Group Title: Molecular Cancer 2003, 2:41
Title: Role of APC and DNA mismatch repair genes in the development of colorectal cancers
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Title: Role of APC and DNA mismatch repair genes in the development of colorectal cancers
Series Title: Molecular Cancer 2003, 2:41
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Roy D
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Molecular Cancer ioMedc


Role of APC and DNA mismatch repair genes in the development of
colorectal cancers
Satya Narayan*1 and Deodutta Roy2

Address: 'Department of Anatomy and Cell Biology and UF Shands Cancer Center, College of Medicine, Academic Research Building, Room R4-
216, 1600 SW Archer Road, University of Florida, Gainesville, FL 32610, USA and 2Environmental Health Sciences, University of Alabama at
Birmingham, 317 Ryals Building, 1665 University Boulevard, Birmingham, AL 35294-0022, USA
Email: Satya Narayan*; Deodutta Roy
* Corresponding author

Published: 12 December 2003
Molecular Cancer 2003, 2:41

Received: 02 December 2003
Accepted: 12 December 2003

This article is available from: I
2003 Narayan and Roy; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in
all media for any purpose, provided this notice is preserved along with the article's original URL.

Colorectal cancer is the third most common cause of cancer-related death in both men and women
in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new
cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10%
of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%.
The main inherited colorectal cancers are the familial adenomatous polyposis (FAP) and the
hereditary nonpolyposis colorectal cancers (HNPCC). The FAP and HNPCC are caused due to
mutations in the adenomatous polyposis coli (APC) and DNA mismatch repair (MMR) genes. The
focus of this review is to summarize the functions of APC and MMR gene products in the
development of colorectal cancers.

Prognosis of colorectal cancer depends on the stage of the
tumor at the time of diagnosis, with surgery being the
most effective treatment. Colorectal cancers develop
through a series of histological distinct stages from "ade-
noma to carcinoma" [1]. Recently, the multi-step model
of colon cancer development by Fearon and Vogelstein [2]
is revised and presented in greater detail to include the
interdependence of different pathways and involvement
of many more gene mutations than before [3,4]. Genes
which are mutated at different stages of colorectal cancer
development include tumor suppressor genes, proto-
oncogenes, DNA repair genes, growth factors and their
receptor genes, cell cycle checkpoint genes, and apoptosis-
related genes (Fig. 1). In contrast to the predictions of
sequential mutation accumulation of genes (Fig. 1), a
recent study presented a quite different view in which the
gene mutation spectrum in a large cohort of colon cancer
patients was correlative to only 6.6% [5]. The heterogene-

ous pattern of tumor mutations found in this study sug-
gests that multiple alternative genetic pathways to
colorectal cancer exist and that the widely accepted
genetic model of cancer development is not representative
of the majority of colorectal tumors. However, it is
believed that mutations in one of these genes set the stage
for initiation and transformation of the normal colonic
epithelial cells. Further accumulation of mutations in
other genes then contributes to the progression of cancer
through adenoma carcinoma metastasis stages. Dur-
ing accumulation of genetic changes, a complex signaling
network is established among inactivated and activated
cellular pathways. Many cells bearing defective signaling
pathways may go through programmed cell death or
apoptosis and may be removed from the normal popula-
tion of cells; however, one of the target cells can go
through the selection process and survive among other
cells by overruling cell cycle checkpoints and abrogating

Page 1 of 15
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I TGFpRII, Bax, MMR genes,
Tcf-4, IGF2R, E2F4 frameshifts

K-Ras DCC p53
~| ^^ Intermediate Late
a Adenoma Smad2 Adenoma

Low grade High grade
dysplasia dysplasia

S Early MHAP/ *- Late
Serrated adenoma adenoma

? progression

Figure I
Model for genetic alterations in the development of colorectal cancer. Based on genetic analysis, at least two path-
ways are characterized in detail, which lead to colon cancer development. One pathway (indicated with red arrows) initiates
with mutations in the adenomatous polyposis (APC) gene and chromosomal instability (CIN) followed by mutations in K-ras,
deleted in colorectal cancer (DCC) and p53 genes. The second pathway (indicated with blue arrows) is initiated by the muta-
tions in the mismatch repair (MMR) genes and microsatellite instability (MSI) followed by mutations in hMSH3, hMSH6, TGFlIR,
IGFIIR, PTEN, BLM, Tcf-4, Bax and E2F4 genes. Other pathways are less characterized, but a high degree of overlapping is
expected among them. At least, seven gene mutations are needed to develop a normal epithelial cell into carcinoma. However,
a cluster of gene and chromosome aberrations such as p15, p16, Bubl, cyclin DI, tPa, CEA, Nm23, MMP, E-cadherin (CDHI),
CD44, 7q, 14q, 22q and 8p are observed in carcinoma and metastatic tumors. ASEF, APC-stimulated guanine nucleotide
exchange factor; DLG, Drosophila discs large; EBI, end-binding protein I, KAP3A, kinesin superfamily-associated protein 3A;
MCR, mutator cluster region; NES, nuclear export signal; NLS, nuclear localization signal; PP2-B56a, protein phosphates 2A
B56a subunit. This figure is adapted from reference [7].

apoptosis pathways. After clonal expansion, the geneti-
cally modified single cell can become a full grown tumor.

Many colon cancer syndromes have been characterized
based upon their phenotypic, histological and genetic
changes. Among them, the most common and highly
studied colon cancer syndromes are familial adenoma-
tous polyposis (FAP) and hereditary nonpolyposis color-
ectal cancers (HNPCC), which are caused by mutations in
the adenomatous polyposis coli (APC) and mismatch
repair (MMR) genes, respectively. Other colon cancer syn-
dromes include Peutz-Jeghers syndrome (PJS), Juvenile
polyposis syndrome (JPS), hereditary mixed polyposis

syndrome (MHAP) and Cowden's syndrome. These syn-
dromes contain hamartomatous polyps and are inherited
in an autosomal dominant fashion. Mutations in serine-
threonine kinase II (STKII) gene, Sma and Mad-related
protein 4/Deleted in pancreatic carcinoma 4 (Smad4/
DPC4) gene, and phsophatase and tension homolog
deleted on chromosome ten (PTEN) gene are linked with
PJS, JPS and Cowden's syndromes, respectively. The inher-
ited syndromes account for only 3-5% of all colon can-
cers, and the rest are the somatic colon cancers in which
both alleles of the tumor suppressor genes are inactivated

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Molecular Cancer 2003, 2


Adenomatous polyposis coli (APC) gene mutations are the
earliest events in the multi-step colorectal cancer
Mutations in the APC gene on chromosome 5q21 locus
are considered as one of the earliest events in the initia-
tion and progression of colorectal cancer. In FAP patients,
allelic mutation of the APC gene followed by a loss of het-
erozygosity (LOH) is a common feature. Notably, muta-
tions in the APC gene also are found in 60 to 80% of
sporadic colorectal cancers and adenomas. FAP patients
with APC mutations are prone to hundreds to thousands
of colorectal adenomas and early onset carcinoma. FAP
patients also are prone to small intestinal adenomas (and
carcinomas), intra-abdominal desmoids and osteomas
tumors (Gardner's syndrome), congenital hypertrophy of
retinal pigment epithelium (CHRPE), fundic gland polyps
in the stomach, pancreas and thyroid, dental abnormali-
ties, and epidermal cysts [6,7]. Mutations in APC are also
associated with malignant brain tumors known as Tur-
cot's syndrome [8]. The APC locus on chromosome 5q21
shows loss of heterozygosity (LOH) in approximately
25% of breast cancers [9]. Approximately 18% of somatic
breast cancers carry APC gene mutations [10]. Further-
more, LOH at the APC gene locus is frequently seen in the
early stages of non-small cell lung cancers [11]. In an ani-
mal model for carcinogen-induced lung tumors, a
decrease in APC gene expression, rather than an increase
in APC mutations, has been shown to be associated with
tumorigenesis [12]. The diverse effects of mutations in
APC gene indicates that this molecule plays a key role in
the regulation of cell growth in a number of colonic and
extra-colonic tissues. It has been suggested that the over-
expression of APC causes a G1/S phase checkpoint in
serum-treated NIH-3T3 cells via components of the pRB
pathway [13]. A role for APC in the G2/M phase transition
is also expected, given that APC is hyperphosphorylated
during M-phase and is a target of the M-phase kinase
p34Cdc2 [14,15]. In a recent study, a direct role of APC in
S and G2 phase arrest of cell cycle has been described in
cell lines immortalized mouse epidermal keratinocytes,
immortalized canine kidney epithelial, mouse squamous
cell carcinoma and skin papilloma, and human colon car-
cimona [16].

APC is expressed constitutively within the normal colonic
epithelium. The APC gene product is a 310 kDa protein
localized in both the cytoplasm and the nucleus. Recently,
it has been shown that the APC gene is inducible, which
is transcriptionally up-regulated by p53 in response to
DNA damage [17-19]. p53 is a negative regulator of nor-
mal cell growth and division. Although mutations in the
p53 gene is also widely present in colorectal cancers, its
consequence on the expression of the wild-type or mutant
APC gene is not clear [2]. In an earlier study, in the ApcM'"i
+ (Min, multiple intestinal neoplasia) mice model, an

increased multiplicity and invasiveness of intestinal ade-
nomas was associated with deficiency for p53 [20]. Also,
the occurrence of desmoid fibromas in these mice was
strongly enhanced by p53 deficiency. The structure of the
APC protein with different protein interaction domains
are given in Figure 2. At the N-terminal site, the APC pro-
tein contains oligomerization and Armadillo-repeat bind-
ing domains; and at the C-terminal site there are EB1 and
tumor suppressor protein DLG binding domains. The
APC protein also contains three 15-amino acids and seven
20-amino acids repeat regions in which the latter is
involved in the negative regulation of B-catenin protein
levels in cells (Fig. 2). The first 20-amino acid-repeat in
the APC gene is located at the 5'-end of the mutation clus-
ter region (MCR). An association has been shown
between severe polyposis phenotype and germ-line muta-
tions in the MCR [21-24]. Selective pressure for an MCR
mutant has been proposed based on the germ-line muta-
tion in FAP [25]. Patients with mutations outside of the
MCR region have a milder phenotype. A recent study has
shown that animals with homozygous truncating muta-
tions at codon 1638 of APC did not develop tumors and
survived through adulthood [26].

It has been reported recently that APC functions as a
nuclear-cytoplasmic shuttling protein and as a B-catenin
chaperone [27-31]. There are at least five APC nuclear
export signals (NESs). Among them, two are Rev-type
NESs located at the N-terminal region and the other three
are located in the 20-amino acid repeat region of B-cat-
enin binding motif [29,32]. The nuclear export of APC is
mediated by CRM1/Exportin receptor pathway [28-
30,33]. The APC protein has two nuclear localization sig-
nals, NLS1 (1767-1772; amino acids GKKKKP) and NLS2
(2048-2053; amino acids PKKKKP). The nuclear localiza-
tion of APC is controlled by post-translational modifica-
tion of NLS residues by protein kinase 2 and/or protein
kinase A [32-34].

APC regulates /-catenin levels through Wnt-signaling
APC may act as a negative regulator of B-catenin signaling
in the transformation of colonic epithelial cells and in
melanoma progression [35-37]. The role of the Wingless/
Wnt signaling pathway has been described in Drosophila,
Xenopus, and in vertebrates. This pathway is important in
organ development, cellular proliferation, morphology,
motility, and the fate of embryonic cells [38-40]. In a sim-
ple model shown in Figure 3, Wingless/Wnt signaling reg-
ulates the assembly of a complex consisting of: axin (and
its homologs Axinl and conducting APC, B-catenin, and
glycogen synthase-3p kinase (GSK3 ). Axin (Axil/conduc-
tin) binds to form a complex with APC, B-catenin, and
GSK3P to promote B-catenin phosphorylation and subse-
quent binding with slim (P-TrCP) which mediates its
ubiquitination and degradation in the proteasome

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Molecular Cancer 2003, 2


Nuclear export signals (NESs)

-- teni binding
p-catenin binding an( down regulationn
Armadillo (1020 1169 aa) (1 42 ;075 aa)
Oligom nation repeat 15 aa repeats O aa peats
do in domain I I

APC (full length) 1 -

binding MCR Axin-binding sites
site (1286 -1514)



Figure 2
Structural features of the APC protein. Most of the mutations in APC occur in the mutator cluster region (MCR) and
create truncated proteins. The truncated proteins contain ASEF and B-catenin binding sites in the armadillo-repeat domain but
looses the B-catenin regulatory activity which is located in the 20-amino acids repeat domain. Somatic mutations are selected
more frequently in FAP patients with germ-line mutations outside of the MCR.

[41,42]. GSK3P regulates this process by phosphorylating
B-catenin, APC and Axin (Axil/conductin) complex [43-
45]. Activation of the Wingless/Wnt signaling pathway
inhibits GSK33 and stabilizes B-catenin [46,47]. Muta-
tions of B-catenin or truncation of APC, which occur both
in colon cancer and melanoma cells, increases the stabil-
ity and transcriptional activity of B-catenin [37,48]. The
stabilized pool of B-catenin associates with members of
the T-cell factor (Tcf)-lymphoid enhancer factor (Lef) fam-
ily of transcription factors. There are four known members
of the Tcf and Lef family in mammals, one of which, the
human Tcf4 gene, is expressed specifically in colon cancer
cells [35]. The B-catenin/Tcf4 complex regulates the proto-
oncogene and cell cycle regulator c-myc [48], the GI/S-reg-
ulating cyclin Dl [49], the gene encoding the matrix-
degrading metalloproteinase, matrysin [50], the AP-1 tran-
scription factors c-jun and fra-1; and the urokinase-type
plasminogen activator receptor gene [51]. From this dis-
cussion, it is clear that the inactivation of APC causes acti-
vation of B-catenin, which results in the constitutive
activation of Tcf/Lef response genes.

In many cases, it has been observed that colon tumors car-
rying mutations in the APC gene also carried increased
levels of c-Myc, a known factor for cellular proliferation.
Recently, a direct link has been established between APC
gene mutation, B-catenin activation and c-myc gene up-
regulation in colon cancer development. The increased

expression of c-Myc through Wnt-signaling pathway up-
regulates the expression of Cdh4 gene, whose product is
responsible for cell cycle regulation in G1 phase [52]. The
c-myc gene encodes a transcription factor of helix-loop-
helix leucine zipper family that binds as a heterodimer
with Max to E boxes (CACGTG) on target promoters, for
example Cdh4 gene, and activates its expression. Max can
also interact with Mad and Mxil and down-regulate c-Myc
target gene expression. It has been suggested that the
increased levels of Cdk4 protein can phosphorylate pRB.
The E2F/DP transcription factor then dissociates from the
hyperphosphorylated pRB, which actively transcribes
genes involved in cell cycle progression through G1 phase.
It is also suggested that the increased levels of Cdk4 may
sequester cell cycle kinase inhibitor p21, p27, and pl6.
This sequestration may account for the ability of c-Myc
overexpression to substitute for p16 deficiency as noted in
mouse fibroblast transformation [53]. These studies thus
establish a link among APC gene mutation, B-catenin sta-
bilization, c-myc gene activation, and Cdk4/cyclin D1/
pRB/pl6 pathway in colorectal cancer development.

APC is involved in actin cytoskeletal integrity, cell-cell
adhesion and cell migration
Actin cytoskeletal integrity is necessary to maintain the
shape and adherence junctions of cells. The imbalance in
actin cytoskeletal integrity can cause disturbance in cell-
cell adhesion and cell migration. The role of APC in actin

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Molecular Cancer 2003, 2


A. Normal colonic epithelial cells B. Colon cancer cells

L .
bl. DV D

depicts the down-regulation of 3-catenin transactivation
activity in normal colonic epithelial cells. -catenin remains in

a complex of Axin/Axil/conductin, APC, GSK33 kinase and
casein kinase I or 2 (CKI or 2). In the absence of Wnt-sign-
aling, GSK33 and CKI or 2 kinases become active and phos-
phorylate 3-catenin at serine and threonine residues in the
N-terminal domain. Axin and APC promote phosphorylation
of B-catenin by acting as a scaffoled protein and bringing
together enzyme(s) and substrate(s). The phosphorylated 3-
catenin then binds with F-box protein 3-TrCP of the Skp I -
Cullin-F-box (SCF) complex of ubiquitin ligases and under-
goes proteasomal degradation. Even though Tcf-Lef tran-
scription factor without 3-catenin may bind to DNA in the
absence of 3-catenin, the repressors and corepressors such
as CtBP (carboxy-terminal binding protein), CBP (CREB-
binding protein), Gro (Groucho), LRP (LDL-receptor-related
protein) bind with Tcf-Lef and repress c-myc or cycling DI gene
expression to control cell cycle progression. Some other
known genes which are regulated by P-catenin/Tcf-Lef path-
way are given here cycling DI, CDHI, Tcf- c-jun, Fra-I,
PPARd, Gastrin, uPAR, MMP7, Conductin, CD44, Id2, Siamois,
Xbra, Twin and Ubx. Panel B shows the role of mutations in
the APC or 3-catenin protein in the regulation of 3-catenin
level and its transactivation property in colon cancer cells.
The mutant P-catenin escapes its degradation through Wnt-
pathway and becomes stabilized in the cytoplasm. The stabi-
lized level of 3-catenin then heterodimerizes with Tcf-Lef
transcription factor and locates into the nucleus, where it
actively transcribes cell cycle related genes causing cellular
proliferation. The binding of b-catenin with Tcf-Lef inhibits
the binding of CtBP, CBP, Gro or LRP and potentiates its
transcriptional activity.

cytoskeletal maintenance is predicted through its interac-
tion with B-catenin. B-catenin establishes a link between
APC and actin by providing a bridge to a-catenin [53]. In
Drosophila, mutations in E-APC have shown to affect the

organization of adherence junctions [54-56]. Another link
of APC with actin is shown through its interaction with
PDZ domain of DLG protein. Since APC co-localizes with
DLG in the cytoplasm in rat colon epithelial cells, the
APC-DLG complex may participate in regulation of cell
cycle progression [57]. Mutant APC lacking the S/TXV
motif for DLG binding exhibits weaker cell cycle blocking
activity at Go/G1 phase than the intact APC [58].

Interaction of APC with B-catenin and through B-catenin
interaction with the members of the cadherin family of
proteins have been implicated in cell-cell adhesion
[24,59]. As shown in Figure 4, the C-terminal domain of
E-cadherin interacts with P- and y-catenin, which associate
with a-catenin and form an E-cadherin complex with
actin cytoskeleton. This complex maintains the stable cell-
cell adhesion [59]. APC becomes a part of the cell-cell
adhesion complex linked with E-cadherin, since it directly
binds with B-catenin, y-catenin, and actin filament
[24,60]. The tyrosine phosphorylation of B-catenin by epi-
dermal growth factor (EGF), hepatocyte growth factor
(HGF) and c-Met receptors is important in modulating
cadherin-catenin complexes from membrane bound form
to free cytosolic form [61]. The phosphorylation of B-cat-
enin at tyrosine residue, which is blocked by tyrosine
phosphatase Pez, is involved in epithelial cell migration
[62]. If the Wnt-pathway and the EGFR or c-Met receptors
pathway are activated at the same time, then the degrada-
tion of B-catenin can be inhibited and it may translocate
to the nucleus, bind to the Lef-Tcf transcription factor, and
down-regulate the transcription of E-cadherin gene,
CDH1, expression [63]. These complex interactions may
finally result in the reduction in E-cadherin-mediated cell-
cell adhesion and proliferation of cells [64-66].

Another important role for APC is assigned in cell migra-
tion. Colonic epithelial cells, derived from a committed
stem cell, divide in the lower two-third of the crypts and
migrate rapidly to the surface to form a single layer. Dur-
ing migration, they are differentiated into absorptive,
secretary, paneth and endocrine cells. The function of a
wild-type APC is necessary in maintaining the direction of
upward movement of these cells along the crypt-villus
axis. Loss of wild-type APC functions due to loss of expres-
sion or mutations affect cell migration. These cells,
instead of migrating upwards towards the gut lumen,
migrate aberrantly or less efficiently towards the crypt
base where they accumulate and form polyps [67]. In due
time, these cells become aneuploid due to defects in chro-
mosome segregation as well as acquiring B-catenin stabi-
lization and activation of genes for cell proliferation. The
mechanisms by which APC might be involved in cell
migration can be understood by its association with the
kinesin superfamily-associated protein KAP3 that has
been established in cell-cell adhesion and migration. It

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Molecular Cancer 2003, 2


A. Normal colonic epithelial cells

B. Colon cancer cells

Figure 4
Mutations in APC gene impair actin cytoskeletal integrity, cell-cell adhesion and cell migration properties of
colon cancer cells. Panel A shows the organization of cell-cell adhesion and cytoskeletal proteins in normal colonic epithe-
lial cells. The APC gene product is shown to bind through its armadillo repeat domain to ASEF. Endogenous APC co-localizes
with ASEF in colonic epithelial cells. APC regulates guanine nucleotide exchange factor (GEF) activity of ASEF and maintains
actin cytoskeletal network, cell-cell adhesion and cell migration. Panel B shows how mutations in the APC gene product may
impair actin cytoskeletal network, cell-cell adhesion and cell migration properties of colon cancer cells. Mutations in APC gene
product disrupt binding with actin and P-catenin; thus it affects actin cytoskeletal network and cell-cell adhesion, respectively.
ASEF binds with most APC mutant proteins containing armadillo repeat sites, stimulates its own GEF activity, and promotes
cell migration.

has been shown that APC, mediated by KAP3, interacts
with kinesin motor proteins which transport it as well as
B-catenin along the microtubules to the growing ends of
the cytoskeletal protruding into motile cell membranes
[68,69]. At the tip of the microtubule, APC interacts with
the end-binding protein EB1 and protein tyrosine phos-
phatase PTP-BL. PTP-BL modulates the steady state levels
of tyrosine phosphorylations of APC associated proteins
such as B-catenin and GSK3B. In fact, GSK3B kinase activ-
ity has been implicated in microtubule dynamics [70,71].

Recently, experimental evidence was presented describing
the mechanisms by which the mutated APC might play a
role in the migration of colorectal tumor cells [72,73]. In

these studies, an interaction of APC has been shown with
APC-stimulated guanine nucleotide exchange factor
(ASEF) that may regulate the actin cytoskeletal network
[72]. APC binds with ASEF and controls its activity. ASEF
is activated in colorectal cancer cells containing truncated
APC. Active ASEF decreases E-cadherin-mediated cell-cell
adhesion and promotes cell migration. Thus, the dynamic
association of APC, EB1, ASEF, catenins, EGFR or c-Met
receptor, PTP-BL and E-cadherin proteins at cell-cell
adherence junctions and microtubule ends play an impor-
tant role in cell-cell communication, cell migration and

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I junctions

_Ca2 Ca+ E-cad


Normal cell growth

Molecular Cancer 2003, 2



Genetic instability in colon cancer progression
Both sporadic and hereditary colorectal cancers exhibit a
defined set of biological and genetic cell heterogeneity
through a series of molecular events. Two specific patho-
logically distinct genetic pathways for colorectal cancer
have been identified the chromosomal instability (CIN)
pathway and the microsatellite instability (MSI) pathway.
The CIN and MSI are associated with two major inherited
syndromes, familial adenomatous polyposis (FAP) and
hereditary non-polyposis colorectal cancer (HNPCC),
respectively. The MSI leads to a 1,000-fold increase in the
rate of subtle DNA changes, whereas CIN enhances the
rate at which gross chromosomal changes occur during
cell division such as chromosome breaks, duplication,
rearrangements, and deletions. The latest findings describ-
ing these two pathways are discussed below.

Mutations in APC gene is associated with chromosomal
instability (CIN)
Aneuploidy, the abnormal number of chromosomes both
quantitatively and qualitatively, is proposed to be a com-
mon characteristic of colon cancer cells. It is believed that
the aneuploidy in colon cancer cells arises during mitosis
through a defective cell division leading to CIN. CIN is a
common feature of about 85% colorectal cancers, and it
has been detected in the smallest adenoma, suggesting
that CIN may occur at very early stages of colorectal cancer
development. The mechanisms) by which CIN is
generated in colon cancer cells is largely unclear. Since the
main feature of CIN is aneuploidy, it has been predicted
that it may arise due to structural changes to the chromo-
somes and abnormal mitosis. In the year 2001, two
different research groups linked mutations in APC gene
with CIN [74,75]. Their results suggested that the wild-
type APC may be involved in maintaining the proper con-
nection of microtubules with chromosomes. APC per-
forms a bridging function between microtubules and
chromosomes. APC binds at the plus end of the microtu-
bule through EB1, stretches it to the chromosomes, and
inserts them into kinetochores after binding with Bub 1.
APC co-localizes to kinetochores and form complexes
with Bub 1 and Bub3, the two mitotic checkpoint proteins
[75]. The successful complex formation may facilitate
proper growth of spindle formation and helps in main-
taining euploidy (Fig. 5A). Once the APC gene is mutated,
the truncated APC protein may loose its ability to bind
with Bubl, and it may become unable to properly main-
tain the attachment of microtubules at kinetochores,
resulting in defect in segregation of chromosomes (Fig.

These studies also pointed out another interesting obser-
vation associated with CIN. It has been found that after
blocking apoptosis in either ApcMi'/+ or Apcl 638T ES cells,
the number of aberrant chromosomes were much greater

than in control ES cells that were unable to undergo apop-
tosis [74]. From these results, a multiple-hit hypothesis of
colorectal cancer development has been suggested. The
chromosome segregation defect in colon cancer cells with
mutated APC gene could lie dormant until an additional
genetic-hit suppresses the mitotic checkpoint or the apop-
tosis of defective cells. In this regard, the proper function-
ing of the hSecurin, a protein which is necessary for the
completion of the anaphase portion of mitosis, is critical
to maintain euploidy, since the loss of hSecurin is often
associated with the loss of chromosomes at a high fre-
quency [76]. Furthermore, telomere dysfunction has been
shown to promote CIN that triggered early carcinogenesis
in the ApcMin/+ Terc-/- mouse models, while telomerase acti-
vation restored genomic stability to a level permissive for
tumor progression [77]. These studies suggested that early
and transient telomere dysfunction is a major mechanism
underlying CIN of human cancer. Thus, multiple factors
may play together in colorectal cancer development from
normal epithelial cells adenoma carcinoma stages.

Mutations in mismatch repair (MMR) genes are associated
with microsatellite instability (MSI)
Microsatellite instability (MSI) is characterized by the size
variation of microsatellites in tumor DNA as compared to
matching normal DNA due to defects in the mismatch
repair (MMR) system. MMR system is critical for the main-
tenance of genomic stability. MMR increases the fidelity of
DNA replication by identifying and excising single-base
mismatches and insertion-deletion loops that may arise
during DNA replication. Thus, the MMR system serves a
DNA damage surveillance function by preventing
incorrect base pairing or avoiding insertion-deletion
loops by slippage of DNA polymerase [78]. Once cells
compromise with these functions, then it may lead to
accumulation of mutations resulting in the initiation of
cancer. The MMR genes are involved in one of the most
prevalent cancer syndromes in humans known as heredi-
tary nonpolyposis colon cancer (HNPCC) or Lynch syn-
drome [79]. HNPCC is characterized as an autosomal
dominant inherited disease with 85-90% gene pene-
trance for early-onset of colorectal carcinoma. HNPCC
accounts for about 5-7% of cases of colorectal carcinoma.
The molecular diagnosis of HNPCC is based on determin-
ing MMR genes for germ-line mutations. There are at least
six different proteins required for the complete MMR sys-
tem. These proteins are hMSH2, hMLH1, hPMS1, hPMS2,
hMSH3 and hMSH6 (GTBP). Recently, mutational germ-
line analysis of hMSH2 and hMLHI genes has been sug-
gested to cause early onset in colorectal cancer patients
[80]. hMSH2 forms a heterodimer with either hMSH6 or
hMSH3 (depending on the type of lesion to be repaired)
and binds to the mismatch site. The complex of hMSH2
with hMSH6 is called hMutSa, and it is required for the
correction of single-base mispairs. The complex of

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Molecular Cancer 2003, 2


A. Cells with wild-type APC

B. Cells with mutated APC

Truncated APC .........."

Figure 5
Chromosomal instability (CIN) in cells carrying mutations in APC gene. Panel A shows a model for the interaction
of APC with plus-end of microtubule through EB I and with kinetochore of chromosome through Bub I in normal colonic epi-
thelial cells. APC can also bind microtubules directly via the C-terminal basic domain. Panel B shows a disruption in the inter-
action between spindle microtubules and kinetochores due to expression of truncated form of APC in colon cancer cells.

hMSH2 with hMSH3 is called hMutS3, and it is required
for the correction of insertion-deletion loops [81]. Subse-
quently, a heterodimer of hMLH1 and hPMS2 proteins
are recruited by hMutSa or hMutSp proteins to the mis-
match recognition complex, which along with other pro-
teins are involved in excision, resynthesis and ligation of
DNA. Mutations in the hMLH1 and hPMS2 have been
found in about 90% of HNPCC cases. Mutations in other
MMR genes have been less frequent in HNPCC patients
[79]. In many sporadic colon cancers, hypermethylation
of the hMLHI gene promoter resulting in its transcrip-
tional silencing has been observed more than mutations.
Both mutations and methylation of hMLHI gene have
been linked with MSI playing a causal role in the initia-
tion ofcolorectal cancer [82-84]. Recently, the CpG island
methylator phenotype (CIMP) pathway has been sug-
gested as a novel mutator pathway that predisposes to

colonic tumorigenesis. In many cases of the sporadic
colon cancers, the MSI can be induced by CIMP, followed
by hMLHI gene's promoter methylation, loss of hMLHI
gene expression, and resultant MMR deficiency [85].
Mutation rate in cells with MMR deficiency are 100-
1,000-fold greater than in normal cells. There are many
targets of MMR gene inactivation; however, the precise
stage of tumorigenesis in which mutation of the wild-type
MMR gene occurs is not clear. There are few well studied
gene targets of MMR, whose mutations in the microsatel-
lite region have been found in gastrointestinal tumors.
These gene targets are the transforming growth factor-P
receptor II (TGF/RII), hMSH3, hMSH6, insulin like
growth factor II receptor (IGFIIR), phosphatase and ten-
sion homologue deleted on chromosome ten gene
(PTEN), caspase 5, Bloom (BLM) syndrome helicase, T-

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Molecular Cancer 2003, 2


cell factor 4 (Tcf-4), the cell cycle regulator E2F4, and
antiapoptotic gene Bax [86-95].

Altered TGFf signaling in MSI cells
In colon cancers, transforming growth factor-P (TGFB)
signaling potently inhibits the growth of normal epithe-
lial cells, since the tumor cells are frequently resistant to
TGFB; they cause pre-neoplastic lesions, increase motility
and spread cancer. The structural basis for TGFB resistance
in colon cancers is defined due to somatic mutations that
inactivate TGFp receptor II (TGFPRII) [86]. In human
colon cancer cell lines with high rates of MSI, the muta-
tions in the TGF/fRII gene was found [96]. These are pri-
marily frameshift mutations that add or delete one or two
adenine bases within or from 10 base-pair poly(A) repeat
in the cysteine-rich coding region (codons 125-128) of
the TGF/fRII gene. Mutations in this region consist of 1- or
2-base deletions or insertions. The other microsatellite
region in the TGF/fRII gene is a poly(GT)3 that was found
with insertion of an extra GT in this region [86,96,97].
These mutations produce truncated proteins that lack the
cytoplasmic domain. As much as 90% of the colorectal
cancers with MSI have the mutated TGFfRII gene. TGF-
PIIR responses are connected with Smads, tumor suppres-
sor gene products, which help to initiate TGFP-mediated
gene transcription (Fig. 6). The transcriptional regulation
of Type 1 plasminogen inhibitor (PAI-1) and cyclin
dependent kinase inhibitor p15 genes are controlled by
TGFp signaling. PAI-1 is the primary inhibitor of tissue-
type plasminogen activator (tPA) and urokinase-type 1
plasminogen activator (uPA) [98]. TGFp inhibits cell pro-
liferation by inducing a GI-phase cell cycle arrest acting
through increased expression of p15 [99]. Thus, the loss of
TGFPIIR or Smad4 can abolish TGFP-signaling and advo-
cate cell proliferation and development of colorectal can-
cer. In a mouse ApcMin/+ mouse model, the combination of
Smad4 and APC gene mutations have been shown to cause
rapid tumor formation of the benign lesions arising only
from the APC gene deficiency [100]. These studies, reiter-
ate the human data that put forward the hypothesis that
mutations in the TGFIIR gene contribute to adenoma -
carcinoma stage progression in colon.

Mutations in IGFIIR gene product augments TGFfPI
signaling in MSI cells
Insulin like growth factor II receptor (IGFIIR) plays a crit-
ical role in cell growth, survival and differentiation;
however, once mutated, these receptors play a major role
in tumorigenesis [101,102]. The IGFIIR/mannose-6 phos-
phate receptor (M6PR) interacts with both IGF-II and
latent TGFp 1 ligands and acts as a tumor growth suppres-
sor [103]. The IGFIIR performs its growth suppressive
function by two different mechanisms. In one case, it
binds and stimulates the plasmin-mediated cleavage and
activation of the latent TGFP1; in the second case, it

participates in the internalization and degradation of its
own ligand IGFII, a mitogen [104]. The IGFIIR gene har-
bors several microsatellites within its coding region; one
of them is poly(G), repeat which is often mutated with 1-
or 2-base pair deletions or insertions. These mutations
produce frameshifts and premature stop codons [89].
Thus, the loss of IGFIIR function due to mutations in MSI
cells may enhance cell proliferation due to accumulation
and activation of IGFII and down-regulation ofTGFP1.

Mutations in PTEN gene causes activation of P13K and
Akt oncogenic signals in MSI cells
The PTEN tumor suppressor gene, also known as mutated
in multiple advanced cancers (MMAC) or TGFP-regulated
and epithelial cell-enriched phosphatase (TEP-1), is
located on chromosome 10q23. A homologous deletion
of this gene has been found in sporadic glioma, endome-
trial, melanoma, prostate, renal, meningioma and small
cell lung carcinomas. Germ-line mutations in PTEN gene
is linked with autosomal dominant inherited cancer
syndromes such as Cowden's disease, Lhermitte-Duclos
disease, and Bannqayan-Zonana syndrome, Bannayan-
Riley-Ruvalcaba syndrome, Proteus syndrome, and Pro-
teus-like syndromes [105-110]. In endometrial and color-
ectal cancers, the mutations in PTEN gene occur in MMR
function-deficient cells [109,111]. The coding region of
the PTEN gene contains several microsallites, among
which a repeat of poly(A), tract in exon 7 and 8 are most
susceptible to frameshift mutations [112]. PTEN is
involved in cell cycle arrest and apoptosis through nega-
tively regulating the survival signaling mediated by phos-
phatidylinositol 3-phsophate (PIP3) kinase (PI3K) and its
down-stream target, a serine/threonine kinase Akt (also
called protein kinase B) [113,114]. Once cells receive sig-
nals through growth factors, hormones, or extracellular
matrix components, the Akt is recruited by PIP3 to the
plasma membrane, where it is phosphorylated at the
Ser473/Thr308 [115]. The phosphorylated Akt is involved
in cell survival, proliferation and migration. Thus, the loss
in PTEN causes elevation of intracellular PIP3 level that
stimulates Akt kianse activity. The active Akt leads to tum-
origenesis due to loss of proliferative and apoptotic con-
trols of cell growth. The genetic data of PTEN gene
mutations found in human tumors were further sup-
ported by animal studies. In a mouse model study, the
PTEN-- homozygous null mice was found to be embry-
onic lethal; however, the PTEN-/+ mice developed
neoplasm in various organs, including breast, prostate,
endometrium, adrenal, lymphoid, skin, colon, thyroid,
liver and thymus [116].

Mutations in E2F4 gene looses cell cycle control in MSI
The ability of pRb to regulate cell cycle progression is
dependent upon its interaction with E2F/DP family of

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Molecular Cancer 2003, 2


A. Normal colonic epithelial cells

TGFP I ......

B. Colon cancer cells

"*'.. h #


hMLH1 gene

Cell membrane

No Smad complex

Nucleus K




Figure 6
TGFP signaling in normal colonic epithelium and cancer cells. Panel A describes the functional pathway of TGFP sig-
naling in normal colonic epithelial cells. TGFp ligand binding with TGFPRII recruits TGF3RI into a tetrameric receptor complex
resulting in transphosphorylation and activation of TGF3RI. After phosphorylation, the TGF3RI becomes an active kinase
which phosphorylates Smad 2 and Smad3. SARA (Smad Anchor for Receptor Activation), a scaffolding protein facilitates inter-
action between Smad2, Smad3 and TGF3RI. Phosphorylated Smad2 and Smad3 allow the formation of homo- and het-
erodimerization complex, including Smad4. The Smad complex then translocates into nucleus, binds with DNA, and stimulates
the expression of target genes including p15 (inhibitor of cell cycle kinase that controls cell cycle into GI phase) and PAI-I
(inhibitor of protease I that degrades extracellular matrix proteins during metastasis). Panel B shows the abnormal pathway
of TGFp signaling in colon cancer cells. Many colon cancer cells with microsatellite instability (MSI) due to defective mismatch
repair (MMR) activity induce mutations in TGF4|RII gene. Often, these are frameshift mutations that insert or delete one or two
adenine bases located within a 10 base-pair polyadenine repeat region (base-pairs 709-718, codons 125-128; referred to as
BAT-RII) of TGF4|RII gene. These mutations encode TGFpRII proteins truncated between 129 and 161 amino acids of the cyto-
plasmic domain which causes functional inactivation of these proteins. Thus, the loss of TGFB signaling may abolish cell cycle
control and induce metastasis of colon cancer cells by inhibiting p15 and PAI-I genes expression, respectively.

transcription factors which are required for the regulation
of a large number of genes involved in cell proliferation
[117-119]. There are at least six E2F proteins which can be
categorized into distinct subgroups that act in direct
opposition to one another to promote either cellular pro-
liferation or cell-cycle exit and terminal differentiation.
The E2F1, E2F2 and E2F3 proteins can be grouped in one
subgroup since they all directly interact with pRb and are

strong transcriptional activators. The E2F4 and E2F5 pro-
teins are comparatively less strong transcriptional activa-
tors or sometimes act as repressors by recruiting the
pocket proteins (BOX 2) and their associated histone-
modifying enzymes. The E2F6 acts as a repressor through
its interaction with Bmil-containing Polycom group of
proteins [120-122]. The E2F4 and E2F5 transcription fac-
tors are also different than their other family members in

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Molecular Cancer 2003, 2



that these factors lack the cyclin A binding domain.
Furthermore, there is another critical feature in the E2F4
gene which is distinct from its family members. The cod-
ing region of the E2F4 gene contains a longer spacer
segment of a repeated sequence of trinucleotide (CAG)I3
that encodes 13 consecutive serine residues; this region is
highly vulnerable to frameshift mutations in MSI cells. In
MMR defective colorectal tumors, mutations in the E2F4
gene have been observed [91]. Thus, defective E2F4 gene
expression in MSI colon cancer cells can possibly promote
cell cycle progression through Go/Gj transition.

Mutations in pro-apoptotic gene Bax in MSI cells
Approximately 50% of the tumors with MSI are found
with mutations in the Bax gene. These mutations produce
frameshift mutations within a coding region of eight
nucleotide stretch of guanine residues (G8) [93,123]. Bax
heterodimerizes with anti-apoptotic protein Bcl2 and
induces apoptosis. In the presence of apoptotic signals,
BH3 domain proteins tBid are activated and bound with
Bax. The interaction of tBid with Bax brings conforma-
tional changes in Bax, and this promotes its translocation
to the mitochondria. Bax oligomerizes and inserts into the
outer mitochondrial membrane where it forms channels
to release cytochrome c (Cyt c) into the cytoplasm. The
released Cyt c forms a complex called apoptosome with
Apaf-1, dATP and pro-caspase 9 [124]. The apoptosome
recruits and processes pro-caspase 9 to form a holoen-
zyme complex, which in turn recruits and activates the
effector caspases leading to apoptosis. Thus, the expres-
sion of mutated Bax protein may fail to release Cyt c and
increase the Bax-Bcl2 ratio resulting in the escape from
apoptosis and inducing initiation of colorectal

Mutations in Bloom (BLM) syndrome helicase gene in MSI
Bloom syndrome (BS) is a rare autosomal recessive inher-
ited cancer predisposition caused by inactivation of the
RecQ family helicase, BLM [125,126]. The genetic disor-
der of the BLM gene is characterized by growth deficiency,
impaired fertility, proportional dwarfism, sun-sensitive
telangiectatic erythema, immunodeficiency and prema-
ture ageing. The function of the BLM helicase is implicated
in reinitiating DNA replication forks that are blocked at
lesions, thereby promoting chromosome stability, which
is characterized by elevated sister chromatid exchanges
(SCEs), as well as chromosomal breaks, deletions, and
rearrangements. Also, BLM helicases perform essential
roles in genetic recombination, transcription, and DNA
repair [127,128]. In all the above systems, the helicase
activities derive energy from hydrolysis of ATP. BLM is a
part of BRCAl-associated genome surveillance complex
(BASC) which includes MSH2, MHS6, MLH1, ATM,
RAD50-MRE11-NBS1 complex, and replication factor C

[129]. BLM also interacts with heterotrimeric, single-
stranded DNA binding protein, replication protein A
(RPA), which after binding, stimulates BLM helicase
activity [130]. The genetic structure of the BLM gene cod-
ing region contains 4,437 base-pairs and encodes 1,417
amino acids. The BLM helicase has different motifs which
are assigned to different activities. Motifs Ia, III and V con-
tain residues that establish direct contact with ssDNA and
motifs I, II-IV and VI form a pocket for binding ATP [131].
It has been observed that many point mutations in BS
patients are confined to these conserved helicase motifs,
thus disrupting its ATPase and helicase activity. DNA
sequencing from the sporadic gastrointestinal tumors
showed abnormal bands caused by a deletion of one ade-
nine residue in the poly-adenine tract (reduction from
nine to eight adenines) in the coding region of the BLM
gene [95,132,133]. Since these tumors had defective MMR
genes, mutations in the BLM gene is directly linked with
MSI and chromosomal instability pathways. Mutations in
BLM gene has been associated with frameshifts in Bax,
hMSH6 and/or hMSH3 with multiple unstable mono- and
trinucleotide repeats located in coding regions that were
significantly higher than that observed in microsatellite
mutator phenotype positive tumors without BLM
frameshifts. From these studies, it has been suggested that
BLM loss of function by MSI in sporadic gastrointestinal
tumors might be an intermediate mutational event, which
may increase the instability of a pre-existent unstable

Mutations in T-cell factor 4 (Tcf-4) gene in MSI cells
In Figure 3, disruption of the APC/B-catenin pathway is
shown as a critical step in the development of colorectal
and other cancers. The oncogenic B-catenin translocates
into nucleus, heterodimerizes with the Tcf/Lef family of
proteins, and transcribes target genes. The transcription
factor Tcf-4 is one of the Tcf-family proteins, which forms
a transcription complex with B-catenin and plays an
important role in maintenance of normal epithelial
growth and development of colorectal tumors [35]. The
Tcf-4/B-catenin transcriptional activity is inhibited by pro-
tein Icat (inhibitor of B-catenin and Tcf-4), which blocks
the interaction between B-catenin and Tcf-4 and thereby
antagonizes Wnt-signaling [134]. Since the APC/Tcf-Lef/
B-catenin pathway plays an important role in colorectal
cancer development, mutational analysis of these genes is
critical to evaluate their functions. The mutations and
their consequences in APC and CTTNB (B-catenin) genes
are discussed earlier, which are characteristics of CIN.
However, the mutations in the Tcf-4 gene were found in
MSI cells [135-137]. In MSI cells, an error-prone poly(A),
monorepeat in exon 17 of the Tcf-4 gene frequently shows
frameshift mutations by deletion of one nucleotide pro-
ducing a short and medium isoform of the Tcf-4 protein.
The transactivating properties of these shorter Tcf-4

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Molecular Cancer 2003, 2


isoforms in the development of colorectal cancers are still
not clear. Recently, it has been suggested that the
frameshift mutations in the poly(A), region of the Tcf-4
gene may in fact produce a gain in function of the shorter
Tcf-4 isoform due to loss of binding sites of the transcrip-
tional suppressor molecules, CtBP or Grg/TLE [138-140].
These mutated Tcf-4 proteins also lose the binding of
chromatin remodeling complexes containing Osa/
Brahma [141]. Thus, based on these studies, it is expected
that mutated Tcf-4 protein may acquire increased tran-
scriptional activity by increased binding affinity for 3-cat-
enin or by facilitating the structural organization of
chromatin [142]. Finally, it has been suggested that muta-
tions in the Tcf-4 gene may not have significant effect and
thus it may contribute marginally to the colorectal car-
cinogenesis or act as passenger mutations [140].

Development of colorectal cancer is a complex and multi-
step process in which several gene defects coordinate with
each other in genotypic and phenotypic outcome. Muta-
tions in many tumor suppressor and proto-oncogenes in
the development of sporadic and hereditary colorectal
cancers are well established; however, their precise role in
this process is still not clear. For example, it is now estab-
lished that mutations in the APC gene may be necessary
for the early onset of FAP. Mutations in the APC gene per-
haps set a stage for mutations in other genes such as K-ras,
DCC, and p53. However, the mechanisms) by which APC
gene mutations may contribute to the accumulation of
mutations in these genes that are associated with the
colon cancer progression remains unclear. Mutations in
the MMR genes can be linked to increased rate of muta-
tions; however, studies indicate that MMR negative cells
develop resistance to apoptosis rather than accumulation
of mutations. Thus, MMR gene mutations and its role in
MSI and apoptosis need further investigation in context
with apoptosis and cell cycle-related genes. These studies
will provide a better understanding of colorectal cancer
development and its intervention by genetic or chemo-
therapeutic means.

Authors' contributions
SN drafted the paper. DR provided suggestions for its
finalization. Both authors read and approved the final ver-
sion of the manuscript.

The work in our laboratory on APC is currently supported to S. N. by NCI-
NIH (CA-09703 I, CA- 100247) and Flight Attendants Medical Research
Institute, Miami, FL. I thank the members of my laboratory for their com-
ments and to Nirupama Gupta and Mary Wall for their critical reading of
this manuscript. I apologize to those colleagues whose work was not cited;
instead, many of them have been accommodated in referenced review

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