, which is formed by
AJs and TJs [27] (Figure 5). These complexes are located in the upper portion of a polarized
epithelial cell and are composed of trans-membrane proteins that interact with molecules in
adjacent cells [142]. In MDA-MB-231 cells, β-catenin is expressed and localized in nuclear.
ERp29 over-expression resulted in an increased expression and membrane localization of Ecadherin and translocation of β-catenin from the nucleus to the cell membrane [27] (Figure 4).
The ERp29-mediated membrane localization of β-catenin facilitates the assembly of Ecadherin/β-catenin complex and formation of AJs [155] (Figure 5).
Intriguingly, ERp29 over-expression led to an increase of TJ components such as ZO-1
and occludin at the membrane and cell–cell junctions in breast cancer cells (Figure 5). The
increased expression of ZO-1 and occludin is regulated at translational level, as ERp29 overexpression did not affect their mRNA levels [27]. The role of ERp29 in ZO-1 protein
expression and trafficking was further demonstrated in the ERp29-knockdown MCF-7 cells.
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Figure 5. ERp29 regulates epithelial cell integrity. Exogenous expression of ERp29 in mesenchymal
MDA-MB-231 breast cancer cells increases expression and membrane distribution of E-cadherin and
translocation of nuclear β-catenin to the membrane to form E-cadherin/β-catenin complex. The levels of
tight junctions proteins ZO-1 and occludin are increased by ERp29 over-expression and their membrane
distribution enhances cell-cell contact of epithelial cells. In addition, ERp29 over-expression in this cell
model up-regulates expression of apical protein Par3 and basolateral protein Scribble, leading to
formation of apical-basolateral cell polarity.
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Translational up-regulation of ZO-1 and occludin by ERp29 in these cell models may
provide a mechanism of how ERp29 induces tumor suppression in breast cancer [24].
Additionally, the formation of cortical actin filaments is critical for the establishment of AJs
and TJs and the regulation of epithelial cell apical–basal polarity [86]. Reorganization of the
actin cytoskeleton induces recruitment of ZO-1 to cell periphery before the assembly of
junctional complexes between adjacent cells [176]. The ERp29-induced restoration of ZO-1
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expression may be associated with actin reorganization. Hence, ERp29 plays a critical role in
restoration of an epithelial-like phenotype by establishing cell–cell contact.
5. ERp29 Restores Cell Polarity
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In line with the role of ERp29 in regulating MET and re-establishment of the epitheliallike phenotype, ERp29 over-expression can restore epithelial polarity [27] (Figure 5). In
mesenchymal MDA-MB-231 and basal-like BT549 cells, ERp29 expression did not affect
mRNA levels of Par3 and Scribble, but increased their protein translation and membrane
distribution. It was reported that Cdc42, a small GTPase, is one of the key regulators
modulating the expression of Par6 and aPKC [177, 178]. and has a critical role in establishing
cell polarity in epithelial cells [179]. However, ERp29 over-expression did not affect both the
expression and localization of Cdc42, Par6 and aPKC, indicating these PAR complex
members are not involved in ERp29-regulated apical polarity. Thus, ERp29 selectively upregulates Par3 protein expression during epithelial morphogenesis. These studies indicate that
in the apical Par complex, Par3, but not the Cdc42/Par6/aPKC, is a downstream target that is
specifically regulated by ERp29.
In addition, ERp29 over-expression did not markedly alter the expression and distribution
of Crumb1, a member of the Crumbs complex [180] (Figure 5), Similar to that observed for
Par3, ERp29 over-expression resulted in a significant increase of protein expression, but not
the mRNA level, of Scribble in both MDA-MB-231 and BT549 cells. Suppression of ERp29
by shRNA in epithelial MCF-7 cells resulted in reduction of these core polarity proteins,
leading to the disruption of cell–cell contact and increased cell spreading. Previous studies
demonstrated that polarity proteins are synthesized in the endoplasmic reticulum, transported
to the Golgi complex and sorted at the trans-Golgi network into distinct apical and basolateral
vesicular routes [181]. Given that ERp29 mediates the folding and secretion of newly
synthesized proteins in the ER system [39], it is plausible that, in addition to increased protein
expression of TJs and the core polarity complex, ERp29 may also have a critical role in
protein trafficking and the maintenance of protein stability to modulate epithelial cell
integrity. In agreement with this, the ERp29-induced tumor suppression in breast cancer cells
is linked to the integrity of apical–basal polarity that is crucial for the prevention of tumor
development [164, 182].
Increased ERp29 expression up-regulates expression and localization of AJs (E-cadherin
and β-catenin), TJs (ZO-1 and occludin) and core polarity proteins (Par3 and Scribble) at
cell–cell junctions. Restoration of these proteins at cell–cell junctions leads to close contact of
cells and establishment of epithelial-like cell features, a phenotypic characteristic of MET
(Figure 5). Consequently, ERp29 has a pivotal role in regulating MET and establishing
epithelial cell integrity in breast cancer.
ERp29 and Resistance to Genotoxic Stress
Recent studies have demonstrated that ERp29 is a novel molecule protecting cells from
the genotoxic stress-induced cell apoptosis [78, 183]. To survive from the stress environment,
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cells have developed a variety of responsive mechanisms to cope with the stress-induced cell
death, such as cell cycle arrest and activation of the DNA repair. In an early study, when cells
were exposed to ionization radiation, ERp29 expression was elevated in several types of
cultured cells [20]. Concomitantly, splicing of XBP-1 mRNA under radiation was increased,
suggesting the involvement of UPR sensor might be a reason to induce ERp29 gene
expression [20]. In nasopharyngeal carcinoma (NPC) cells, ERp29 knockdown attenuated
radio-resistance of NPC CNE-1 cells, whereas ERp29 over-expression enhanced radioresistance of NPC CNE-2 cells. This was reflected by the experimental data showing that
ERp29 knockdown in CNE-1 cells increased radiation-induced cell apoptosis, while ERp29
over-expression in CNE-2 cells reduced radiation-induced cell apoptosis. Clearly, ERp29
could potentiate resistance to radiation in NPC cells [183].
Doxorubicin (DOX) is one of the conventional chemotherapeutic drugs for cancer
intervention via the intercalation of DNA and subsequent activation of the tumor suppressor
p53 [184]. While most of cancer cells are sensitive to DOX and eventually killed by this drug,
some cells develop an adaptive response to DOX-induced genotoxic stress and survive from
stress. Clinically, chemo-resistance of cancer cells is a predominant cause of cancer
recurrence after long-term treatment. It has been found that DOX induced ERp29 expression
and ERp29 expression is causally linked to resistance against DOX by a mechanism that
requires PERK [185]. PERK activation promotes the phosphorylation of a general translation
factor eIF2α and attenuates translation of global proteins including cyclin D1 [73], thereby
resulting in inhibition of cell cycle. Apparently, the DOX-induced ERp29 facilitates cell's
response to genotoxic stress that ultimately results in an resistance against chemotherapy by
DOX. Indeed, recent studies further supported ERp29‘s role for cell survival under genotoxic
stress condition. When ERp29 was over-expressed in MDA-MB-231 cells, these cells showed
a significant resistant to DOX treatment, whereas knockdown of ERp29 in MCF-7 cells led to
an enhanced sensitivity of these cells to DOX [78]. Mechanistic studies revealed a critical
role of up-regulated Hsp27 in the ERp29-induced DOX resistance in these cell models. In
addition, the ERp29-induced activation of ER stress-related XBP-1/p58IPK cell survival
pathway also plays a pivotal role in this aspect [77]. In support of this, silencing of p58IPK in
MCF-7 cells and ERp29-overexpressing MDA-MB-231 clones re-sensitizes them to DOX by
activating ATF4/CHOP/caspase-3 pro-apoptotic signaling [77].
Interestingly, in addition to these identified downstream molecules that involve in
ERp29-induced DOX resistance, ERp29 over-expression in MDA-MB-231 cells significantly
up-regulated the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA
repair protein through facilitating dissociation of p53/mSin3A/HDAC1 transcription
repressors via suppressing p53 (unpublished data). MGMT repairs the mutagenic and
cytotoxic interstrand DNA cross-links via rapidly reversing alkylation, including methylation,
at the O6 position of guanine by transferring the alkyl group to the active site of the enzyme
[186]. In addition to DNA repair function, MGMT plays a role in integrating DNA
damage/repair-related signals with replication, cell cycle progression and genomic stability
[187, 188]. Hence, MGMT is also an important factor in ERp29-induced anti-genotoxic stress
and cell survival. The ERp29-upregulated DNA repair pathway cause resistance to chemoand radiotherapy, and thus targeting this pathway might have a potential to develop
alternative strategy for efficient treatment of chemo- and /or radio-resistant cancer cells.
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Clinical Implication
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1. ERp29 and Tumorigenesis
2. ERp29 and Metastasis
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The role of ERp29 in carcinogenesis has been studied with inconsistent accounts. For
example, ERp29 was found to be highly expressed in some primary tumors, e.g., basal cell
carcinoma and lung cancer [23, 24, 189, 190]. In lung tumors, ERp29 expression varied
within and between tumor stages and inversely correlated with tumor progression [23].
Similarly, a tissue array study in 98 breast tumors showed that ERp29 expression was found
to be down-regulated in tumors with a more aggressive phenotype [24]. These results indicate
a negative association of ERp29 expression with tumor progression, at least in breast and lung
tumors. However, the correlation of abnormal ERp29 expression to tumor progression in
epithelial cancers needs to be extensively assessed in large cohort of clinical specimens.
Shnyder et al reported a significant importance of ERp29 during the histogenetic stage of
tumorigenesis in which ERp29 was noticeably over-expressed in all epithelial cancers
investigated, and its expression correlated with the rates of cancer growth and lactogenesis
[23]. As such, ERp29 expression in epithelial cells could be a factor leading to tumor
histogenesis. Recent studies revealed that ERp29 was significantly expressed in radioresistant nasopharyngeal carcinoma (NPC) tissues compared to radio-sensitive NPC tissues,
indicating a potential role ERp29 in radio-resistance in NPC tumors [191]. ERp29‘s role in
resistance to radiotherapy has been well established in cell lines [22].
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Recent studies showed that increased ERp29 expression has been found in highly
metastatic cancer cells. A proteomics study identified that ERp29 was significantly increased
in the highly metastatic variant of parental MDA-MB-231 cells compared to the parental cells
[192]. Similarly, ERp29 was found to be one of the proteins that were highly expressed in the
metastatic tissues compared to the primary uveal melanoma tissues [189]. These results may
implicate an important role of ERp29 in cancer cell metastasis and disease recurrence. Indeed,
high expression of ERp29 in breast tumors strongly associated with reduced relapse time of
disease and short survival time of patients (unpublished data). The ERp29-enhanced
metastasis is probably related to its role in driving MET in cancer cells [24]. The role of MET
in facilitating distant metastasis has been clinically recognized by the observation that MET is
able to reversibly convert the disseminated mesenchymal cancer cells to an epithelial cell
state [193]. ERp29 may have a critical role in promoting distant metastasis during cancer
progression, although this needs to be investigated further. Consequently, understanding the
association of ERp29 with disease recurrence and distant metastasis is of significance in
assessing its prognostic value in clinical applications.
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Daohai Zhang
3. Tumor Microenvironment Affects MET and Metastasis
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Tumor microenvironment is an important factor in regulating cancer metastasis via MET
[194, 195]. The interplays between tumor cells, host cells, and the extracellular matrix in
tumor ecosystem endow cancer cells with malignant properties, leading to metastatic
dissemination. Interestingly, Shyder et al reported that the expression of ERp29 was
significantly affected by the culture mode [23], where ERp29 expression was significantly
increased in xenografts compared with the same cell types cultured in monolayer or spheroid
condition. This indicates that ERp29 could be physiologically regulated in tumor ecosystem.
However, it is uncertain whether the increased expression of ERp29 in tumor ecosystem
could facilitate MET process, similar to that observed in exogenously ERp29-overexpressed
MDA-MB-231 cells [24]. It should be noted that PC3 prostate cancer cells underwent a MET
in a three-dimensional culture system [196]. In another study, it was reported that DU145 and
PC3 prostate cancer cells expressed high level of E-cadherin when co-cultured with
hepatocytes and these carcinoma cells bound to hepatocytes in an E-cadherin-dependent
manner [197]. Similarly, when MDA-MB-231 cells were co-cultured with hepatocytes, Ecadherin was re-expressed, resulting in an increased chemo-resistance [198]. In vivo studies
demonstrated that MDA-MB-231 cells formed E-cadherin-negative primary tumors, but
showed a re-activated E-cadherin expression in lung metastatic site via MET, suggesting an
effect of the microenvironment on cells at the metastatic site [199]. Although the tumor
microenvironment-induced MET and metastasis is a complex process, investigating the
involvement of ERp29 in MET and metastasis may enhance our knowledge and
understanding of its biological and pathological functions in cancer progression.
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Conclusion
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Accumulating evidence supports a critical role of ERp29 in cancer cell survival and
metastasis. However, the current controversy regarding the role of ERp29 in different cancers
further emphasizes its importance in understanding its pathological regulation in
tumorigenesis. Therefore, it is important to carefully examine the role of ERp29 in each
tumor type. The current data from breast cancer cells supports that ERp29 can function as a
tumor suppressive protein, in terms of suppression of cell growth and primary tumor
formation and inhibition of signaling pathways that facilitate EMT. Nevertheless, the
significant role of ERp29 in cell survival against drugs, induction of cell differentiation and
potential promotion of metastasis may lead us to re-assess its function in cancer progression,
particularly in metastasis. It is of importance to explore in detail the ERp29‘s role in cancer as
a ―friend or foe‖ and elucidate its clinical significance in breast cancer and other epithelial
cancers. Targeting ERp29 and/or its downstream molecules might be an alternative molecular
therapeutics for metastatic cancer treatment.
321
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ISBN: 978-1-62808-665-2
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Chapter 16
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Divergent Roles for Tumor Suppressor
Genes in Cancer
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Marina Trombetta-Lima1, Thiago Jacomasso2,
Sheila Maria Brochado Winnischofer2 and Mari Cleide Sogayar1
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Biochemistry Department, Chemistry Institute, University of São Paulo,
São Paulo, Brazil
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Biochemistry and Molecular Biology Department, Federal University of Paraná,
Paraná, Brazil
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Abstract
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Tumor suppressor genes or anti-oncogenes are defined as genes which control cell
growth and may lead to the development of cancer upon mutation or deregulated
expression. Several tumor suppressor genes that were initially taken as great promises for
the clinic, such as the tissue inhibitor of matrix metalloproteinases TIMP-1, have been
shown to have more complex roles and even, in some models, to be constitutive markers
of tumor aggressiveness. Contrasting roles of a certain gene associated with the same
molecular pathway might be explained by the multifuncionality of its products through:
(i) epigenetic modifications; (ii) balance between different isoforms displaying different
properties, especially since alternative splicing is observed in more than 90% of the
human genes; (iii) post-translational modifications, such as methylation, phosphorylation
and glycosylation; and/or (iv) differences in the microenvironment and molecular context
under different physiological and pathological conditions. Here, we focus on analyzing
cases of tumor suppressor genes multifunctionality acting to promote divergent roles of
these genes in tumorigenesis, tumor progression and metastasis, thereby imposing a great
challenge to translation of these genes as clinical tools. Support: FAPESP, CNPq,
BNDES, MS-DECIT, MCTI.
Tumor suppressor genes, also known as anti-oncogenes, are wild type alleles playing
roles in processes such as: modulation of cell proliferation, differentiation, angiogenesis,
invasion, and apoptosis, among others. Classically, the hallmark of these functionally distinct
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genes is the fact that their loss or functional inactivation has an oncogenic outcome. This class
of regulatory genes was first identified using different approaches such as: in vitro phenotypic
reversion from malignant to normal of tumor cell lines that were transfected with normal cell
transcripts; fusion of normal and malignant cells; and induction of terminal differentiation of
malignant cells [1-4]. Since their first characterization, tumor suppressor genes became a
great promise to the clinic, but in several cases the use of these genes as therapeutic tools
revealed to be a great challenge. Here, we discuss the factors which influence tumor
suppressor genes dual function and role in tumorigenesis and tumor progression, namely: (i)
epigenetic modifications; (ii) alternative splicing; (iii) post-translational modifications; and
(iv) differences in the microenvironment and molecular context under different physiological
and pathological conditions.
I. Epigenetic Modifications
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Alterations in the epigenetic state of a cell lead to hereditable changes in gene expression
without involving modifications in the DNA sequence. Epigenetic mechanisms comprise
changes in microRNAs expression and affinity, DNA methylation status, and histone
modifications. Taken together, these tools compose an orchestrated control of gene
expression leading to different physiological and pathological processes such as cancer [5].
MicroRNAs (miRNAs, miRs) are short non-coding RNA sequences (18-25nt when
maturated), which integrate the RNA-Induced Silencing Complex (RISC) cascade resulting in
silencing of mRNAs which display a complementary sequence to the ‗seed‘ portion of the
miR, 2-7 nt long near the 5‘ extremity, that usually targets the mRNA at its 3´-UTR [6,7]. As
coding genes, miRs can either act as tumor suppressor or display a pro-tumoral activity. In
this context, Single Nucleotide Polymorphism (SNP) may have an important impact not only
in miR expression, but, also, in miRs target sequence specificity and affinity, and, thus, in its
function [8] (Figure 1A). This is the case of the Rs11614913 polymorphism in hsa-miR196a2, shown to alter mature microRNA expression and function [9] and described to
suppress proliferation and invasion capacity of breast cancer cells, having among its targets,
the tumor protein p63 [10]. The Rs11614913 polymorphism is associated to susceptibility to
breast and lung cancer and to severe toxicity in lung cancer patients submitted to a platinumbased treatment [9,11], highlighting the importance of SNPs to miRs‘ structures and target
recognition.
The transcriptional activity of a DNA region is considered to be a reflection of its
methylation status. Methylation occurs in the carbon 5 position of cytosine residues, generally
followed by a guanine residue, being mediated by methyltransferases. Highly methylated
regions are found in the heterochromatin and in transcriptionally silenced regions, frequently
occurring in genes displaying promoter regions rich in CpG dinucleotides, the so called CpG
Islands [5]. In tumors, promoter regions of tumor suppressor genes which contain CpG
Islands are found to be hypermethylated (Figure 1B). It is important to highlight that different
tumors display different patterns of CpG methylation aberrations, suggesting specific and fine
tuned controlling mechanisms that upon disruption may give rise to different alteration
profiles [5,12]. Several genes involved in chemosensitivity have been described to display an
aberrant methylation profile in different types of cancer, as for example: the O6-
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methylguanine-DNA Methyltransferase (MGMT,) gene, involved in DNA repair, reduces the
toxicity of alkylating agents, the methylation of its promoter is associated with better response
to temozolomide [5,13-17]; on the other hand, promoter hypermethylation of the
Transforming Growth Factor-Beta-Inducible gene-h3 (TGFBI) gene, involved in microtubule
stabilization through integrin signaling, is associated with resistance to paclitaxel in ovarian
cancer [18,19]; also associated with chemoresistence is the promoter hypermetylation of
Transcription Factor AP-2 Epsilon (TFAP2E) in colorectal cancer [20,21]. Therefore,
Methyltransferase family members cannot be classified as neither tumor suppressors nor
oncogenes.
DICER
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Degradation
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mRNA
Target mRNA
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Figure 1. (Continued).
Target mRNA
Target mRNA
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Figure 1. Epigenetic mechanisms influencing genes fate. miRNAs, which may act as tumor suppressors
due to the fact that their mRNA targets are subject to SNPs which may alter their target recognition
efficiency and specificity, thereby altering their function (A). In cancer, many tumor suppressor genes
containing CpG Islands in their promoters are hypermethylated, silencing their expression (B). At the
same time, hypomethylation in repeated DNA regions causes opening of the chromatin and DNA
breakage, accounting for many of the chromosomal aberrations observed in cancer (C).
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Histones are subject to modifications of different natures in their N-terminal, which may
be acetylated, methylated, ubiquitinilated, ribosylated and phosphorylated [5]. While
acetylation in the surroundings of the transcription start sites leads to an open chromatin
configuration and is often correlated to an active transcriptional state, histone modifications
have a complex pattern that may contribute to activate or silence transcription of a gene
displaying a crucial impact in cancer progression. Examples of such phenomenon are: the
inhibition of histone deacetylation, resulting in a higher level of MGMT transcription in
glioblastoma xenographs, with similar MGMT promoter methylation status, results in a higher
chemoresistance to temozolomide [13]; similarly, in the Multidrug Resistance 1 (MDR1),
gene of the ATP-Binding Cassette (ABC) family, which is closely related to chemoresistance
by effluxing the drug molecules from the cells, promoter methylation is dependent on the
Mixed Lineage Leukemia 1 (MLL1) protein, a histone methyltransferase specific for H3K4
[22]. DNA methylation in mammals, combined with histone modifications such as H3K9, is
predominant in repeated regions, such as transposable elements and satellite DNA, present in
the heterochromatin. Methylation, and thus transcriptional silencing, of these regions is
thought to be involved in maintenance of genomic stability and integrity as a host defense
mechanism[5]. Global methylation levels are diminished in tumor cells, when compared to
healthy tissues. Hypomethylation in repeated DNA regions causes opening of the chromatin
and DNA breakage, accounting for many of the chromosomal aberrations observed in cancer
[5,23] (Figure 1C).
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The use of therapies that explore epigenetic mechanisms is of extreme interest due to the
importance of altered epigenetic profiles to the onset of cancer. Inhibitors of
methyltransferases, such as 5-aza-2'deoxycytidine (Decitabine) and 5-Azacytidine (Vidaza),
have shown promising clinical results and their use has been approved by the Food and Drug
Administration (FDA). These Methytransferases inhibitors may be used either as a single
drug for treatment, as, for instance, in clinical trials targeting myelodysplastic syndrome and
acute myeloid leukemia in older patients or patients that are not prone to a more intensive
treatment, which have shown significant improvement [24-31]; or synergistically, when
combined with other drugs, such as romiplostim, aclacinomycin/cytarabine and vorinostat,
showing prognostic improvement for different tumor types [32-35]. However, the
promiscuous activity of methyltransferases represents a challenge to their successful
implementation. Genome wide hypomethylation promoted by 5-aza-2'deoxycytidine was
shown to induce trinucleotide CAG·CTG repeat instability in hamster CHO cells and in
human cells from myotonic dystrophy patients. In the latter, destabilization of repeat tracks in
the Dystrophia-Myotonica Protein Kinase (DMPK) gene was observed [36,37]. This kind of
genomic instability is observed in several neurodegenerative diseases, such as Huntington´s
disease, type 1 myotonic dystrophy and different spinocerebellar ataxias [36-41]. The
potentiality of genomic instability, as a consequence of treatment with Methyltransferases
inhibitors, is controversial. In animal models, hypomethylation has been associated to
chromosomal instability and tumor induction, and different types of human tumors are known
to be associated with global hypomethylation and chromosomal instability, such as colorectal
cancer and leukemias [42-48]. At the same time, it has been reported that patients with
myelodysplastic syndrome treated with 5-aza-2'deoxycytidine did not display higher
chromosomal instability, when compared to the cohorts that did not received this specific
treatment [49]. Yang et al. and Lengauer highlighted that caution should be taken to transpose
the data obtained upon 5-aza-2'deoxycytidine treatment consequences in humans, since the
different results may be explained by the different models used. Nevertheless, some aspects to
be taken into consideration are that methylation status of the treatment appears to be transient
and according to tumor type the resulting genomic instability could either promote tumor
progression and/or induce tumorigenesis in a second site; or, in the case of excessive
chromosomal instability, contribute to the anti-tumoral efficacy of the treatment, leading the
tumor cells to death [12,49-51]. Epigenetic manipulation revealed to be a useful tool for the
clinic and further studies aiming to access its side effects both at short and long term and to
refine its specificity will amplify the range of tumor types which may be susceptible to this
approach.
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Pre-mRNA alternative splicing is a major source of proteomic diversity in eukaryotic
cells. It is estimated that more than 90% of human multiexon genes generate at least two
different transcripts [52]. Alternative selection of exon-intron boundaries during pre-mRNA
maturation may cause the protein products to present different – or even antagonizing –
functions (Figure 2). Splicing events associated with cancer and other diseases have been
reported and extensively reviewed [18,53-57].
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Figure 2. Alternative splicing influencing gene fate. Isolated or combined: exon skipping, inclusion and
alternative selection of exon-intron boundaries during pre-mRNA maturation can cause the protein
products to present different – or even antagonizing – functions (A). The balance between the
alternative isoforms may influence the outcome of different pathways, thereby leading to a pathological
condition, such as cancer (B).
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Regarding cancer biology, alternative splicing plays a crucial role in determining whether
some genes will ultimately generate an oncoprotein or a tumor suppressor protein. The set of
exons present in the mature mRNA population through alternative splicing is affected by
oncogenic signaling pathways, such as Ras/PI3K/Akt [58,59] Ras/Raf/MEK/ERK [60] and cMyc [61], which activate downstream non-specific splice regulators, promoting changes in
the splice pattern of several genes to activate global splicing programs [53]. The most studied
splicing regulators are those of the serine-arginine (SR) class of proteins. These factors bind
to exonic splicing enhancer (ESE) elements in the pre-mRNA to favor either exon inclusion
or exclusion during splicing. One member of this family in particular, namely, SRSF1, has
been widely implicated in pro-tumoral splicing events, appearing to be a point of convergence
of oncogenic signaling cascades [18]. Overexpression of SRSF1 alone was shown to be
sufficient to transform normal cells, whereas its RNAi-mediated knockdown attenuated the
aggressive phenotype of tumor cells [62,63]. Despite its oncogene-like behavior, SRSF1induced splicing shows great functional variety, producing either pro- or anti-tumoral
isoforms, depending on the gene. The mRNA variants population from a given gene may also
be affected by SNPs and point mutations in splice sites or cis-acting elements on the premRNA, disrupting their recognition by trans-regulatory factors [56].
Alternatively spliced isoforms of tumor suppressors may either lack tumor suppressor
activity or generate antagonistic, pro-tumoral products. One example of antagonistic splice
variants is the Krüppel-like zinc finger transcription factor 6 gene (Klf6). KLF6 promotes
growth suppression by several mechanisms (reviewed in [64]), including up-regulation of p21
in a p53-independent manner [65,66] and inhibition of Cyclin D1 binding to CDK4 [67]. Its
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inactivation has been described in gastric, prostate and lung cancers, glioblastomas, and
hepatocellular carcinomas [68-72]. A relatively frequent SNP in prostate cancer was shown to
up-regulate alternatively spliced variants of the Klf6 gene by creating a novel binding site for
SRSF5 (also known as SRp40) [73]. One of these variants, named KLF6 SV1, was shown to
be incapable of up-regulating p21 and reducing cell proliferation [74], and its overexpression
increased prostate cancer progression and metastasis [75]. Moreover, RNAi knock down of
KLF6 SV1 reduced proliferation, tumor growth and angiogenesis [59]. It was later
demonstrated that KLF6 SV1 levels were also increased by Ras signaling, via PI3K/Akt and
SRSF1, and that, in fact, up-regulation of this transcript was responsible for part of the
growth promoting effects of Ras activation [59].
Antagonistic splice isoforms are also involved in regulation of angiogenesis, an important
process for tumor establishment. Several angiogenesis-related genes present alternatively
spliced isoforms with opposite effects on neovascularization of tumors as well as of normal
tissues [76]. The vascular endothelial growth factor A (VEGFA), in particular, represents an
interesting example. Vegfa transcript splicing originates several different isoforms. VEGFA165
is secreted by tumor cells in response to oxygen deprivation. It binds to and activates
receptors (mostly VEGFR2), along with a co-receptor (neuropilin-1 – NRP1), in endothelial
cells, stimulating tumor vascularization. Another isoform, VEGF165b, results from the choice
of an alternative last exon, denoted exon 8b. The amino acid sequence differs from VEGF165
only in the last six residues [77]. Despite the apparently small difference, VEGFA165b binds
to VEGFR2, but is unable to interact with NRP1 and, consequently, is incapable of activating
the receptor and any downstream signaling cascades [78]. Pro-angiogenic splicing of VEGFA
is favored when normal cells are treated with the IGF1 and TNFα growth factors, whereas
TGFβ1 treatment up-regulates anti-angiogenic isoforms. SRSF1 was shown to drive the
inclusion of the proximal exon 8a, generating pro-angiogenic VEGFAxxx isoforms, while
SRSF6 (SRp55) promotes inclusion of the distal exon 8b, forming anti-angiogenic isoforms,
VEGFAxxxb [79]. VEGFA165b is the predominant form in many normal tissues, and splicing
in corresponding tumor cells is often shifted towards VEGFA165 predominance [77]. The
existence of an anti-angiogenic isoform of VEGFA turned out to be the main reason why the
promising anti-VEGFA treatment was ineffective. Bevacizumab, a monoclonal antibody
against VEGFAxxx, also inactivates the anti-angiogenic VEGFAxxxb isoforms, thus failing to
improve survival, as shown by tests in patients with metastatic renal cancer [80].
Recombinant VEGFA165b is becoming a potential therapeutic alternative [76]. Interestingly,
many other factors involved in angiogenesis produce antagonistic alternatively spliced
isoforms, such as VEGFR1, VEGFR2 and NRP1, and these receptors have at least one
soluble isoform that acts as a ligand trap [76]. This illustrates how alternative splicing is
deeply involved in regulation of physiological and pathological processes.
Resistance to cell death is yet another hallmark of cancer regulated by alternative splicing
modulation. The Bcl-X gene is a classic example of how alternative splicing is involved in
concerted cellular responses. This gene encodes one short, pro-apoptotic (Bcl-XS) and one
long, anti-apoptotic isoform (Bcl-XL). Both forms are involved in the intrinsic apoptosis
pathway; oppositely regulating mitochondrial outer membrane permeabilization, a necessary
step towards the release of cytochrome c to the cytosol, and assembly of the apoptosome [81].
Other Bcl-2 family members, such as Mcl1, as well as many other apoptosis-related factors,
are also regulated by alternative splicing, and generate both pro- and anti-apoptotic isoforms
(reviewed in [82]). The Ich1 gene codes for the long and the short isoforms of Caspase-2 [83].
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SRSF2-mediated exon 9 skipping produces the long form, termed CASP2L [84], which is an
initiator caspase that promotes apoptosis by activating subsequent pro-caspases. Exon 9
inclusion induces a frame shift, creating a premature stop-codon, producing an mRNA
encoding CASP2S. When overexpressed, CASP2S seems to protect cells from apoptosis [83].
One final example of antagonistic splice isoforms related to cancer arises from the lens
epithelium-derived growth factor p75 (LEDGF/p75). LEDGF/p75 is a transcription factor
associated with increased tumor metastatic potential, resistance to oxidative stress and
chemoresistance [85-87]. It activates the stress response and anti-oxidant genes to promote
cell survival. An alternatively spliced form, LEDGF/p52, shares the N-terminal amino acid
residues with the p75 isoform, but has a different C-terminus. Upon caspase-3-mediated
cleavage of an N-terminal domain, p52 inhibits p75 transactivation of stress-related and antioxidant proteins, and induces apoptosis of tumor cells [86].
III. Post-translational Modifications
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Protein post-translational modifications (PTMs) play an important role in intracellular
signaling pathways, by covalent addition of functional groups or by proteolytic cleavage of
precursor proteins. About 5% of the genome in higher eukaryotes encodes enzymes that carry
out posttranslational modifications. So far, more than 200 PTMs have been characterized
[88], including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation,
acetylation, lipidation and proteolysis. These modifications increase the functional diversity
of proteins, regulating its activity, localization and interaction with other cellular molecules
and may promote a distinct and sometimes opposite role, in normal cells and under
pathological conditions (Figure 3).
The Cip/Kip (Cdk interacting protein/Kinase inhibitory protein) family of cyclindependent kinases inhibitors (CKIs) includes three members, namely: p21Cip1/Waf1/Sdi1 (p21,
encoded by cdkn1a) [89-91], p27Kip1 (p27, cdkn1b) [92-95], and p57Kip2 (p57, cdkn1c)
[95,96]. Classically, these proteins were considered as tumor suppressors due to their ability
to bind to and inhibit several cyclin-CDK complexes, and hence, cell-cycle progression.
However, several studies suggest that these proteins are involved in other cellular process
beyond cell cycle regulation, and could be considered as oncogenic targets under specific
situations. It has been described that post-translational modifications, in special distinct
phosphorylation patterns, promotes alteration in the Cip/Kip proteins functions by modulating
their subcellular localization, protein-protein interactions, and stability. The Cip/Kip proteins
exhibit completely different nuclear and cytoplasmatic functions [97,98], being considered
tumor suppressors in the nucleus, but becoming oncogenic when localized in the cytoplasm.
The p27 protein has been well characterized as a Cdk2 inhibitor in G0 and early G1
phase. Multiple residues of p27 may be phosphorylated, modulating its cell cycle regulatory
function. Phosphorylation of p27 at threonine 187 by cyclin E/cdk2 destabilizes p27 [99-101],
and activates SCFSKP2-mediated p27 proteolysis during the G1 to S phase transition
[102,103]. During cell cycle progression, p27 plays a second function by promoting both
cyclin D1-Cdk4 and Cdk6 complexes assembly and nuclear localization [104], a mechanism
which is also mediated by the different phosphorylation patterns of p27 in the early G1 phase
[105,106]. However, phosphorylation of p27 at threonine 157 by protein kinase B (PKB, also
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known as Akt) [107-109] impairs the nuclear import of p27, leading to its cytoplasmic
mislocalization. Cytoplasmic p27 has been associated to oncogenic functions (independently
of its ability to inhibit Cdks) and is considered as a poor prognostic indicator in several types
of tumors, including breast [107], hepatocellular [110], acute myelogenous leukemia (AML)
[111], colon [112] and ovarian cancer [113]. More recently, the p27 nuclear/cytoplasmic ratio
showed significant predictive value when breast cancer patients were treated with
anthracycline-based chemotherapy [114].
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Figure 3. Post-translation modifications influencing gene fate. Post-translation modifications increase
the functional diversity of proteins, regulating their activity, cell localization and interaction with other
cellular molecules, thereby exerting a distinct role when compared with the unmodified protein.
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Nagahara and collaborators showed, for the first time, that high cytoplasmic p27 levels
promote cell migration in human hepatocellular carcinoma model [115]. The cytoplasmic role
of p27 in promoting cell migration involves phosphorylation at threonine 198, that regulates
its stability [116,117] and promotes the ability of cytoplasmic p27 bind to RhoA, which
disrupts RhoA–ROCK activation, leading to increased cell motility [118,119]. It has been
described that RSK1 (p90 ribosomal S6 kinase), a downstream effector of both Ras/MAPK
and PI3K signaling pathways, promotes p27 phosphorylation at T198 [119], suggesting a link
between the increased cell motility induced by cytoplasmic p27 and intracellular pathways
commonly altered in human cancer.
As p27, the p21 and p57 proteins appear to have cytoplasmic functions independent of
their cell cycle-inhibitors roles. The p21 protein may be phosphorylated at threonine 145 by
PKB [120-123] and at serine 153 by protein kinase C (PKC), both of which leading to its
cytoplasmic localization [119,124,125]. Cytoplasmic p21 has also been associated to
oncogenic functions and resistance to anticancer drugs [126-129]. In the cytoplasm, p21
promotes anti-apoptotic functions by inhibition of pro-apoptotic caspases or apoptosisregulating kinases [130-132]. p21 may also bind to ROCK1, inhibiting its kinase activity,
disrupting actin stress fibers stability [133]. HER2/neu overexpression and cytoplasmic p21
were associated with poor prognosis in breast cancer patients, showing that phosphorylation
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of p21 at threonine 145 could be considered as a prognostic biomarker [134]. Furthermore, it
has been shown that cytoplasmic phosphorylated p21 contributes to mammary tumorigenesis
and lung metastasis in vivo [135].
On the other hand, genes which are classically described as oncogenes may also mediate
tumor-suppressive functions through a phosphorylation switch. A recent and interesting
example is the transcription factor c-Myc, classically linked to cell proliferation, being a
hallmark for human tumorigenesis [136]. c-Myc can both activate and repress transcription of
its target genes, according to interaction with its different binding partners (ex. Max, Myz-1,
etc) [136]. It has been described that Pak2 kinase phosphorylates MYC at three sites
(threonine 358, serine373 and threonine 400) of the C-terminal domain and represses the
functions of Myc that lead to cellular transformation by abrogating c-Myc/Max dimerization
and its binding to E boxes in the cell [137]. More recently, in leukemia cells, it has been
shown that c-Myc binds to retinoic acid receptor α (RARα) and represses the expression of
RAR-dependent targets required for differentiation, a mechanism that leads to increased
leukemia malignancy. However, after PAK-2-mediated c-Myc phosphorylation, the cMyc/RARα complex is able to activate transcription of those same genes to promote cellular
differentiation. This effect is E-box-independent and inhibits tumor invasion [138,139].
One link between alternative splicing and post-translational modifications related to
cancer arises from the p75 lens epithelium-derived growth factor (LEDGF/p75), which is a
transcription factor associated with increased tumor metastatic potential, resistance to
oxidative stress and chemoresistance [85,86]. It activates the stress response and anti-oxidant
genes to promote cell survival. An alternatively spliced form, LEDGF/p52, shares the Nterminal amino acid residues with the p75 isoform, but has a different C-terminus. Upon
caspase-3-mediated proteolytic cleavage of an N-terminal domain, p52 inhibits p75
transactivation of stress-related proteins and induces apoptosis of tumor cells [86]. It was also
shown that the p75/p52 balance is involved in chemoresistance in acute myelogenous
leukemia [140].
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and Molecular Context under Different
Physiological and Pathological Conditions
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The microenvironment surrounding the tumor cell, comprising the extracellular matrix,
stromal cells, and infiltrated immune cells, plays a crucial role in tumor progression (Figure
4). Genes which are classically considered as tumor suppressors, may act as oncogenes in an
altered microenvironment (revised in 141-143]. One example is E-Cadherin that encodes a
protein involved in cell-cell adhedion type junctions in epithelial tissues. E-Cadherin loss is
associated with epithelial mesenchymal transition and increased invasive behavior [141].
Despite this classical tumor suppressor role, higher expression of E-Caderin was associated
with poor prognosis in breast cancer patients, and more aggressive behavior in breast and
ovarian cancer [141]. It has been described that under hypoxia, in tumor cells that retain its
expression, E-Cadherin is required for cell growth and invasiveness [141,144].
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Figure 4. Microenvironment influencing gene fate. The microenvironment surrounding the tumor cell,
comprising extracellular matrix components, interaction with other tumor and stromal cells, and
infiltrated immune cells, influence both cell fate and protein role in the different contexts.
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One interesting case that exemplifies the different roles played by tumor suppressor genes
is the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1). In addition to its role as an inhibitor
of Matrix Metalloproteases (MMPs), TIMP-1 binding to CD63 is implicated in cell growth
inhibition and apoptosis [145]. On the other hand, TIMP-1 is also described to promote cell
division in different cell types, such as keratinocytes, mesenchymal and tumor cell lines
(reviewed in 145). TIMP-1 is considered as a malignancy marker for several kinds of tumors,
such as liver, breast, prostate, and glioblastoma [146-148]. Currently, patents are available for
the use of TIMP-1 as a malignancy marker for cancer in general (EP 1619206 A3), colon
cancer diagnosis (EP 1439393 A3), colorectal cancer (EP 2145190 A1), and the measurement
of its glycosylation pattern alterations related to tumorigenesis and metastasis (EP 1972939
B1).
Tumor suppressor genes generate multifunctional proteins or RNAs with complex roles
in physiological and pathological processes, highlighting the importance of the molecular
context to determine the pro- or anti-tumoral function of such players. This is classically
illustrated by the TIMP-1 metalloproteinase inhibitor and by the fine-tuned balance between
its p75 and p52 isoforms, whose balance and post-translational modifications have
implications in tumor cells survival fate. As discussed, there is growing evidence that the
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Final Remarks
344
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nc
.
cancer context is dependent on the epigenetic state of the cells, alternative splicing processes,
post-translational modifications and microenvironment, among other factors, which were not
addressed here, such as inflammatory and redox states. Therefore, there may be room for a
discussion of the concept of ―tumor suppressor genes‖, in order to prevent it from becoming
imprecise and utopic. The examples explored here remind us that the clinical applications of
these genes must be approached with careful foresight and should be uniquely examined in
each case.
he
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,I
nc
.
N
ov
a
Sc
i
en
ce
Pu
bl
absorption spectra, 8, 15
accelerator, 9
access, 337
accounting, 66, 136, 156, 257, 336
acetylation, 23, 27, 33, 40, 42, 43, 122, 137, 139,
153, 164, 168, 238, 336, 340
acid, 1, 7, 8, 12, 52, 58, 76, 112, 140, 196, 211, 255,
269, 342
active site, 235, 318
acute leukemia, 171
acute lymphoblastic leukemia, 123, 144
acute myelogenous leukemia, 181, 341, 342, 351,
353
acute myeloid leukemia, 160, 220, 229, 337, 346
acute promyelocytic leukemia, 76
AD, v, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 127, 171, 226
ADAM, 45, 46, 47, 50, 52, 56, 58, 62
adaptation, 67
adenine, 58
adenocarcinoma, 22, 39, 114, 115, 116, 118, 127,
205, 219, 221, 229, 244, 250, 279
adenoma, 115, 128, 261, 277, 283
adenomatous polyposis coli, 219, 253, 259, 260, 261,
312
adenovirus, 2, 14, 23, 26, 41, 82, 83, 84, 87, 88, 89,
90, 91, 92, 93, 94, 95, 99, 100, 101, 102, 103,
130, 135, 161, 223, 224, 230, 232, 282, 289, 292,
295, 296, 353
adhesion(s), 29, 46, 48, 52, 73, 107, 125, 154, 196,
198, 202, 203, 204, 238, 246, 256, 271, 272, 273,
274, 282, 308, 313, 314, 315, 325
adhesion properties, 271
adipocyte, 241
adipose, 150
adipose tissue, 174, 250
ADR, 230
adrenal gland, 3, 4, 11
adults, 54, 111, 197, 236, 270
adverse effects, 15
age, 187, 256, 258, 277, 284
aggregation, 304
aggressive behavior, 342
aggressiveness, 117, 156, 284, 309, 333
aging process, 274
airway epithelial cells, 247
alanine, 254
aldolase, 321
alkylation, 318
allele, 16, 143, 144, 150, 162, 175, 245
allelic loss, 2, 21, 23, 29, 198, 200
alopecia, 113
alternative medicine, 12, 15
alters, 135
amino, 3, 4, 8, 11, 52, 66, 106, 112, 113, 119, 120,
131, 186, 196, 210, 234, 235, 254, 255, 265, 268,
270, 325, 339, 340, 342
amino acid(s), 3, 4, 8, 11, 52, 66, 106, 112, 113, 119,
120, 131, 196, 210, 234, 254, 255, 265, 268, 325,
339, 340, 342
amyotrophic lateral sclerosis, 178
anastomosis, 257
anchorage, 199
anchoring, 29
androgen, 158, 172, 180
anemia, 49, 184, 186, 191, 192
angiogenesis, 20, 23, 26, 27, 31, 34, 39, 40, 43, 46,
53, 62, 68, 69, 70, 71, 74, 91, 92, 99, 142, 207,
223, 231, 232, 239, 270, 271, 272, 282, 333, 339,
349
anhidrotic ectodermal dysplasia, 114
is
A
he
rs
Index
356
Index
B
ce
en
Sc
i
a
ov
N
nc
.
autosomal recessive, 50, 59
avian, 145, 205
axons, 198, 199, 200, 201, 203, 205, 206
is
he
rs
,I
bacterial infection, 208
basal cell carcinoma, 22, 36, 217, 319
basal lamina, 197
basal layer, 109, 118
base pair, 66, 184, 254
basement membrane, 25, 62
BD, 203, 228, 229, 280
beneficial effect, 162
benefits, 162, 188
benign, 66, 116, 127, 230, 246, 256, 257, 272
benign tumors, 256, 257
biliary tract, 269
bing, 41
bioavailability, 61
biochemistry, 32, 34, 249, 258
bioinformatics, 125, 163
biological activity(ies), 12, 354
biological processes, 18, 67, 129, 131, 152, 163, 236,
304
biological roles, 75, 226
biomarkers, 29, 31, 39, 56, 59, 242, 245
biopsy, 127, 276
biosynthesis, 121, 140, 159, 302, 324
biotechnology, 190
birds, 249
births, 66
bladder cancer, 37, 103, 116, 126, 127, 221, 222,
230, 268, 309, 327
bleeding, 48
bleeding time, 54
blindness, 125
blood, 20, 184, 189, 223, 271
blood supply, 142
blood vessels, 223
body mass index (BMI), 275, 351
body size, 32
bonds, 11, 304
bone, 92, 101, 212, 236, 275, 309, 325
bone cancer, 222
bone form, 212
bone marrow, 92, 101, 275
bone tumors, 222
brachydactyly, 50, 53
brain, 34, 58, 66, 145, 146, 150, 175, 197, 202, 203,
205, 210, 234, 240, 254, 323
brain tumor, 21, 23, 29, 30, 43, 54, 98, 218
branching, 138
Pu
bl
anhydrase, 69, 71
anisotropy, 8
annotation, 245
antagonism, 226
anti-apoptotic role, 119
antibody, 20, 88
anti-cancer, 99, 103, 156, 160, 161, 162, 163, 223,
278
anticancer drug, 121, 221, 223, 341
antigen, 28, 32, 123, 127, 128, 289, 292, 295, 296
antioxidant, 142, 179, 321
antisense, 199, 282, 353
antitumor, 41, 74, 81, 82, 83, 84, 85, 86, 87, 88, 90,
91, 92, 93, 94, 95, 102, 103, 182
aorta, 62
APC, vi, 28, 219, 253, 254, 255, 256, 257, 258, 259,
260, 261, 312, 327
APL, 122
aplasia, 113
apoptotic mechanisms, 270
apoptotic pathways, 26
Arabidopsis thaliana, 13
arginine, 103, 112, 338
argon, 7
arrest, 19, 26, 31, 43, 74, 81, 82, 83, 85, 88, 90, 92,
95, 99, 110, 123, 125, 132, 133, 134, 139, 141,
143, 144, 146, 148, 154, 156, 157, 180, 219, 236,
237, 246, 266, 267, 270, 271, 274, 284, 292, 293,
297, 298, 301, 306, 307, 318, 330, 350, 351
arteriovenous malformation, 250
artery, 54, 58, 61, 62
arthritis, 46, 59, 60, 61
ascorbate, 1, 3, 8, 12, 13, 14, 15, 16, 67
ascorbic acid, 12, 13, 15
aspiration, 126
assessment, 36, 189, 193
asthma, 52, 59, 62
astrocytes, 58, 63, 347
astrocytoma, 36, 218
astrogliosis, 60
asymmetry, 250
ataxia, 130, 170, 347
atherosclerosis, 46
atherosclerotic plaque, 63
ATO, 122
ATP, 12, 13, 140, 169, 336
atrophy, 133, 134, 135, 166
attachment, 273, 308
attractant, 198, 201
autism, 241, 242, 250
autoimmunity, 173, 236
autosomal dominant, 66, 111, 112, 113, 234, 240,
253
357
Index
N
ov
a
Sc
i
en
ce
nc
.
,I
Pu
bl
Ca2+, 209, 312, 313
CAF, 130, 158
calcium, 46, 49, 226
caloric restriction, 169
calorie, 169
cancer cells, 12, 13, 17, 24, 25, 26, 27, 30, 31, 63,
71, 74, 83, 85, 86, 87, 88, 91, 93, 94, 95, 99, 101,
103, 121, 145, 146, 149, 151, 152, 156, 157, 158,
159, 160, 161, 162, 163, 181, 208, 219, 223, 248,
264, 272, 274, 277, 286, 301, 303, 308, 309, 310,
311, 318, 319, 320, 326, 327, 330
cancer death, 260
cancer progression, 34, 97, 103, 129, 148, 199, 263,
308, 319, 320, 327, 336, 339, 354
cancer stem cells, 87, 156, 313, 328
cancer therapy, 18, 27, 32, 41, 59, 78, 95, 96, 102,
132, 162, 181, 207, 220, 225, 286, 330
cancerous cells, 7, 274
candidates, 2, 28, 46, 144, 188, 242
capillary, 150, 176, 231
capsule, 198
carbohydrate, 284
carbon, 334
carbon dioxide, 326
carboxyl, 239, 243, 269, 281
carcinogenesis, 21, 28, 30, 37, 56, 70, 72, 76, 118,
136, 152, 176, 196, 199, 200, 207, 208, 218, 219,
220, 221, 222, 233, 240, 242, 270, 301, 309, 319,
352
carcinoma, 13, 22, 23, 24, 25, 26, 27, 38, 39, 40, 42,
54, 62, 63, 76, 77, 88, 109, 114, 115, 116, 117,
118, 128, 202, 205, 212, 215, 217, 219, 220, 221,
222, 224, 227, 230, 232, 249, 263, 268, 269, 272,
276, 277, 283, 284, 285, 306, 308, 309, 315, 320,
324, 325, 328, 329, 330, 331, 344
cardiac muscle, 133
cardiomyopathy, 275
cartilage, 46, 48, 49, 59, 61, 62
cascades, 148, 270, 312, 326, 338, 339
casein, 130, 136, 312
Caspase-8, 26
caspases, 290, 293, 340, 341
catalysis, 324
catalytic activity, 52, 235, 238
cattle, 48
he
rs
C
CBP, 78, 130, 133, 137, 139, 142, 164, 168, 238
CD8+, 220
CD95, 92, 101, 123, 155
CDK inhibitor, 264, 265, 278, 279, 291, 292, 298,
351
cDNA, 2, 15, 37, 172, 254, 273, 296
cell biology, 32, 42, 87, 177, 191, 205, 206, 250,
279, 283, 350, 352, 353
cell culture, 111
cell cycle, 12, 13, 19, 20, 23, 26, 28, 31, 32, 35, 40,
43, 74, 81, 82, 83, 85, 88, 90, 92, 95, 98, 105,
108, 109, 110, 120, 123, 124, 125, 132, 133, 134,
139, 141, 142, 143, 144, 146, 148, 152, 154, 156,
157, 165, 190, 208, 219, 236, 237, 242, 245, 246,
256, 260, 263, 264, 265, 266, 267, 269, 273, 274,
277, 281, 283, 284, 285, 287, 289, 290, 291, 292,
293, 294, 295, 297, 298, 299, 301, 312, 318, 340,
341, 350
cell death, 24, 30, 40, 81, 82, 83, 84, 85, 86, 87, 88,
90, 91, 92, 93, 94, 95, 102, 103, 106, 123, 139,
142, 165, 178, 195, 198, 226, 264, 270, 272, 274,
278, 282, 299, 318, 339, 349, 353
cell differentiation, 108, 153, 177, 179, 207, 287,
288, 313, 320, 324, 329
cell division, 105, 290, 343
cell fate, 84, 95, 98, 128, 129, 131, 208, 324, 343
cell invasion, 25, 27, 30, 46, 55, 78, 114, 329
cell invasiveness, 25
cell line(s), 20, 21, 23, 27, 30, 39, 40, 41, 43, 61, 63,
71, 73, 74, 99, 100, 109, 122, 123, 141, 143, 152,
159, 162, 171, 181, 198, 218, 219, 220, 221, 222,
233, 239, 254, 270, 271, 272, 273, 274, 281, 294,
309, 319, 322, 327, 330, 331, 334, 343
cell movement, 315, 325
cell signaling, 354
cell size, 133, 134, 239
cell surface, 205, 209, 211, 233, 239, 242, 272, 302,
303
cellular energy, 141, 157, 169
cellular homeostasis, 105, 304
cellular regulation, 208
central nervous system (CNS), 54, 58, 59, 200, 202,
205, 227, 236
centrosome, 184, 190
ceramide, 94
cerebellum, 240
cerebral cortex, 242
cervical cancer, 212, 227, 229, 276
cervical intraepithelial neoplasia, 217
cervix, 109, 114, 117, 220, 225, 229, 285
challenges, 294
chaperones, 301, 302, 304, 305, 322
checks and balances, 264
is
breakdown, 49
breast carcinoma, 26, 117, 127, 160, 161, 204, 269,
295, 309, 315, 325
bronchial epithelium, 16, 118
bystander effect, 91, 92, 101
358
Index
is
he
rs
,I
nc
.
coding, 81, 82, 84, 85, 145, 195, 198, 199, 200, 210,
236, 254, 255, 260, 268, 309, 326, 334
codon, 21, 66, 113, 210, 255, 257, 260, 340
codon 158, 255
colectomy, 257
collagen, 45, 46, 48, 52, 53, 63, 72, 76, 77, 127, 326
colon, 150, 153, 176, 205, 210, 240, 255, 261, 269,
328, 329, 341
colon cancer, 21, 86, 88, 94, 97, 99, 187, 228, 253,
257, 258, 260, 272, 277, 343, 351
colon carcinogenesis, 258, 261
colonisation, 330
colonization, 309, 310, 327, 328
colonoscopy, 257
colorectal adenocarcinoma, 152, 175, 219, 284
colorectal cancer, 29, 37, 42, 145, 151, 152, 161,
172, 195, 196, 198, 199, 200, 202, 203, 204, 205,
206, 219, 223, 226, 231, 253, 254, 256, 257, 258,
259, 261, 268, 272, 277, 335, 337, 343, 345, 347,
349
combination therapy, 26, 90, 92, 99, 294
combined effect, 26
commercial, 100
commissure, 198, 200
common symptoms, 234, 240
communication, 46, 188
competition, 142
complement, 52
complementarity, 71
complexity, 123, 129, 131, 226, 297, 347
complications, 257
composition, 56, 106
compounds, 11, 74, 83, 84, 160
conductance, 303, 323
configuration, 336
conjugation, 113, 120
connective tissue, 257
consensus, 133, 139, 144, 149, 157, 164
conserving, 192
construction, 120
contradiction, 277
control group, 56
controversial, 149, 200, 201, 337
controversies, 199
contusion, 58
convergence, 338
cooperation, 94, 178
coordination, 10, 20, 291
corepressor, 139
corpus callosum, 198, 200
correlation(s), 13, 21, 29, 30, 34, 54, 115, 118, 125,
126, 147, 159, 249, 285, 319
cortex, 120
N
ov
a
Sc
i
en
ce
Pu
bl
chemical, 1, 7, 9, 11, 83, 84, 90, 108, 160, 161, 181,
208, 321
chemical properties, 1, 11
chemokine receptor, 69
chemoprevention, 184, 189, 230
chemotherapeutic agent, 17, 24, 30, 31, 42, 88, 99,
160, 162, 208, 270, 271, 294, 353
chemotherapy, 30, 31, 81, 82, 85, 88, 89, 156, 220,
318, 330, 341, 344, 346, 354
chicken, 202, 211, 234
childhood, 34, 287, 288
children, 178, 287
chimpanzee, 211
chloroplast, 204
CHO cells, 2, 154, 337
chondrocyte, 54
chondroitin sulfate, 48, 54, 58, 60
chondrosarcoma, 59, 63
choroid, 218
chromatid, 191
chromatography, 7, 330
chromosomal instability, 258, 337, 345, 347
chromosome, 2, 13, 14, 16, 18, 23, 38, 39, 66, 67,
71, 78, 106, 107, 113, 118, 125, 153, 162, 172,
175, 184, 196, 198, 200, 202, 204, 205, 206, 210,
233, 234, 235, 243, 246, 254, 256, 259, 264, 265,
288
chromosome 10, 172, 233, 234, 235, 243, 246
chronic lymphocytic leukemia, 76
cigarette smoke, 60
cilia, 72, 73, 76
cilium, 73, 78, 79
circulation, 54, 271
cirrhosis, 219, 228
CIS, 215
classes, 177
classification, 16, 47, 115, 126, 218, 226, 227
cleaning, 53
cleavage, 4, 47, 51, 54, 60, 61, 71, 212, 271, 282,
305, 308, 340, 342, 350
cleft lip, 112, 113, 114
cleft palate, 112, 113
clients, 189
clinical application, 86, 319, 344
clinical assessment, 265
clinical oncology, 36, 39, 192, 193, 202
clinical presentation, 113, 125, 126
clinical trials, 337
clonality, 206
clone, 273
cloning, 2, 60, 63, 66, 253, 258, 296, 345
closure, 281
359
Index
ce
D
a
Sc
i
en
damages, 54, 133, 142, 294
database, 82, 96, 173, 191, 257, 295
deacetylation, 41, 85, 137, 139, 146, 150, 168, 293,
336, 345
deaths, 208
defects, 49, 50, 54, 108, 111, 120, 150, 175, 191,
200, 264, 270, 289, 323, 328
deficiency, 52, 54, 111, 125, 271
deformation, 11, 13
degenerate, 61
degradation, 25, 27, 28, 49, 54, 56, 59, 60, 61, 62,
67, 71, 74, 75, 85, 91, 119, 120, 121, 132, 134,
135, 136, 137, 138, 139, 142, 145, 146, 147, 148,
163, 166, 167, 168, 212, 236, 238, 245, 247, 248,
255, 256, 257, 264, 271, 279, 286, 292, 298, 302,
304, 306, 323, 351
degradation mechanism, 136
DEL, 241
Delta, 124, 323
dendritic cell, 82, 92, 101, 220
dephosphorylation, 161, 234, 237, 238, 244, 297
deposition, 79
ov
N
is
he
rs
,I
nc
.
deprivation, 67, 135, 189, 339
depth, 219
deregulation, 131, 148, 163, 208, 218, 236, 249, 288,
294
derivatives, 109, 160
dermatology, 250, 281
dermis, 48
destruction, 57, 258
detectable, 228
detection, 24, 126, 195, 198, 200, 230, 242, 328
detoxification, 132, 138, 140, 142, 156, 157, 297,
302
developmental process, 208
diabetes, 46, 173, 241, 250
diagnostic criteria, 66, 249
diagnostic markers, 264
differential diagnosis, 115
diffusion, 313
dihydrolipoic acid (DHLA), 1, 6, 7, 8
dimerization, 32, 303, 305, 342
diploid, 96, 146, 268, 284
disclosure, 220
discs, 329
disease progression, 301
diseases, 46, 48, 49, 52, 57, 62, 72, 75, 111, 233,
236, 241, 242, 256, 285, 321, 337
disintegrin, 45, 46, 52, 58, 60, 61
disorder, 70, 82, 113, 125, 153, 173, 240, 255, 257
dissociation, 318
distribution, 164, 167, 200, 206, 308, 314, 316, 317,
350
diversification, 131, 171
diversity, 269, 337, 340, 341
DNA breakage, 336
DNA damage, 19, 24, 33, 72, 81, 82, 83, 85, 98, 106,
111, 120, 121, 123, 124, 133, 136, 141, 142, 157,
170, 182, 184, 186, 190, 191, 263, 264, 265, 270,
271, 274, 297, 305, 318, 345
DNA polymerase, 290, 291
DNA repair, 19, 20, 27, 31, 131, 132, 133, 134, 139,
141, 142, 144, 152, 156, 157, 166, 183, 184, 190,
191, 208, 269, 318, 335, 346
dogs, 62
domain structure, 350
donors, 4
dopamine, 14, 15, 302, 321
dopaminergic, 248
dosage, 168
down-regulation, 21, 23, 25, 26, 30, 40, 56, 72, 101,
124, 129, 133, 145, 147, 149, 151, 153, 154, 170,
181, 207, 219, 220, 223
Drosophila, 131, 164, 196, 200, 202, 204, 205, 206,
245, 255, 314
Pu
bl
cost, 188
counseling, 183, 187, 188, 193, 250
counterbalance, 155
covering, 132
crystal structure, 234
CSCs, 130, 156, 313
CSF, 220
cues, 158, 200, 202, 205
culture, 72, 273, 283, 320
curcumin, 31, 56
cure, 220
cycling, 31, 239
cyclins, 190, 264, 265, 291
cysteine, 50, 52, 211, 302, 303, 311, 322
cystic fibrosis, 303, 323
cytochrome, 1, 2, 3, 4, 11, 12, 13, 14, 15, 16, 26, 27,
339
cytochromes, 9
cytokines, 54, 57, 72, 75, 270
cytology, 126
cytoplasm, 3, 26, 28, 29, 119, 134, 135, 138, 145,
148, 184, 212, 256, 264, 277, 279, 303, 312, 340,
341
cytoplasmic tail, 198
cytosine, 58, 334
cytoskeleton, 311, 313, 316, 327, 328
cytostatic drugs, 13
cytotoxicity, 13, 156
360
Index
N
ov
a
Sc
i
en
ce
nc
.
,I
Pu
bl
Eastern Europe, 187
E-cadherin, 72, 73, 76, 77, 115, 256, 260, 308, 309,
310, 311, 312, 313, 315, 316, 317, 320, 325, 326,
327, 329, 331, 354
ECM, 25, 27, 45, 46, 48, 49, 52, 69, 72, 311
ECM degradation, 54, 73
ecosystem, 320
ectoderm, 110
ectodermal dysplasia, 108, 111, 113, 124, 125
egg, 46
Ehlers-Danlos syndrome, 48
electron(s), 1, 3, 4, 5, 9, 10, 11, 12, 14, 15, 16
electron state, 10
electrophoresis, 330
elucidation, 28, 136, 323
e-mail, 263
embryogenesis, 78, 109, 131, 146, 162, 206, 212,
236, 293, 308
embryonic stem cells, 87, 98, 255
emphysema, 60
enamel, 116, 321
encoding, 32, 60, 71, 101, 131, 133, 153, 173, 230,
232, 234, 256, 280, 296, 304, 309, 340, 348
endocrine, 324
endocrinology, 278
endometrial carcinoma, 116, 204, 206, 273, 283
endonuclease, 302
endothelial cells, 60, 157, 179, 223, 231, 272, 282,
339, 352
endothelium, 48, 54, 219, 223, 231
energy, 10, 67, 141, 168, 250, 352, 354
energy expenditure, 250
England, 280
enrollment, 194
environment, 2, 10, 56, 73, 251, 271, 317
environmental factors, 208
environmental stimuli, 159
environmental stress, 270
enzymatic activity, 139
enzyme(s), 13, 27, 45, 46, 48, 50, 52, 53, 61, 63,
121, 138, 140, 142, 159, 290, 302, 318, 324, 340
ependymal, 218
epidemiology, 39, 230
epidermis, 108, 109, 116, 205, 231
he
rs
E
epigenetic levels, 17, 24
epigenetic modification, 333, 334, 344
epigenetic silencing, 132, 229, 230, 235, 242, 275
epithelia, 28, 109, 149, 153, 176, 197, 277, 283, 303,
330
epithelial cells, 73, 109, 111, 114, 116, 125, 132,
146, 147, 148, 149, 150, 151, 152, 158, 159, 173,
191, 225, 273, 285, 314, 316, 317, 319, 321, 322,
323, 325, 328, 329, 330, 350
epithelial ovarian cancer, 100, 174, 229
epithelium, 76, 108, 116, 117, 118, 218, 254, 256,
257, 276, 314, 326, 329, 340, 342, 350, 353
EPR, 1, 10, 16
Epstein-Barr virus, 279
equilibrium, 14
erosion, 108
erythrocytes, 49
erythrocytosis, 74, 78
erythropoietin, 71
esophageal atresia, 150, 176
esophageal cancer, 24, 56, 101, 204, 219, 268
esophagus, 109, 219
estrogen, 34, 117, 159, 181, 184, 189, 246, 344
ethnic groups, 187
etiology, 72
EU, 12
eukaryotic, 130, 140, 302, 304, 337
eukaryotic cell, 302, 337
evidence, 13, 17, 24, 72, 73, 74, 81, 82, 87, 89, 108,
121, 129, 136, 138, 139, 142, 143, 144, 145, 146,
147, 151, 154, 156, 157, 158, 159, 162, 178, 199,
200, 223, 225, 248, 257, 269, 273, 275, 301, 304,
314, 315, 320, 325, 327, 331, 343, 344, 347
evolution, 31, 164, 177, 257, 345
excision, 33, 43
excitation, 11
exclusion, 135, 160, 161, 167, 338
execution, 165
exons, 18, 66, 67, 106, 107, 113, 119, 184, 196, 210,
234, 235, 254, 255, 266, 268, 288, 338
experimental autoimmune encephalomyelitis, 58
exporter, 160
exposure, 208, 270, 271
extinction, 155
extracellular matrix, 27, 45, 46, 50, 52, 53, 54, 57,
60, 69, 79, 174, 246, 273, 311, 320, 342, 343
extracts, 160
is
drug delivery, 99, 294
drug resistance, 162, 179, 324, 344
drug targets, 32, 202, 206
drugs, 75, 94, 121, 122, 163, 273, 318, 320, 337
dysplasia, 50, 61, 112, 118, 126, 150, 153, 176, 219,
234, 276, 285
F
families, 46, 56, 66, 112, 113, 192, 251, 257, 260,
265, 270, 278, 291, 297, 326
family history, 187, 193
361
Index
is
he
rs
,I
nc
.
gene regulation, 164
gene silencing, 149, 151, 163, 290, 297
gene therapy, 17, 24, 25, 26, 27, 41, 81, 82, 83, 85,
86, 87, 88, 89, 91, 92, 94, 95, 96, 97, 99, 100,
101, 232, 258, 283, 344
gene transfer, 2, 26, 84, 86, 87, 88, 89, 92, 94, 99,
100, 101, 181, 231
genetic alteration, 2, 26, 36, 81, 82, 144
genetic background, 153
genetic counselling, 193
genetic defect, 65, 72
genetic disease, 188
genetic disorders, 150
genetic engineering, 83
genetic mutations, 257
genetic screening, 160
genetic testing, 187, 188, 189, 193
genetics, 31, 32, 164, 192, 193, 204, 205, 227, 228,
243, 245, 246, 247, 249, 250, 251, 258, 259, 260,
279, 280, 346, 347
genitourinary tract, 112
genome, 42, 63, 71, 155, 163, 204, 244, 294, 329,
340, 347
genomic instability, 111, 152, 337
genomic stability, 32, 141, 151, 152, 155, 159, 287,
288, 318, 336
genomics, 164, 245
genotoxic stresses, 81, 82, 83
genotype, 125, 202, 249
germ cells, 246
germ line, 257, 269
germline mutations, 66, 111, 125, 153, 193, 240,
269, 270, 280
gerontology, 279
gland, 74, 113
glial cells, 54, 63
glioblastoma, 23, 24, 29, 30, 33, 40, 41, 43, 54, 103,
109, 146, 165, 218, 240, 244, 336, 343, 345, 349,
353, 354
glioblastoma multiforme, 103
glioma, 31, 38, 43, 54, 55, 61, 88, 98, 99, 100, 103,
209, 212, 218, 223, 226, 272, 282
glucocorticoid, 131, 135, 166
gluconeogenesis, 135, 166
glucose, 69, 71, 130, 133, 135, 150, 153, 166, 174,
176, 302
glucose tolerance, 151
glucoside, 4
glutamate, 234
glutamine, 113
glutathione, 7
glycans, 274
glycerol, 4
en
ce
Pu
bl
family members, 24, 32, 52, 96, 123, 129, 131, 140,
164, 171, 178, 187, 189, 207, 209, 236, 265, 267,
310, 335, 339
FDA, 75, 337
ferric ion, 2, 14
fertilization, 303
FHIT gene, 91
fiber(s), 102, 135, 166, 311, 327, 341
fibroblast growth factor, 52, 158, 309
fibroblasts, 20, 52, 60, 63, 94, 96, 130, 146, 150,
152, 158, 159, 180, 205, 232, 268, 271, 282, 284,
327, 350
fibrosis, 52, 74, 76, 308, 323
fidelity, 191
filament, 28, 186
financial support, 12
fish, 18, 268
flexibility, 255
fluid, 73
follicle(s), 109, 153, 171, 177
food, 225
Food and Drug Administration, 89, 337
force, 354
forebrain, 284
formation, 25, 29, 42, 48, 54, 56, 60, 72, 73, 74, 78,
107, 110, 117, 119, 123, 142, 143, 162, 172, 183,
184, 186, 192, 199, 200, 212, 223, 224, 231, 236,
237, 242, 247, 256, 261, 269, 270, 279, 289, 302,
311, 312, 314, 315, 316, 320, 351, 353
FOX family transcription, 129
fragility, 108
fragments, 59, 61, 62
freedom, 11
fumarate hydratase, 71
functional analysis, 322
fusion, 94, 112, 144, 145, 160, 161, 172, 303, 322,
334, 352
Sc
i
G
N
ov
a
GABA, 130, 135
gallbladder, 102
ganglion, 200
ganglioneuroma, 218
gastrointestinal tract, 153, 197, 236, 270
gastrulation, 226
gel, 273, 330
gene amplification, 146, 173
gene expression, 12, 18, 31, 35, 37, 56, 60, 62, 63,
69, 71, 76, 89, 114, 116, 117, 119, 139, 143, 146,
150, 154, 162, 171, 178, 218, 236, 254, 264, 270,
273, 275, 306, 309, 318, 324, 329, 334
gene promoter, 56, 68, 89, 94, 178, 260, 273, 315
362
Index
H
a
Sc
i
en
ce
hair, 108, 112
hair follicle, 109, 116
HCC, 21, 30, 219, 274
HDAC, 18, 24, 27, 28, 82, 94, 287, 290, 294
HE, 125, 126, 229, 280, 284
head and neck cancer, 38, 40, 59, 61
health, 164, 189, 190, 248
health problems, 188
heart attack, 46
heart failure, 275
heat shock protein, 304, 306
Helicobacter pylori, 225
hematology, 34, 346
hematopoietic stem cells, 143, 170, 275
hematopoietic system, 75
heme, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 16
hemihypertrophy, 250
hemorrhage, 175
hemostasis, 48
hepatocellular cancer, 245
hepatocellular carcinoma, 21, 36, 37, 39, 63, 89, 100,
102, 114, 116, 146, 212, 219, 228, 273, 284, 339,
341, 348, 349, 351
hepatocytes, 320, 331
hepatoma, 247
heterochromatin, 334, 336
heterogeneity, 208, 212, 243, 322, 345
highly anisotropic low-spin (HALS), 1, 10
ov
N
is
he
rs
,I
nc
.
hippocampus, 120, 242
histidine, 3, 9, 14
histogenesis, 319
histone, 18, 20, 27, 33, 34, 40, 41, 42, 43, 82, 94,
103, 110, 124, 133, 150, 163, 168, 174, 236, 275,
290, 293, 297, 298, 300, 334, 336, 345
histone deacetylase, 18, 34, 41, 42, 82, 94, 103, 133,
163, 168, 174, 236, 290, 293, 297, 298, 300
histones, 27, 42
history, 187, 210, 253, 258
HM, 102, 172, 173, 193, 203, 206, 231
homeostasis, 133, 141, 150, 158, 164, 170, 180, 208,
256, 301, 302, 305
homocysteine, 302, 321
hormone, 20, 34, 74, 177
host, 56, 94, 127, 276, 320, 336
HPV, 115, 276, 285, 296
hTERT, 82, 89, 90, 100
HTLV, 225
hub, 313
human brain, 36, 37, 172, 243
human development, 245
human genome, 67, 184, 268
Hungary, 1, 12
Hunter, 350
hybrid, 73
hybridization, 18, 29, 150, 175
hydrogen, 7, 12, 13, 14, 269
hydrogen bonds, 269
hydrogen peroxide, 7, 12, 13
hydrogenase, 14
hydrolysis, 211
hypermethylation, 17, 71, 206, 212, 218, 219, 221,
226, 227, 228, 229, 230, 265, 269, 275, 277, 335,
345
hyperplasia, 149, 217, 222
hypersensitivity, 32, 250
hypertension, 60
hypertrophy, 240, 248, 257
hypoplasia, 113, 125
hypothalamus, 203
hypothesis, 52, 83, 117, 288
hypoxia, 20, 27, 34, 39, 46, 53, 58, 65, 66, 67, 68,
71, 74, 76, 77, 78, 79, 130, 142, 170, 171, 323,
342, 354
hypoxia inducible factor (HIF), 20, 27, 34, 39, 41,
46, 53, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 130, 142, 170, 171
Pu
bl
glycine, 113
glycogen, 73, 76, 257, 302, 312
glycolysis, 74, 170
glycoproteins, 311
glycosylation, 211, 274, 333, 340, 343
grades, 26, 36, 218
grants, 95
granules, 13
GRAS, 177
Great Britain, 37
growth arrest, 27, 72, 86, 97, 301, 303, 305, 306,
307, 308, 322, 325
growth factor, 39, 50, 52, 53, 68, 69, 70, 72, 75, 77,
82, 99, 120, 130, 134, 135, 136, 140, 141, 150,
157, 158, 160, 166, 168, 174, 180, 181, 208, 209,
235, 238, 239, 246, 247, 248, 270, 306, 339, 340,
342, 349, 350
GTPases, 208, 314
guanine, 318, 334
guardian, 98, 244
guidance, 29, 195, 196, 197, 198, 199, 200, 201, 202,
203, 206
I
iatrogenic, 21
ID, 174, 281
363
Index
is
he
rs
,I
nc
.
inhibitor, 30, 31, 32, 34, 35, 36, 37, 39, 40, 41, 42,
46, 52, 59, 76, 94, 96, 103, 110, 121, 124, 130,
133, 141, 143, 147, 157, 158, 160, 161, 181, 182,
219, 224, 225, 226, 227, 229, 230, 264, 268, 271,
275, 278, 279, 281, 284, 285, 287, 289, 291, 294,
296, 305, 333, 340, 343, 347, 350, 351, 354
initiation, 21, 23, 26, 28, 30, 39, 66, 77, 85, 105, 110,
130, 131, 140, 152, 198, 240, 254, 263, 274, 304,
312, 324
injections, 26
injury, 58, 61, 175, 176
innate immunity, 20, 35
inositol, 302
INS, 241
insertion, 112, 257
insulin, 120, 130, 135, 140, 151, 166, 167, 168, 169,
170, 180, 246, 247, 250, 296
insulin sensitivity, 166, 170, 250
insulin signaling, 167, 169, 180
integration, 321, 323
integrin, 63, 114, 115, 128, 176, 247, 272, 273, 282,
306, 307, 335
integrins, 272, 273
integrity, 48, 49, 141, 143, 165, 236, 242, 244, 256,
264, 294, 314, 316, 317, 322, 327, 336
interface, 165, 200
interference, 245
internalization, 209
intervention, 75, 129, 131, 258, 313, 318
intestine, 147, 150, 153, 174
intracellular calcium, 302
intron, 106, 113, 144, 199, 306, 337, 338
introns, 18
invasive cancer, 219
ionization, 318
ionizing radiation, 162, 191
ipsilateral, 187
Ireland, 37
iron, 2, 9, 10, 14, 15, 67
irradiation, 94, 192, 244
islands, 112, 273, 283
isolation, 112
issues, 144, 156, 193, 243, 328
N
ov
a
Sc
i
en
ce
Pu
bl
identification, 14, 30, 38, 42, 60, 66, 115, 117, 126,
160, 169, 187, 196, 205, 220, 233, 234, 263, 270,
296, 324, 328, 353
identity, 107, 326
idiopathic, 21
IL-8, 23, 26, 27
immune response, 20, 92, 131
immunity, 20
immunofluorescence, 2
immunoglobulin, 196, 202
immunoglobulin superfamily, 196
immunohistochemistry, 236, 276
immunoprecipitation, 315
immunoreactivity, 118
immunosuppression, 147
immunotherapy, 158
in situ hybridization, 236
in transition, 325
in vitro, 2, 14, 17, 24, 25, 26, 31, 58, 62, 70, 88, 98,
102, 116, 119, 122, 138, 139, 142, 145, 150, 159,
160, 161, 162, 168, 179, 199, 229, 232, 234, 246,
249, 256, 269, 270, 297, 334, 354
in vivo, 2, 14, 26, 54, 58, 59, 62, 70, 72, 88, 92, 99,
115, 122, 125, 139, 143, 145, 152, 154, 157, 159,
160, 161, 162, 168, 170, 172, 173, 174, 178, 192,
200, 248, 256, 261, 269, 270, 273, 274, 283, 326,
330, 342, 349, 350
incidence, 32, 66, 188, 189, 194, 220, 221, 227, 230,
257, 299
India, 295
indirect effect, 131
individuals, 189, 193, 194, 250, 280
inducer, 159, 209, 212, 287, 294
inducible protein, 305, 324
induction, 13, 20, 24, 40, 61, 82, 83, 84, 85, 86, 87,
88, 90, 93, 94, 103, 109, 122, 134, 141, 143, 156,
159, 160, 161, 165, 175, 179, 182, 208, 218, 219,
221, 224, 226, 232, 236, 240, 271, 274, 277, 283,
284, 293, 298, 299, 300, 320, 334, 337, 346, 347
induction chemotherapy, 346
infection, 88, 90, 92, 93, 94, 304, 322, 323
infertility, 21, 153
inflammation, 53, 60, 62, 74, 278, 283, 308
inflammatory cells, 62
inflammatory disease, 173
inheritance, 182, 189, 257
inhibition, 9, 12, 13, 20, 23, 26, 28, 38, 40, 62, 71,
74, 87, 122, 123, 129, 140, 141, 145, 146, 147,
156, 157, 160, 162, 165, 166, 167, 169, 170, 174,
181, 182, 198, 224, 226, 231, 238, 244, 248, 263,
264, 265, 269, 272, 273, 274, 278, 280, 281, 282,
283, 292, 298, 306, 308, 314, 318, 320, 328, 336,
338, 341, 343, 348, 349, 350, 351, 352, 353
J
Japan, 1, 12, 16, 81, 95, 207, 251, 345
Jordan, 164
K
keratin, 128
364
Index
N
ov
a
Sc
i
en
ce
nc
.
,I
Pu
bl
lactation, 302
landscapes, 163, 280
large intestine, 256, 257
larvae, 314, 315
larynx, 118
LDL, 226, 311
lead, 28, 46, 48, 50, 52, 53, 54, 82, 83, 85, 117, 119,
129, 144, 147, 149, 183, 200, 257, 258, 278, 308,
320, 333, 334, 342
learning, 242
learning disabilities, 242
leiomyoma, 285
lens, 340, 342, 350
lesions, 37, 66, 114, 118, 127, 222, 257, 260, 272,
276, 277
leucine, 19, 20, 146, 306
leukemia, 21, 36, 130, 132, 144, 145, 156, 157, 160,
161, 212, 215, 220, 229, 236, 268, 269, 342, 346,
347, 353, 354
life quality, 189
life sciences, 33, 34, 202
lifestyle changes, 224
lifetime, 183, 189, 236, 240, 257
ligand, 13, 92, 101, 115, 128, 133, 165, 182, 195,
197, 198, 200, 201, 204, 209, 239, 273, 339
light, 17, 130, 135, 144, 146, 156, 157, 160, 162,
163, 282
lipid metabolism, 170
lipids, 237, 302, 314
liposomes, 88
liquid chromatography, 191
liver, 2, 112, 147, 150, 153, 158, 170, 175, 176, 178,
184, 210, 219, 236, 269, 323, 343, 348, 354
liver cancer, 158, 178, 219
liver metastases, 354
localization, 18, 28, 29, 37, 40, 58, 59, 109, 118,
130, 135, 136, 138, 139, 141, 145, 149, 184, 190,
212, 237, 238, 239, 248, 254, 277, 278, 279, 282,
283, 311, 314, 315, 316, 317, 340, 341, 351, 352,
353
he
rs
L
loci, 31, 38, 58, 63, 145
locus, 18, 21, 23, 26, 30, 32, 36, 53, 109, 124, 153,
154, 173, 174, 204, 218, 227, 246, 259, 263, 265,
266, 268, 269, 270, 275, 278, 279
longevity, 129, 131, 138, 139, 142, 156, 165, 168,
169
loss of heterozygosity (LOH), 17, 20, 21, 22, 23, 26,
30, 31, 153, 195, 196, 198, 200, 204, 206, 218,
265, 269, 328, 347, 349
luciferase, 273
lung cancer, 2, 14, 15, 16, 21, 23, 25, 26, 37, 38, 39,
40, 41, 88, 99, 100, 101, 103, 126, 127, 158, 159,
160, 161, 180, 206, 212, 217, 222, 227, 231, 271,
282, 319, 334, 339, 344, 348
lung metastases, 108
Luo, 37, 39, 97, 102, 228, 244, 245, 298, 344
lymph, 126, 218, 219, 221, 222, 224, 271
lymph node, 21, 26, 117, 152, 184, 227, 272, 309,
325
lymphocytes, 158
lymphoid, 21, 23, 34, 130, 144, 260, 312
lymphoid tissue, 158
lymphoma, 114, 115, 130, 133, 143, 158, 277, 346
lysine, 139
lysosome, 135
is
keratinocytes, 110, 116, 124, 126, 283, 343, 350
kidney, 2, 54, 70, 71, 73, 77, 78, 79, 145, 147, 160,
161, 210, 315
kidney failure, 73
kidneys, 54
kill, 13, 101
killer cells, 92
kinase activity, 37, 141, 145, 154, 237, 265, 269,
281, 292, 341, 350
kinetics, 275
M
machinery, 14, 136, 141, 298, 299, 302
macrophages, 286
magnetic resonance imaging, 189
majority, 66, 71, 72, 75, 162, 184, 257, 280
malignancy, 29, 36, 37, 152, 175, 206, 218, 221,
222, 277, 342, 343
malignant cells, 92, 103, 334
malignant melanoma, 23, 28, 30, 149, 222, 231, 280
malignant tissues, 202
malignant tumors, 66, 82, 95, 277
mammalian cells, 135, 150, 191, 324, 346, 352
mammalian tissues, 302
mammals, 109, 131, 133, 140, 157, 165, 196, 314,
336
mammography, 184, 189
management, 75, 125, 126, 193, 345
manipulation, 337
MAPK/ERK, 182
mapping, 38, 39, 61, 296
mass, 1, 166, 207, 250, 330, 351
mass spectrometry, 330
mastectomy, 184, 189, 193
materials, 188
matrix, 11, 45, 46, 49, 52, 55, 56, 59, 61, 63, 77, 78,
109, 155, 256, 271, 333
365
Index
is
he
rs
,I
nc
.
migratory properties, 308
Ministry of Education, 95
mitochondria, 23, 26, 27, 66, 157, 167, 190, 274, 353
mitogen, 70, 130, 137, 247, 270, 327, 351
mitosis, 152, 190, 259, 264, 284, 290
MMP(s), 25, 27, 45, 46, 47, 50, 56, 72, 73, 325, 343
MMP-2, 25, 27, 73
MMP-9, 25, 27, 56, 73
model system, 140, 160
models, 25, 59, 83, 108, 143, 153, 180, 182, 191,
200, 224, 249, 270, 277, 294, 295, 308, 316, 318,
333, 337
modifications, 20, 137, 139, 153, 169, 333, 334, 336,
340, 341, 342, 343, 350
modules, 46, 157
mole, 8
molecular biology, 34, 280, 281
molecular mass, 3
molecular oxygen, 67
molecular pathway, 184, 333
molecular weight, 128
molecules, 10, 12, 17, 27, 28, 29, 31, 39, 49, 52, 53,
74, 154, 160, 196, 203, 207, 238, 253, 270, 273,
282, 302, 316, 318, 320, 328, 336, 340, 341
monoclonal antibody, 92, 325, 339
monolayer, 320
monosomy, 204
Moon, 226
morphogenesis, 53, 109, 110, 123, 124, 150, 159,
200, 207, 227, 301, 303, 317
morphology, 108, 311
mortality, 126, 230, 278
mosaic, 241, 250
motif, 3, 5, 16, 18, 19, 20, 32, 35, 45, 52, 58, 120,
125, 133, 186, 235, 240, 255, 269, 278, 289, 296,
301, 302, 306
motor neurons, 178
MR, 98, 101, 128, 167, 168, 172, 182, 206, 251, 281,
284, 296
mRNA(s), 2, 20, 21, 22, 23, 25, 26, 29, 30, 32, 34,
38, 43, 52, 54, 59, 66, 71, 107, 126, 127, 140,
141, 144, 145, 146, 147, 149, 150, 164, 197, 210,
212, 218, 219, 221, 222, 223, 228, 236, 245, 254,
271, 288, 304, 305, 306, 307, 309, 311, 316, 317,
318, 321, 334, 336, 337, 338, 340, 345, 348, 349,
350
mtDNA, 274
mucosa, 115, 118, 158, 254, 329
multiple myeloma, 62, 97, 122, 280
multiplication, 256
muscle atrophy, 134, 166
musculoskeletal, 48
mutagenesis, 12, 14
N
ov
a
Sc
i
en
ce
Pu
bl
matrix metalloproteinase, 45, 46, 56, 59, 63, 78, 155,
333
MB, 26, 159, 166, 192, 224, 225, 251, 284, 286, 299,
307, 308, 309, 310, 311, 312, 313, 316, 317, 318,
319, 320
MCP, 191
median, 112
medical, 187, 249, 250, 251
medicine, 201, 243, 249, 250, 282, 286, 345, 351,
352, 354
MEK, 132, 138, 145, 147, 148, 162, 182, 247, 275,
276, 338
melanoma, 20, 21, 25, 28, 30, 33, 35, 36, 38, 39, 43,
62, 96, 157, 161, 181, 222, 223, 234, 244, 268,
269, 270, 272, 273, 280, 281, 292, 319, 330, 345
melting, 61, 63
membranes, 3, 9, 15, 16, 273, 306
mental retardation, 112
mesenchyme, 150, 175, 329
mesoderm, 150, 175
mesothelioma, 114, 212, 222
messages, 243
messenger RNA, 288
meta-analysis, 53, 202, 295
metabolism, 15, 68, 69, 70, 71, 129, 131, 133, 134,
135, 141, 150, 164, 169, 179, 239, 278, 325, 354
metalloproteinase, 45, 46, 60, 61, 62, 256, 343, 354
metastasis, 26, 27, 29, 30, 46, 56, 58, 71, 72, 98, 100,
108, 114, 116, 117, 118, 126, 127, 131, 132, 152,
159, 163, 175, 196, 200, 205, 218, 219, 220, 221,
222, 224, 227, 231, 232, 271, 272, 273, 283, 301,
309, 310, 319, 320, 325, 326, 329, 333, 339, 342,
343, 349,352, 353
metastatic cancer, 319, 320
methylation, 21, 54, 61, 62, 63, 85, 97, 150, 199,
207, 208, 212, 218, 219, 220, 221, 222, 224, 228,
229, 230, 234, 236, 265, 269, 273, 280, 285, 318,
327, 333, 334, 336, 337, 340, 345, 347
mice, 18, 19, 20, 26, 33, 48, 49, 52, 60, 62, 63, 74,
76, 77, 83, 96, 105, 108, 109, 116, 120, 143, 147,
149, 150, 153, 154, 157, 159, 166, 171, 172, 175,
176, 198, 199, 200, 203, 205, 222, 223, 224, 234,
241, 242, 243, 250, 261, 270, 273, 281, 323, 346,
347
microarray technology, 114
microRNA, 20, 35, 76, 82, 84, 97, 98, 124, 132, 145,
163, 235, 242, 245, 326, 327, 334
microscopy, 2
midbrain, 200
migration, 19, 20, 25, 27, 29, 35, 39, 43, 59, 60, 114,
126, 131, 149, 150, 158, 174, 176, 200, 219, 222,
227, 237, 242, 246, 247, 256, 272, 273, 282, 283,
299, 308, 327, 328, 329, 341, 352
366
Index
nucleotides, 7, 85
nucleus, 18, 26, 28, 29, 42, 56, 66, 69, 71, 94, 106,
119, 135, 138, 152, 161, 166, 184, 190, 209, 237,
239, 242, 256, 274, 277, 305, 306, 313, 316, 340
null, 74, 77, 109, 122, 140, 160, 162, 175, 176, 271
nutrient, 135
nutrition, 223
N
obesity, 208
obstruction, 113
occlusion, 58, 61, 62
olfactory nerve, 203
oligomerization, 106, 107, 110, 119, 254, 255, 305,
322
oligomers, 303
oligosaccharide, 274
oncogenes, 28, 71, 110, 129, 131, 133, 147, 201,
208, 225, 236, 257, 279, 291, 333, 335, 342
oncogenesis, 42, 108, 146, 225, 238, 288
oncoproteins, 148, 158, 163, 167, 289, 292, 296,
297, 298, 350
oocyte, 303, 322
ophthalmologist, 66
opportunities, 25, 75, 163, 229, 282
optimization, 191
oral cancers, 21
oral cavity, 114, 118, 272, 277
organ, 150, 169, 208, 236, 243, 308
organelle(s), 135, 212, 274, 301, 302, 304, 324
organism, 167
organize, 54
organs, 2, 46, 54, 65, 114, 150, 158, 218, 223, 236
ornithine, 121, 258
osteoarthritis, 46, 48, 59
osteoporosis, 189
ovarian cancer, 25, 36, 88, 132, 147, 148, 149, 154,
156, 183, 184, 186, 187, 188, 189, 191, 192, 193,
220, 221, 229, 276, 327, 335, 341, 342, 345, 352
ovarian failure, 153, 177, 178, 179
ovarian tumor, 21, 153, 154, 155
ovaries, 220
overlap, 70, 125
ovulation, 46, 53
oxidation, 4, 7, 15, 238
oxidative stress, 72, 134, 136, 138, 154, 155, 156,
165, 167, 170, 180, 325, 340, 342, 350
oxygen, 66, 67, 71, 74, 78, 79, 142, 170, 171, 223,
251, 339
N
ov
a
Sc
i
en
ce
,I
he
rs
is
Pu
bl
NAD, 7, 139, 168
naming, 18
nasopharyngeal carcinoma, 54, 318, 319, 322, 330
nasopharynx, 114, 116

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