Alsnlasnf

REVIEWS
APC, SIGNAL TRANSDUCTION

AND GENETIC INSTABILITY IN
COLORECTAL CANCER
Riccardo Fodde*, Ron Smits* and Hans Clevers
Colorectal cancer arises through a gradual series of histological changes, each of which is
accompanied by a specific genetic alteration. In general, an intestinal cell needs to comply with
two essential requirements to develop into a cancer: it must acquire selective advantage to allow
for the initial clonal expansion, and genetic instability to allow for multiple hits in other genes
that are responsible for tumour progression and malignant transformation. Inactivation of APC
the gene responsible for most cases of colorectal cancer might fulfil both requirements.
* Department of Human
and Clinical Genetics,
and Center for Biomedical
Genetics, Leiden University
Medical Center,
P.O. Box 9503, 2300 RA
Leiden, The Netherlands.
Department of
Immunology, and Center
for Biomedical Genetics,
University Medical Center
Utrecht, P.O. Box 85500,
3508 GA Utrecht,
The Netherlands.
Correspondence to R.F.
e-mail: r.fodde@lumc.nl
The adenomatous polyposis coli (APC) gene can be

regarded as the gene for colorectal cancer. Germ-line
mutations in the APC gene result in familial adenomatous polyposis (FAP)1, one of the principal hereditary
predispositions to colorectal cancer. Somatic APC mutations are also found in most sporadic colorectal
tumours2. Analysis of APC and its protein product has
revealed a broad spectrum of functions, ranging from
control of the WNT signal transduction pathway, to cell
adhesion, migration and apoptosis, and chromosomal
segregation at mitosis35. Inactivation of APC function
seems to underlie both tumour initiation (selective
advantage through constitutive activation of the WNT
signal transduction pathway) and promotion (enhanced
mutation rate through chromosomal instability) in the
large bowel. This is relevant in view of the ongoing
controversy about the function of selective advantage
versus genetic instability in tumour initiation and progression68. Is tumorigenesis an evolutionary process that
is mainly driven by selection, or does an increased mutation rate itself cause a tumour to arise from a normal cell?
How does loss of APC function influence these processes,
and can we reconcile the selective advantage and genetic
instability theories in colorectal cancer?
The adenomacarcinoma sequence
Colorectal cancer is one of the most common malignancies among populations in the United States and
Western Europe, and one of the leading causes of

worldwide morbidity and mortality due to cancer. In
the United States alone, approximately 140,000 new
cases are registered each year, and its mortality exceeds
50,000 (REF. 9). In Europe, about 213,000 new cases and
110,000 deaths are reported each year10. The lifetime
colorectal cancer risk in the general population is 5%,
but this figure rises dramatically with age: by the age of
70, approximately half the Western population will
have developed an adenoma. In general, the incidence
of colorectal cancer is high in developed countries,
with incidence rates varying up to 20-fold between
high- and low-risk geographical areas throughout the
world10. These variations are likely to result mainly
from environmental and dietary factors.
Colorectal carcinomas arise through a series of
well-characterized histopathological changes (BOX 1)
as a result of specific genetic hits in a handful of
oncogenes and tumour-suppressor genes. At least four
sequential genetic changes need to occur to ensure
colorectal cancer evolution (BOX 1). One oncogene
(KRAS) and three tumour-suppressor genes (APC,
SMAD4 and TP53) are the main targets of these
genetic changes. The dominant or recessive nature of
these genes predicts that at least seven mutations are
required: one oncogenic mutation at KRAS and six
additional ones to inactivate both alleles of the above
tumour-suppressor genes1,11. The tumour-suppressor
NATURE REVIEWS | C ANCER
VOLUME 1 | OCTOBER 2001 | 5 5
2001 Macmillan Magazines Ltd
REVIEWS
Box 1 | Histopathology of colorectal cancer

Intestinal epithelial crypts
Aberrant crypt focus
APC
Adenoma
KRAS
Other oncogenes?
Carcinoma
SMAD2/SMAD4
TP53
Chromosome
Chromosome
18q LOH
17p LOH
Nuclear -catenin levels and chromosomal instabilty
Throughout the large intestine, a monolayer of epithelial cells lines tubular glands or crypts. These are invaginations
of the epithelium that effectively increase the surface area of the colon and rectum. The small number of stem cells
located at the base of each crypt give rise, by asymmetric division, to four cellular types: columnar absorptive cells,
goblet cells (mucus-secreting), and neuroepithelial cells, and paneth cells, which are occasionally present at the base
of crypts in the caecum and ascending colon. Dividing cells are located in the lower third of the crypt, whereas the
differentiated types are found in the upper two-thirds. The cells continuously migrate upwards and are eventually
exfoliated into the lumen by an apoptotic mechanism. The process of epithelial renewal takes 36 days142. Cell
mitotic rates in the colonic epithelium equal loss rates. Intestinal tumours are the result of an increase in this
gain:loss ratio. The earliest manifestations of colorectal neoplasia are the aberrant crypt foci or ACF143 only visible
by methylene blue staining or by microscopy. ACF usually encompass few crypts and can be composed either of cells
of normal morphology (nondysplastic), or dysplastic cells (see figure). The latter are more likely to progress to
become a polyp a benign tumour mass that protrudes into the lumen from the intestinal epithelium144 (see
figure). Polyps, like their ACF counterparts, can be of two types: hyperplastic (nondysplastic) and adenomatous
(dysplastic). Hyperplastic polyps preserve normal architecture and cellular morphology, whereas adenomatous
polyps are characterized by abnormalities in both inter- and intracellular organization. The epithelium is organized
in multiple layers, nuclei are enlarged, and their alignment at the basal membrane is lost (see figure). (Figures
reproduced with permission from WebPath, courtesy of Edward C. Klatt M.D., Florida State University College of
Medicine, and from REF. 144 (1998) Massachusetts Medical Society.)
DYSPLASIA
Disordered growth: that is,

an abnormally organized cell.
The alterations include size,
shape (pleomorphism),
hypochromatic nuclei, and
also architectural orientation
of adult cells, generally
representing a premalignant
stage.
LOSS OF HETEROZYGOSITY
(LOH). In cells that carry a

mutated allele of a tumoursuppressor gene, the gene
becomes fully inactivated when
the cell loses a large part of the
chromosome carrying the
wild-type allele. Regions with
high frequency of LOH are
believed to harbour tumoursuppressor genes.
56
gene mutations are found in most tumours, whereas

KRAS mutations are found in approximately 50%
of cases.
Mutations in the APC gene are the earliest genetic
alteration in the genesis of colorectal tumours and
seem to be required to initiate clonal evolution2. The
temporal order, rather than the accumulation, of
these genetic changes along the tumorigenic process
is of paramount importance for both tumour morphology and the likelihood of tumour progression.
APC mutations are found in the very earliest stages of
the adenomacarcinoma sequence, known as aberrant crypt foci (ACF; see BOX 1), and are closely associated with the degree of DYSPLASIA of these small
lesions12,13. Both allelic losses and point mutations
have been found in chromosome 5q21 where APC is
localized2,14,15.
Acquired, activating KRAS mutations are found in
approximately 50% of sporadic colorectal adenomas
and carcinomas1618. Notably, KRAS mutations are also
found in a large proportion of nondysplastic lesions
that have a more limited potential to progress to larger
tumours, such as nondysplastic ACF and hyperplastic
polyps12,19. Apparently, the synergistic action of the

mutated APC and KRAS genes underlies clonal expansion and dysplasia in the nascent colorectal tumour.
However, the observation that 50% of colorectal cancers do not have KRAS mutations indicates that mutations in other unknown oncogenes might be involved
in a substantial proportion of cases. Alternatively, other
epigenetic and/or mutation mechanisms might promote APC-driven tumorigenesis in the absence of the
activation of KRAS or other oncogenes.
LOSS OF HETEROZYGOSITY (LOH) studies have been pivotal in the identification of the tumour-suppressor
genes that are involved in colorectal tumorigenesis18,2022. LOH in chromosomes 5q, 8p, 17p and 18q
was found at high frequencies, presumably centered
around the following tumour suppressor genes: APC
in 5q, TP53 in 17p, and SMAD4 in chromosome 18q.
Loss of chromosome 17p is found in approximately
75% of colorectal carcinomas, but infrequently in
benign lesions, indicating that loss of p53 function is
involved in late progression rather than initiation18,23.
Interestingly, LOH of chromosome 17p is nearly
always associated with the presence of missense
| OCTOBER 2001 | VOLUME 1
www.nature.com/reviews/cancer
REVIEWS
Box 2 | The TGF- signal transduction pathway in colorectal cancer
Cofactor
TGF-
Mutations in members
Plasma membrane
TGF- receptor
of the transforming
II I
growth factor- (TGF-)
ATP
ADP
signalling pathway are
thought to have a ratelimiting role in
R-SMAD
R-SMAD
P
colorectal cancer26.
co-SMAD
Depending on the tissue
type, TGF- can
SMAD
Nuclear
stimulate or inhibit cell
Nuclear membrane
pore
proliferation,
differentiation, motility,
adhesion or
co-SMAD
Transcription
Ubiquitylation
apoptosis153. In the
of target genes
R-SMAD
and degradation
colorectum, TGF- and
DNA
related cytokines exert a
potent inhibitory effect
Cellular response:
on cell growth. This is
Differentiation
Growth inhibition
mediated by a
Deposition of extracellular matrix
heterodimeric receptor
Apoptosis
(comprising a type I and
a type II subunit), which
is a ligand-activated Ser/Thr kinase, and the substrates of these receptors, the SMAD
proteins. Binding of TGF- to its receptor activates the type I receptor kinase activity,
which phosphorylates receptor-specific SMADs (R-SMADs), such as SMAD2.
Phosphorylated R-SMADs are then translocated to the nucleus by a co-SMAD such as
SMAD4. The SMAD complex, in conjunction with other DNA-binding proteins, then
activates (or represses) transcription of specific target genes. Although some of these
have been identified (p21, PAI1, JUNB), the intestine-specific TGF- targets have not
yet been investigated.
In general, tumours acquire TGF- resistance at a relatively late stage26. But germ-line
mutations in SMAD4 and BMPR1A (encoding another receptor in the TGF- pathway)
have been identified in juvenile polyposis. This is an inherited predisposition to
hamartomatous polyps, lesions in which the affected tissue is essentially normal, but is
arranged in a disorganized fashion145,146. More evidence of SMAD genes being involved
in intestinal tumour initiation and progression comes from genetically modified mice:
Smad3- and Smad4-mutant mice develop intestinal cancer147149, whereas
Apc+/716/Smad4+/ compound heterozygous mice develop intestinal polyps that are
more malignant than those in the simple Apc+/716 heterozygotes150. Although
previously regarded as innocuous lesions, colorectal hyperplastic polyps are now
recognized as an important pre-cancerous condition151.
Molecular genetic analysis of hereditary non-polyposis colorectal cancer (HNPCC)
has underscored the rate-limiting function of TGF- signalling in colorectal cancer.
The coding sequence of the type II TGF- receptor contains an (A)8 repeat that makes
it a preferential target for mutations in mismatch repair (MMR)-deficient tumours.
Indeed, frameshift mutations of this repeat are found in most tumours with
microsatellite instability (MIN)152. (Figure reproduced with permission from REF. 153
(2000) Macmillan Magazines Ltd.)
KNUDSONS TWO-HIT-MODEL
Proto-oncogenes become active

oncogenes by a single gain-offunction (activating) mutation,
whereas two inactivating hits
(mutations) are required to
achieve loss of function in a
tumour-suppressor gene.
mutations in the remaining TP53 allele, in line with

KNUDSONS TWO-HIT MODEL for tumour-suppressor
genes24. Similar LOH frequencies that is, approximately 50% of large adenomas and 75% of malignant
colorectal cancers have been found for chromosome 18q (REFS 18,2022). The identification of the
tumour-suppressor gene(s) around which 18q losses
are centred has been challenging, as several candidate
genes map to this chromosomal segment. The
SMAD2 (JV18-1/MADR2) and SMAD4 genes are
now regarded as the most likely tumour suppressors.
SMAD4, also known as DPC4 (deleted in pancreatic

cancer)25, encodes a key signalling molecule within
the growth-suppressing transforming growth factor
(TGF-) pathway26, (BOX 2). Loss of SMAD4 function, by LOH, deletions and point mutations, has
been found in pancreatic and colorectal carcinomas25,27. SMAD2 also maps to chromosome 18q, and
encodes another member of the TGF- pathway that
is specifically mutated in a subset of colorectal carcinomas28. Although additional studies will be necessary to elucidate precisely the role of these and other
chromosome 18q genes in cancer, SMAD4 and
SMAD2 are likely to represent the main tumour-suppressor genes, inactivation of which drives malignant
progression of colorectal tumours (BOX 2).
The analysis of genetic alterations of these tumour
suppressors and oncogenes at different stages of the
adenomacarcinoma sequence has allowed Vogelstein
and colleagues to develop a model for the clonal evolution of colorectal tumours by the acquisition of sequential mutations11 (BOX 1). At the start of the process, inactivation of both alleles of the APC gene triggers the
adenomatous process by providing the nascent tumour
cell with a selective advantage to allow clonal expansion.
However, an additional oncogenic mutation often in
KRAS is required for adenoma growth and progression. Subsequent clonal expansion and malignant transformation is driven by additional mutations and allelic
losses in TP53, SMAD4 and other 18q tumour-suppressor genes. Although additional mutations in as yet
unidentified genes might be necessary for colorectal
cancer development, the model predicts that at least
seven genetic hits are required. This is reflected by the
long time 2040 years that the whole process
takes to develop an invasive carcinoma from the smallest observed lesion, the ACF. The situation seems to be
different in mice, in which no somatic mutations in the
Trp53 (mouse TP53) and Kras genes were found in
intestinal tumours derived from Apc-mutant animals29
(see also BOX 3). This might be explained by physiological and genetic differences between humans and mice
(for example, the shorter lifespan in mice, and/or the
inbred genetic backgrounds in which the morbid
phenotypes of targeted mouse mutations are analysed).
Selection or genetic instability? The clonal evolution
model is in agreement with the concept of selection
being the main driving force behind tumour initiation
and progression7. However, it has been argued that
endogenous mutation rates are not sufficient to allow
the high number of genetic hits found in human cancers, implying that genetic instability, in the form of a
mutator phenotype, is an essential requirement for
cancer to occur8,30. A third hypothesis was proposed by
Duesberg and colleagues31,32, according to which aneuploidy, rather than gene mutations, can explain genetic
instability, as it generates abnormal chromosomes in an
autocatalytic fashion, thereby effectively unbalancing
large numbers of genes and explaining the complex
changes in cellular phenotypes observed in cancer.
Colorectal cancer offers a unique model to show how
REVIEWS
Box 3 | APC mouse models

E E
E I
EI
NH2
Structure of gene product
*** * *
**
COOH
Wild-type Apc
Mouse models
Tumour burden Extra-intestinal

tumour burden
in GI tract
Apc+/716
>100
Apc+/Min
>100
+/
Apc+/1638N
56
+++
Apc+/1638T
Tumour free
Tumour free
E E
E E
(12%)
E E
MISMATCH REPAIR
A system to repair base-pair

mismatches that can occur,
for instance, during DNA
replication. Mutations in genes
that encode components of the
mismatch-repair machinery
result in microsatellite
instability (MIN).
BUB1
This gene encodes one of the

components of a complex that
localizes to chromosomal
kinetochores and mediates the
mitotic-spindle checkpoint.
This checkpoint inhibits the
metaphase to anaphase
transition until all the
chromosomes are properly
attached to the mitotic spindle.
58
Mouse models for intestinal cancer have been generated by introducing specific germ-line mutations in the mouse Apc
gene29. The availability of these mice on the same inbred genetic background (C57BL/6J (B6)) allows the comparison
of their phenotypes and the establishment of precise genotypephenotype correlations. Analysis of these mice has also
provided important clues to the function of APC in homeostasis and tumorigenesis.
The best-known model is the ApcMin mouse, which was generated by random ethylnitrosourea mutagenesis. ApcMin
carries a nonsense mutation in codon 850 of the Apc gene, leading to a truncated Apc polypeptide of approximately 95
kDa. Heterozygous Apc+/Min (Min) animals develop more than 100 intestinal tumours per animal, mainly located in the
upper gastrointestinal (GI) tract. The wild-type Apc allele is lost, together with the entire chromosome 18 in most of
the lesions. ApcMin mice also develop some extra-intestinal tumours.
The Apc716 mouse model was generated by introducing a neomycin cassette in Apc codon 716, resulting in a
truncated protein of approximately 80 kDa. Similar to Min, this mutation leads to many upper GI adenomas, but
extra-intestinal lesions have also been reported.
The Apc1638N mouse was generated by introducing the neomycin gene in codon 1638, in the transcriptional
orientation opposite to that of Apc. For unknown reasons, homozygous Apc+/1638N cell lines contain very low amounts
(12%) of the expected 182 kDa truncated protein. Apc+/1638N mice develop, on average, 56 intestinal tumours per
mouse, and a broad spectrum of extra-intestinal manifestations, including multifocal desmoids and cutaneous cysts.
A similar targeting construct was used to generate the Apc1638T mouse, but the cassette is inserted in codon 1638 in
the same transcriptional orientation as Apc. This leads to expression of a truncated 182 kDa Apc polypeptide in a
1:1 ratio with the wild-type 312 kDa protein. The truncated protein lacks the binding region for tubulin, EB1-like
proteins and DLG (Drosophila discs large). Apc+/1638T mice do not develop tumours. Moreover, unlike all the models
discussed above, homozygosity for Apc1638T is compatible with adult life. Most importantly, homozygous Apc638T
animals do not show any increased tumour susceptibility. E, nuclear export signal; I, nuclear import signal
(Figure adapted from REF. 29. (2001), with permission from Elsevier Science.
different types of genetic instability contribute to

tumour initiation and progression. Two forms of genetic instability have been described in colorectal cancers33:
microsatellite instability (MIN) and chromosomal
instability (CIN). MIN has been widely studied in many
tumour types and is due to defects in the DNA MISMATCH
REPAIR (MMR) machinery. This results in a mutator phenotype at the nucleotide level, and a consequent instability of repetitive sequences such as microsatellites34,35.
Sporadic MIN tumours account for approximately 15%
of all colorectal cancers. Notably, most MIN tumours
are near-diploid and have nucleotide mutation rates
23-fold higher than normal cells3638. CIN is the hallmark of most colorectal cancers, but is incompletely
understood at the molecular level. CIN tumours have a

defect in chromosome segregation, which manifests as
both qualitative and quantitative variations in chromosome numbers among cells from individual clones39,40.
CIN might result from loss of the mitotic checkpoint
gene BUB1 (REF. 41) or from TP53 mutations42. However,
41,
BUB1 mutations are very rare in colorectal carcinomas
and TP53 mutations and LOH occur too late to account
for CIN18, as aneuploid changes have been observed in
very early stages of adenoma4345. More recently, we and
others have found that mutations in APC can give rise
to chromosomal instability4,5 and are likely to elicit CIN
with the synergistic cooperation of other somatically
acquired mutations.
REVIEWS
E E
E I
EI
NH2
COOH
*** * *
**
Mutation cluster region

Oligomerization
DLG/PTP-BL binding
-catenin binding (15-amino-acid repeat)
Armadillo repeat
Nuclear export signal
-catenin downregulaion (20-amino-acid repeat)
Microtubule binding
Nuclear import signal
SAMP (axin/conductin binding)
CDK consensus phosphorylation

site (p34cdc2 binding?)
EB1/RP1 binding
Figure 1 | The adenomatous polyposis coli (APC) protein. Conserved regions, such as the Armadillo repeats, and regions
that interact with other proteins, including tubulin, the microtubule-associated protein EB1, discs large (DLG), -catenin and
axin/conductin, are shown. APC also contains several consensus sites for phosphorylation by p34cdc2, five nuclear export signals
(E) and two nuclear import signals (I). Most somatic mutations occur in the mutation cluster region. Most of these mutations lead
to truncated proteins.
SCF (FOR SKP/CULLIN/F-BOX)

COMPLEXES
In these so-called E3-ubiquitin

ligase complexes, F-box
proteins recognize substrates,
after which ubiquitin is
enzymatically transferred by
the complex to a lysine residue
on the substrate. The ubiquitin
tail marks the substrate protein
for degradation by the
proteasome.
TCF
Transcription factors of the

T-cell factor/lymphoidenhancer factor (LEF) family
that bind to DNA HMG boxes
and constitute the most
downstream components of
the WNT pathway. Mammals
possess four TCF/LEF genes,
whereas worms and flies
each carry one Tcf gene in
their genome.
ADHERENS JUNCTIONS
Intercellular adhesion
structures that tightly seal the
lateral spaces between cells in
simple epithelia. They contain
the intercellular adhesion
molecule E-cadherin, and and -catenin.
Cahill et al.46 have proposed a model to reconcile

genetic instability and Darwinian selection in
tumours. Studies in bacteria have shown that fitness is
achieved through a balance between the positive and
deleterious cellular effects of genetic variability
caused by mutations to allow survival in a selective
environment. At one end of the spectrum, absence of
genetic instability does not allow enough cells to pass
through the first selection barrier along the multistep
pathway to cancer. At the other end, excessive genetic
instability leads to widespread DNA damage, activation of apoptotic pathways and cell death. Only a
moderate form of genetic instability is tolerated by the
cell. Genetic instability accompanies the tumour along
its progression route as it continuously ensures sufficient genetic variability to overcome additional selection barriers46. This just-right instability model might
be active at early stages of colorectal tumorigenesis45,47.
Functional aspects of APC
APC was identified by positional cloning of the FAP

locus48,49. Subsequently, most sporadic colorectal
tumours were found to harbour mutations in both
APC alleles50. The sequence of the large (312 kDa)
APC protein (FIG. 1) did not allow specific predictions
about its intracellular function. The first functional
clues were provided by the identification of -catenin
as a binding partner of APC51,52. Mammalian -catenin
had been cloned as an essential intracellular component of cadherin adhesion complexes, whereas its
Drosophila homologue, termed Armadillo, had been
identified as a downstream component of the
Wingless/Wnt signalling pathway.
The WNT signalling pathway has been assembled
from combined work in flies, frogs and mammals (FIG.
2). In unstimulated cells, free -catenin is destabilized
after binding to the so-called destruction complex,
which consists of the scaffolding proteins axin and conductin, glycogen synthase kinase 3 (GSK3) and
APC5356. The 15- and 20-amino-acid repeats in APC
mediate binding and downregulation of -catenin
respectively, whereas the three SAMP repeats (Ser-AlaMet-Pro) that are interspersed among the 20-aminoacid repeats allow interaction with axin/conductin.
GSK3 is believed to phosphorylate four critical Ser/Thr
residues in the amino terminus of -catenin57. This targets -catenin/Armadillo for ubiquitylation by an SCF
complex, that contains the F-box protein TrCP58,59, and
for subsequent proteasomal degradation.
The serpentine Frizzled receptors60 cooperate with
the low-density lipoprotein-receptor-related protein
LRP6 to bind WNT factors and to inactivate GSK3 in
the destruction complex. This inactivation process is
not well understood, but involves the intracellular protein Dishevelled56. WNT-activated LRP6 might also
directly bind and inhibit axin. As a consequence of
WNT signalling, -catenin becomes stabilized and
shuttles to the nucleus. Once in the nucleus, -catenin
binds to DNA-binding proteins of the T-cell-factor
(TCF) family, to serve as an essential co-activator of transcription61,62. The activity of TCF is tightly controlled,
as TCFs are complexed with potent corepressors, such
as Groucho63,64, in the absence of WNT signalling.
It is not clear why the destruction complex contains
two different scaffolding proteins, APC and axin/conductin. One hypothesis states that they perform distinct
functions: APC might serve to carry -catenin to the
destruction complex, whereas axin might be the central
co-ordinator of the activity of the complex65,66. APC
harbours nuclear localization signals (NLS) as well as
nuclear export signals (NES), although there is still
debate as to which NES sequences are dominant.
Although the presence of NLS and NES sequences indicates that APC is involved in mediating nuclear transport of -catenin, regulation of -catenin nuclear
export/import might be more complex as other factors
are known to be required67.
APC has other functions in addition to controlling
the WNT pathway (FIG. 2). In this regard, -catenin functions not only as a WNT transducer, but also as an essential component of ADHERENS JUNCTIONS, where it provides
the link between E-cadherin and -catenin, which, in
REVIEWS
LRP6
Frizzled
b
Frizzled
LRP6
WNT
Destruction complex
P
GSK3
Dishevelled
-cat
APC
Axin/conductin
GBP
SCF complex
-TrCP
E-cadherin
-cat
-cat
-cat
-cat
APC
Axin/conductin
-cat
Actin cytoskeleton
Adherens
junction
Polyubiquitin
Groucho
Proteasome
-cat
GSK3
-cat
-cat
TCF
No transcription
TCF
Transcription
Figure 2 | The WNT signalling pathway. a | In the absence of a WNT signal, the level of free intracellular -catenin is minimized
by sending it for degradation in the proteasome. Free cytoplasmic -catenin, which is in equilibrium with -catenin at adherens
junctions, is recruited to a destruction complex containing APC, axin/conductin and glycogen synthase kinase 3 (GSK3).
GSK3 phosphorylates -catenin, allowing it to be recognized by an SCF complex containing the F-box protein -TrCP. Other
proteins in the SCF complex catalyse the addition of a polyubiquitin chain to -catenin, allowing -catenin to be recognized and
degraded by the proteasome. Consequently, -catenin cannot reach the nucleus, and cannot co-activate TCF-responsive
genes. Groucho, a corepressor, also prevents the activation of TCF-responsive genes in the absence of -catenin. b | In the
presence of WNT, its receptor, Frizzled, in complex with LRP6, is activated. This leads to a poorly understood signalling cascade
in which Dishevelled activates GBP an inhibitor of GSK3. Consequently, -catenin cannot be targeted for destruction and is
free to diffuse into the nucleus, where it acts as a co-activator for TCF-responsive genes.
turn, binds actin and actin-associated proteins68. APC

might, therefore, control intercellular adhesion by regulating the stability and subcellular localization of catenin. In addition, APC associates with the microtubule cytoskeleton directly69,70. This function involves its
carboxyl terminus and is unrelated to its capacity to regulate the WNT signalling pathway. Accordingly, APC
concentrates in granules at the growing ends of microtubules7173. A domain in the extreme C terminus of APC
mediates binding to microtubule-associated proteins of
the EB/RP family7476. Genetic evidence in yeast implies
important roles for EB/RP proteins in mitosis: the budding yeast homologue Bim1 controls microtubule stability and positioning of the mitotic spindle, and the fission
yeast homologue Mal3 was identified in a screen for
chromosome missegregation77.
APC in cancer
Selective advantage and the second hit. APC, then,

encodes a multifunctional protein that might participate in several cellular processes, such as cell adhesion
and migration, signal transduction, microtubule
assembly and chromosome segregation. However,
despite the fact that each of these roles is potentially
linked with cancer, it seems that the main tumoursuppressing function of APC resides in its capacity to
properly regulate intracellular -catenin levels7880.
60
Moreover, although most colorectal tumours carry

mutations in APC, those with an intact APC gene contain activating mutations of -catenin that alter functionally significant phosphorylation sites79,81.
Mutations in other members of the WNT pathway,
including conductin82 and axin83,84, have also been
shown to be associated with cancer.
Most APC mutations result in truncated proteins
that lack all axin/conductin-binding motifs and a variable number of the 20-amino-acid repeats that are
associated with the downregulation of intracellular catenin levels85,86. In FAP, germ-line mutations are scattered throughout the 5 half of the APC gene; by contrast, most somatic mutations are clustered between
codons 1286 and 1513, the so-called mutation cluster
region (MCR)8587. In agreement with Knudsons twohit hypothesis, inactivation of both APC alleles can be
detected in most intestinal tumours at early stages of
tumour development2,85. However, in its original formulation, Knudsons hypothesis postulates that the two
hits represent independent mutation events, the end
result of which is loss of tumour-suppressing function.
Detailed mutation analysis of tumours from patients
with FAP and Apc+/ mice (BOX 3) has shown that APC
does not entirely follow this model8890. The position
and type of the second hit in FAP polyps depends on
the localization of the germ-line mutation. This is
REVIEWS
Second hit
Germ-line mutation
1
or
or shorter
LOH
or (less frequently)
0
2
or (less frequently)
LOH
Oligomerization
-catenin binding (15-amino-acid repeat)
Armadillo repeat
-catenin downregulation (20-amino-acid repeat)
Figure 3 | Interdependence of germ-line and somatic APC mutations in FAP polyps. Both the position and type of the
second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germ-line mutation. The germ-line
and somatic mutations are represented by their resulting truncated protein. Numbers to the right of the proteins refer to the
number of 20-amino-acid repeats still present in the truncated protein. Germ-line mutations leading to a truncated protein
lacking all the 20-amino-acid repeats acquire somatic hits, resulting in truncated proteins harbouring one or two repeats. If the
germ-line mutation generates a truncated protein retaining only one repeat, most second hits have allelic loss of the wild-type
allele or, less frequently, point mutations resulting in short truncated proteins. On the other hand, if the germ-line mutation results
in a truncated protein with two 20-amino-acid repeats, most somatic hits result in short truncated proteins. In all cases, at least
one of the mutations leaves a truncated protein retaining one or two 20-amino-acid repeats. LOH, loss of heterozygosity.
unexpected in a two-hit scenario , in which any loss-offunction mutation would result in tumour formation.
Apparently, somatic APC mutations are selected based
on the growth advantage they provide to the tumour
cell. However, the limited point-mutation data available
from this original study did not allow the authors to
propose a selection mechanism based on a specific
functional motif within APC 88. An Apc mouse model
specifically engineered to enrich for point mutations as
the main second-hit mechanism has allowed us to
identify the motif that is selectively lost: somatic Apc
mutations are selected that remove all the 20-aminoacid repeats, thereby completely inactivating the catenin-downregulating activity of Apc90. In contrast, in
most human colorectal adenomas, truncated APC proteins are selected that retain one or two 20-amino-acidrepeats. Recently, Albuquerque et al. (C. Albuquerque,
C. Breukel, R. van der Luijt, P. Fidalgo, P. Lage, F. J. M.
Slors, C. Nobre Leitao, R. F. and R. S., unpublished
observations) have analysed in more detail the APC
second hit in more than 100 polyps from several different FAP patients. These results show that the dependence between germ-line mutation and the resulting
spectrum of somatic mutations that successfully lead to
tumour formation is more complex than suggested
previously. Specific APC genotypes are selected during
tumour formation on the basis of the specific level of
residual -catenin downregulating activity that they
can still exert, rather than on the complete inactivation
of this signal transduction regulatory function of APC
(FIG. 3). According to this just-right signalling model,
the signalling function of APC must be impaired to a
specific degree to allow sufficient accumulation of
nuclear -catenin and activation of the downstream

target genes that are relevant for tumour formation in
the intestine. Excessive -catenin accumulation in the
nucleus has been shown to result in programmed cell
death91, and is therefore unlikely to be selected for during tumour formation. The reasons for the species-specific difference between somatic APC mutations in
humans and mice might relate to the absence of
acquired Kras and Trp53 mutations in Apc-mutant
mouse tumours29 and/or to other differences in the
Wnt-signalling downstream response.
Downstream targets of APC/-catenin signalling. The
first two identified downstream targets of the APC/catenin pathway, MYC and cyclin D1, are clearly relevant in tumour formation because of their role in proliferation, apoptosis and cell-cycle progression9294.
Changes in the normal expression pattern of MYC and
cyclin D1 are likely to affect normal intestinal epithelial
renewal by increasing the overall proliferation rate. In
fact, several studies have reported increased numbers of
cycling cells in colorectal tumours9598. The products of
other WNT target genes, such as matrilysin99,100, CD44
(REF. 101), MYC itself102 and the urokinase-type plasminogen activator receptor103, seem more likely to be
involved in tumour promotion rather than initiation.
In the normal intestinal epithelium, nuclear catenin expression is higher in the proliferative compartment, whereas it is decreased in the upper twothirds of the crypt (M. Kielman and R. F., unpublished
observations). Conversely, cytoplasmic APC staining is
markedly increased in post-replicative cells within the
upper portions of the crypt, indicating an increased
REVIEWS
a
APC
-catenin
Villus
Crypt
Figure 4 | Models of colorectal tumorigenesis. a | The

gastrointestinal epithelium contains crypts and villi. Cell
division occurs in the crypts, and cells gradually move up
the crypt walls as they differentiate, eventually being
sloughed off into the gut lumen. APC is highly expressed in
differentiated intestinal epithelial cells (IECs), and its levels
gradually increase as differentiating IECs move up the
cryptvillus axis. Conversely, levels of -catenin decline as
IECs move up the cryptvillus axis. Two models to explain
the origin of colorectal cancer are b | the bottom-up, and
c | the top-down model. In the bottom-up model,
transformation is initiated in a stem cell at the base of the
crypt (arrow), which proliferates, replacing the normal
mucosa with transformed cells from the bottom up. In the
top-down model, transformation is initiated in a fully
differentiated cell, which proliferates and replaces the
normal mucosa from the top down.
62
level of expression with maturation, whereas it is virtually absent in the crypt region where cells are actively
dividing104,105. This pattern of expression is in agreement with the role of -catenin signalling in maintaining stem-cell properties and controlling differentiation
in the intestine106. In the bowel, TCF4 is the main transcription factor that transduces -catenin signals in the
nucleus107. Tcf4/ mice cannot sustain an intestinal
stem-cell compartment, indicating that activation of
downstream targets such as MYC, TCF1, and cyclin D1
are required to maintain proliferative capacity106,108110.
Moving upwards along the cryptvillus axis, an
increase of APC expression counteracts -catenin signalling and allows differentiation. Activation of catenin signalling by APC mutation is therefore likely
to result in an enlargement of the stem-cell compartment and diminished differentiation. Indeed, it is generally accepted that colorectal tumours arise from the
stem cells that are located at the base of the crypts.
However, a recent study has shown that, at early stages
of the neoplastic process, dysplastic cells reside at the
luminal surface of otherwise normal crypts111. These
luminal dysplastic cells carry APC mutations not present in stem cells located at the base of the same crypts.
Although other explanations of these data are still plausible, one model predicts that dysplastic cells spread laterally by replacing the normal pre-existing crypts. This
top-down process has important implications for
understanding the mechanisms underlying tumour
formation in the colorectum (FIG. 4). Whereas activation
of MYC and cyclin D1 in luminal post-replicative cells
is still likely to release their cell-cycle block and stimulate cell proliferation, it is not clear whether de-differentiation of these cells occurs. Intestinal tumours from
both Apc and -catenin mutant mice show a high proportion of undifferentiated crypt-like cells with large
numbers of dividing cells, although all differentiated
cell types are still present112,113.
APC and chromosomal instability. Recent studies have
shown that the C terminus of APC is involved in maintaining chromosomal stability during mitosis4,5. APC
localizes to the kinetochore of metaphase chromosomes,
and this localization is likely to be dependent on the interaction between APC and EB1. Accordingly, Apc-mutant
cells have an abundance of spindle microtubules that fail
to connect to kinetochores and are characterized by CIN
(FIG. 5). The CIN phenotype is also recapitulated by the
induced expression of the EB1-binding domain of APC
in near-diploid colorectal cancer cells4. However, the failure of APC to localize at the kinetochores in mutant cell
lines might also result from the inability of truncated
APC to bind microtubules, and might not be directly
related to EB1 binding. Moreover, Apc/ cells have supernumerary centrosomes, a defect possibly unrelated to the
kinetochore-capture function of APC4. Two types of
chromosomal abnormalities occur in mouse Apc-deficient cells: quantitative changes (near-tetraploidy), presumably arising from non-disjunction defects, and structural rearrangements (chromosomal translocations),
resulting from chromosomal breakage and reunion.
REVIEWS
Notably, both processes seem to occur in most cells. This
is in agreement with a dual function for APC in mitosis:
the proper attachment of the mitotic spindle to the dividing chromosomes at the kinetochore, and the regulation
of centrosome duplication through its interaction with
tubulin and the centrosome5. Whereas loss of the former
function will lead to non-disjunction and tetraploidy,
defects of the latter will result in mitotic cells with multipolar spindles that exert multidirectional forces on the
kinetochore, resulting in chromosomal breakage and
fragmentation114. Thiagalingam et al.40 have observed a
similar dichotomy in a detailed study of chromosomal
losses in human colorectal cancers. Approximately 40%
of the losses were predicted to result from mitotic nondisjuction, but in more than half of the cases, fusions
between different chromosomes were observed that are
likely to derive from double-strand breaks and interchromosomal recombination. However, it should be pointed
out that the data showing that loss of APC function can
elicit CIN were obtained in mouse embryonic stem (ES)
cells, a cellular type in which p53 checkpoint pathways are
compromised by factors affecting p53 nuclear localization and by loss of downstream cell-cycle arrest factors115.
In differentiated cell types, mutations in APC might not
suffice to determine chromosomal instability without the
synergistic cooperation of mutations in other genes.
Notably, the mouse Apc1638T mutation truncates the C terminus of APC that is responsible for its CIN-related function, without affecting the -catenin regulatory motifs. It
is, therefore, important to dissect the role of the two
domains in tumour initiation and progression80.
Homozygous Apc1638T ES cells are CIN4. However, the
corresponding mice are viable and tumour free, which
underscores the importance of the selective advantage
provided by loss of -catenin control in tumour formation, and argues against the ability of chromosomal
instability to initiate the neoplastic process4,80.
Darwinian selection versus genetic instability
A general picture is emerging from the analysis of the

essential roles of the WNT signal transduction pathway in providing selective advantage to the nascent
tumour cell, and of genetic instability to ensure
tumour progression and malignant transformation:
the APC gene, because it encompasses both functions,
has a central, initiating and promoting role in colorectal cancer. Its inactivation, and the resulting constitutive activation of the WNT pathway, provides a strong
selective advantage by affecting cell proliferation,
migration, apoptosis, and, possibly, differentiation of
the intestinal stem cell. Subsequently, mutation of
KRAS or another synergistic event might allow the
mutant APC to induce CIN and accelerate tumour
progression along the adenomacarcinoma sequence.
Several genetic studies in humans and mice have
shown that selection of specific APC mutations is based
on the growth advantage that they provide to the
tumour cell. Specific APC genotypes (the combination
of the two hits in both alleles) are selected during
tumour formation, based on the specific level of residual
-catenin regulating activity that they can still exert. This
a Wild-type cells
Centrosome
APC EB1
Kinetochore
Microtubules
b Apc-mutant cells
Centrosome
APC EB1
Kinetochore
Microtubules
Figure 5 | The mitotic spindle in relation to APC and CIN.

a | In APC wild-type cells, APC accumulates at the
kinetochore, where it may facilitate the binding of spindle
microtubules to the kinetochore by interacting with the
microtubule-associated protein EB1. b | In cells that express a
truncated form of APC, the interaction between kinetochores
and spindle microtubules is disrupted, leading to chromosomal
instability (CIN). APC, adenomatous polyposis coli.
just-right signalling is likely to result in the differential

expression of a subset of the WNT downstream targets
that provide a growth advantage to the APC-mutant
intestinal cell.
The role of APC mutation in deregulating the
WNT pathway does not necessarily need to be limited
to the initial stages of the adenomacarcinoma
sequence. Nuclear -catenin staining strongly correlates with tumour size and dysplasia102,116, and high
levels of nuclear -catenin have been found at the
invasion fronts of adenocarcinomas117. So, the progression from an early adenoma to an invasive carcinoma is associated with a progressive increase of
nuclear -catenin levels. Accordingly, several downstream targets of the APC/-catenin signalling pathway, such as MYC, matrilysin, CD44, and the urokinase-type plasminogen activator receptor, show
similar correlations with tumour progression and
have been implicated in tumour invasion and metastasis99103. Moreover, mutations in other genes that are
not directly involved in the WNT pathway might also
affect signal transduction. For example, loss of E-cadherin is often observed in epithelial tumours and is
generally correlated with cell-adhesion defects.
However, because of its direct interaction with catenin, E-cadherin loss could result in an increase of
the cytoplasmic pool of -catenin that is available for
nuclear signalling. Accordingly, both genetic and biochemical evidence has recently been provided to show
REVIEWS
ALLELIC IMBALANCE
The hallmark of chromosomal

instability at the molecular
level, representing losses or
gains of specific chromosomal
regions.
64
that the growth-suppressor activity of E-cadherin is

adhesion independent and results from inhibition of
the -catenin/TCF signalling pathway, and that loss of
E-cadherin expression can contribute to upregulation
of this pathway in human cancers118,119.
An important issue in the current discussion on the
role of selection versus genetic instability in colorectal
cancer is the stage at which CIN originates. Does
genetic instability precede mutations at rate-limiting
oncogenes and tumour-suppressor genes, or does it
simply underlie tumour progression? Several studies
have shown that genetic instability, measured as ALLELIC
IMBALANCES in specific chromosomal regions, occurs at
very early stages of tumorigenesis4345,47. The question
then arises as to whether loss of APC function can elicit CIN from the start of tumour formation, or whether
additional synergistic mutations are required.
Several of the oncogenes and tumour-suppressor
genes known to be altered along the adenomacarcinoma sequence cooperate in promoting genomic instability, and might be synergistic partners of APC in eliciting
CIN. In the nascent tumour cell, APC-driven chromosomal instability is likely to be suppressed by the cellcycle and mitotic checkpoint machinery. Both the high
incidence of KRAS mutations in ACF and early adenomas, and the presence of chromosomal instability at
early stages of the adenomacarcinoma sequence, support a role for this oncogene in genetic instability.
Several previous studies have reported a strong correlation between KRAS mutations and aneuploidization120123. At the molecular level, KRAS may induce
genomic instability via the mitogen-activated protein
kinase (MAPK) pathway and/or by affecting G1 and
G2/M cell-cycle transit times and apoptosis124,125.
Moreover, KRAS activation also affects MDM2 transcriptional regulation, accounting for the observation
that cells transformed by KRAS are more resistant to
DNA-damage-induced, p53-dependent apoptosis126.
At later progression stages, mutations and LOH in the
other known members of the adenomacarcinoma pathway, such as p53, members of the TGF- pathway, and
even MYC, are likely to cooperate progressively with APC
to promote and enhance CIN42,127131. So, CIN will allow a
stepwise malignant progression through a gradual
increase of numerical and structural chromosomal
rearrangements. Notably, Samowitz et al.132 have reported
that small adenomas with -catenin mutations do not
seem to be as likely to progress to larger adenomas and
invasive carcinomas as adenomas with APC mutations.
This indicates that although tumour initiation either by
loss of APC or by oncogenic -catenin mutations is functionally equivalent (by constitutive activation of the
WNT signalling pathway), inactivation of the additional
APC functions in chromosomal stability namely the
ability to capture kinetochores and stabilize the centrosome during the establishment of the mitotic spindle
underlies malignant progression in the colorectum.
We propose that the multifunctional nature of APC
confers a rate-limiting role in tumour initiation and progression to this colorectal cancer suppressor gene. Loss of
-catenin regulation by APC provides the intestinal cell
with a selective advantage, allowing the initial clonal

expansion. At this stage, CIN caused by loss of the C-terminal functional motifs of APC is either latent or of low
penetrance, owing to surveillance by the cell-cycle and
mitotic checkpoint machinery. The early activation of
the KRAS and MYC oncogenes triggers CIN and the
subsequent allelic imbalances in chromosomes 17p and
18q. Additional synergisms between APC and other
tumour-suppressor genes in eliciting aneuploidy and
CIN will progressively lead to malignant transformation
and metastasis. Hence, the combined signalling and
genetic stability functions make APC a true gatekeeper,
inactivation of which provides the intestinal cell with a
growth-selective advantage and genetic instability that
are just right to successfully trigger tumorigenesis.
MIN versus CIN tumours
The above model for APC-driven tumorigenesis is supported, rather than contrasted, by the analysis of the
DNA mismatch-repair tumour suppressor genes that are
involved in hereditary and sporadic colorectal cancer.
Hereditary non-polyposis colorectal cancer (HNPCC) is
the main inherited predisposition to colorectal cancer
and is caused by germ-line mutations of MMR genes,
namely MSH2, MLH1, MSH6 and PMS2 (REF. 133).
HNPCC tumours are invariably MIN, and this has been
an essential element of the mutator phenotype theory,
according to which an increased mutation rate underlies
tumorigenesis, even in the absence of a selective advantage. However, MMR-deficient cells have been shown to
be resistant to mutagens through alteration of their apoptotic response, which could represent an obvious selective
advantage134,135. Recent studies from Rick Fishel and colleagues136,137 have suggested a signalling role for MMR
based on an A-protein molecular switch, similar to the
classical G-protein molecular switch in the KRAS oncogene. In this scenario, mismatched nucleotides provoke
exchange of ADP for ATP and conformational transitions
that allow the MMR complexes to complex with DNA
and exert their repair function. Locally high concentrations of ATP-bound MMR complexes might signal
directly to the apoptotic machinery as the result of excessive DNA damage134. MSH2 and MSH6 also associate
with other tumour- suppressor and DNA-damage-repair
proteins in a large complex named BASC (BRCA1-associated genome surveillance complex), which presumably
functions as a sensor for DNA damage138. The concept of
MMR signalling is reminiscent of the dual role of APC.
MMR inactivation desensitizes the intestinal cell to DNAdamage-induced apoptosis, allowing initial clonal expansion. The mutator phenotype will then progressively lead
to accumulation of additional somatic mutations, thereby underlying tumour progression137,139.
Loss of mismatch-repair function seems to counteract CIN, as many MIN cell lines are near-diploid even
in the presence of APC and TP53 mutations140.
However, overexpression of the C-terminal EB1-binding domain of APC conferred chromosomal instability
in a near-diploid MIN colorectal cancer cell line by
inducing spindle abnormalities similar to those
observed in Apc-mutant ES cells4,5.
REVIEWS
Conclusions
Over the last two decades, we have witnessed several

important breakthroughs in understanding the molecular basis of colorectal cancer. Mutations in the APC
gene, because of its multifunctional nature, make an
important contribution to tumour initiation and progression in the large bowel. Likewise, the DNA mismatch-repair genes and many other tumour-susceptibility genes encode multifunctional proteins. The
proposed gatekeepercaretaker distinction for cancer
genes141 might need one main adjustment: the true cancer gatekeepers are those genes that have both gatekeeping and caretaking functions. When mutated, these
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Kinzler, K. W. & Vogelstein, B. Lessons from hereditary

colorectal cancer. Cell 87, 159170 (1996).
Powell, S. M. et al. APC mutations occur early during
colorectal tumorigenesis. Nature 359, 235237 (1992).
Analysis of sporadic colorectal tumours showed that
APC mutations are present in the earliest tumour
stages, and that the frequency of such mutations
remains constant as tumours progress from benign
to malignant stages.
Polakis, P. The adenomatous polyposis coli (APC) tumor
suppressor. Biochim. Biophys. Acta 1332, F127F147
(1997).
Fodde, R. et al. Mutations in the APC tumour suppressor
gene cause chromosomal instability. Nature Cell Biol. 3,
433438 (2001).
APC-mutant embryonic stem cells have extensive
chromosomal, centrosomal and spindle aberrations,
providing evidence for a role of APC in CIN. APC
accumulates at the kinetochore during mitosis.
Kaplan, K. B. et al. A role for the Adenomatous polyposis
coli protein in chromosome segregation. Nature Cell Biol.
3, 429432 (2001).
Cells carrying a truncated APC gene (ApcMin) are
defective in chromosome segregation. During
mitosis, APC localizes to the ends of microtubules
embedded in kinetochores and forms a complex with
the checkpoint proteins BUB1 and BUB3. In vitro,
APC is a high-affinity substrate for BUB kinases.
Boland, C. R. & Ricciardiello, L. How many mutations does
it take to make a tumor? Proc. Natl Acad. Sci. USA 96,
1467514677 (1999).
Tomlinson, I. & Bodmer, W. Selection, the mutation rate
and cancer: ensuring that the tail does not wag the dog.
Nature Med. 5, 1112 (1999).
Loeb, L. A. A mutator phenotype in cancer. Cancer Res.
61, 32303239 (2001).
Greenlee, R. T., Murray, T., Bolden, S. & Wingo, P. A. Cancer
statistics, 2000. CA Cancer J. Clin. 50, 733 (2000).
Pisani, P., Parkin, D. M., Bray, F. & Ferlay, J. Estimates of
the worldwide mortality from 25 cancers in 1990. Int. J.
Cancer 83, 1829 (1999).
Fearon, E. R. & Vogelstein, B. A genetic model for
colorectal tumorigenesis. Cell 61, 759767 (1990).
Colorectal tumour development and progression
results from the accumulation of somatic genetic
alterations (mutations) in both oncogenes and
tumour-suppressor genes. Mutations in four to five
genes may be necessary for the development of a
malignant tumour; fewer changes may suffice for
benign tumour formation.
Jen, J. et al. Molecular determinants of dysplasia in
colorectal lesions. Cancer Res. 54, 55235526 (1994).
Smith, A. J. et al. Somatic APC and KRAS codon 12
mutations in aberrant crypt foci from human colons.
Cancer Res. 54, 55275530 (1994).
Okamoto, M. et al. Loss of constitutional heterozygosity in
colon carcinoma from patients with familial polyposis coli.
Nature 331, 273277 (1988).
Solomon, E. et al. Chromosome 5 allele loss in human
colorectal carcinomas. Nature 328, 616619 (1987).
Following the localization of the FAP gene, sporadic
colorectal adenocarcinomas were analysed for LOH
in chromosome 5q. At least 20% of these tumours
had lost one of the alleles present in matched
normal tissue. This indicated that LOH at
chromosome 5q might be a crucial step in the
progression of colorectal cancer.
genes can provide the cell with the selective growth

advantage as well as the genetic instability necessary to
allow tumour initiation and progression.
How does the above notion affect our diagnostic and
therapeutic approach to colorectal cancer? The elucidation of the molecular and cellular mechanisms underlying APC-driven tumorigenesis will point out which cellular functions (proliferation, apoptosis, cell migration
and differentiation) are affected and how specific geneexpression profiles are associated with poor clinical
prognosis. Also, the identification of downstream targets
of the WNT/-catenin pathway will open the way for
tailor-made preventive and therapeutic intervention.
16. Forrester, K., Almoguera, C., Han, K., Grizzle, W. E. &

Perucho, M. Detection of high incidence of KRAS
oncogenes during human colon tumorigenesis. Nature
327, 298303 (1987).
17. Bos, J. L. et al. Prevalence of ras gene mutations in human
colorectal cancers. Nature 327, 293297 (1987).
References 16 and 17 are the first two papers to
show a high incidence of KRAS gene mutations in
human colorectal cancers, mainly in codon 12, and
that the mutations usually precede the development
of malignancy.
18. Vogelstein, B. et al. Genetic alterations during colorectaltumor development. N. Engl. J. Med. 319, 525532
(1988).
19. Pretlow, T. P., Brasitus, T. A., Fulton, N. C., Cheyer, C. &
Kaplan, E. L. KRAS mutations in putative preneoplastic
lesions in human colon. J. Natl Cancer Inst. 85,
20042007 (1993).
20. Fearon, E. R., Hamilton, S. R. & Vogelstein, B. Clonal
analysis of human colorectal tumors. Science 238,
193197 (1987).
Analysis of the clonal composition of human
colorectal tumours by means of Xlinked restrictionfragment length polymorphisms. The results support
a monoclonal origin for colorectal neoplasms, and
indicate that a gene on the short arm of
chromosome 17 might be associated with
progression from the benign to the malignant state.
21. Law, D. J. et al. Concerted nonsyntenic allelic loss in
human colorectal carcinoma. Science 241, 961965
(1988).
22. Vogelstein, B. et al. Allelotype of colorectal carcinomas.
Science 244, 207211 (1989).
23. Rodrigues, N. R. et al. p53 mutations in colorectal
cancer. Proc. Natl Acad. Sci. USA 87, 75557559
(1990).
24. Baker, S. J. et al. Chromosome 17 deletions and p53 gene
mutations in colorectal carcinomas. Science 244,
217221 (1989).
25. Hahn, S. A. et al. DPC4, a candidate tumor suppressor
gene at human chromosome 18q21.1. Science 271,
350353 (1996).
26. Blobe, G. C., Schiemann, W. P. & Lodish, H. F. Role of
transforming growth factor in human disease.
N. Engl. J. Med. 342, 13501358 (2000).
27. Thiagalingam, S. et al. Evaluation of candidate tumour
suppressor genes on chromosome 18 in colorectal
cancers. Nature Genet. 13, 343346 (1996).
28. Eppert, K. et al. MADR2 maps to 18q21 and encodes a
TGF-regulated MAD-related protein that is functionally
mutated in colorectal carcinoma. Cell 86, 543552 (1996).
29. Fodde, R. & Smits, R. Disease model: familial
adenomatous polyposis. Trends Mol. Med. 7, 369373
(2001).
30. Loeb, L. A., Springgate, C. F. & Battula, N. Errors in DNA
replication as a basis of malignant changes. Cancer Res.
34, 23112321 (1974).
31. Duesberg, P., Rausch, C., Rasnick, D. & Hehlmann, R.
Genetic instability of cancer cells is proportional to their
degree of aneuploidy. Proc. Natl Acad. Sci. USA 95,
1369213697 (1998).
32. Rasnick, D. & Duesberg, P. H. How aneuploidy affects
metabolic control and causes cancer. Biochem. J. 340,
621630 (1999).
33. Lengauer, C., Kinzler, K. W. & Vogelstein, B. Genetic
instabilities in human cancers. Nature 396, 643649
(1998).
34. Ionov, Y., Peinado, M. A., Malkhosyan, S., Shibata, D. &

Perucho, M. Ubiquitous somatic mutations in simple
repeated sequences reveal a new mechanism for colonic
carcinogenesis. Nature 363, 558561 (1993).
The first report showing that approximately 10% of
colorectal carcinomas carry somatic deletions at
simple sequence repeats. Tumours with these
mutations show distinctive genotypic and
phenotypic features. These mutations reflect a
previously undescribed form of carcinogenesis in
the colon mediated by a mutation in a DNA
replication factor that results in reduced fidelity for
replication or repair (a mutator mutation).
35. Thibodeau, S. N., Bren, G. & Schaid, D. Microsatellite
instability in cancer of the proximal colon. Science 260,
816819 (1993).
36. Parsons, R. et al. Hypermutability and mismatch repair
deficiency in RER+ tumor cells. Cell 75, 12271236
(1993).
37. Bhattacharyya, N. P., Skandalis, A., Ganesh, A., Groden,
J. & Meuth, M. Mutator phenotypes in human colorectal
carcinoma cell lines. Proc. Natl Acad. Sci. USA 91,
63196323 (1994).
38. Eshleman, J. R. et al. Increased mutation rate at the HPRT
locus accompanies microsatellite instability in colon
cancer. Oncogene 10, 3337 (1995).
39. Lengauer, C., Kinzler, K. W. & Vogelstein, B. Genetic
instability in colorectal cancers. Nature 386, 623627
(1997).
Colorectal tumours without microsatellite instability
show a defect in chromosome segregation, resulting
in gains or losses. This form of chromosomal
instability persists throughout the lifetime of the
tumour cell and is not simply related to chromosome
number. Whereas microsatellite instability is a
recessive trait, chromosomal instability seems to be
dominant.
40. Thiagalingam, S. et al. Mechanisms underlying losses of
heterozygosity in human colorectal cancers. Proc. Natl
Acad. Sci. USA 98, 26982702 (2001).
41. Cahill, D. P. et al. Mutations of mitotic checkpoint genes in
human cancers. Nature 392, 300303 (1998).
The authors show that CIN is associated with the
loss of function of a mitotic checkpoint. Moreover,
in a few cases, loss of this checkpoint was
associated with the mutational inactivation of a
human homologue of the BUB1 gene that controls
mitotic checkpoints and chromosome segregation
in yeast.
42. Fukasawa, K., Choi, T., Kuriyama, R., Rulong, S. & Vande
Woude, G. F. Abnormal centrosome amplification in the
absence of p53. Science 271, 17441747 (1996).
43. Giaretti, W. A model of DNA aneuploidization and
evolution in colorectal cancer. Lab. Invest. 71, 904910
(1994).
44. Bardi, G. et al. Cytogenetic comparisons of synchronous
carcinomas and polyps in patients with colorectal cancer.
Br. J. Cancer 76, 765769 (1997).
45. Shih, I. M. et al. Evidence that genetic instability occurs at
an early stage of colorectal tumorigenesis. Cancer Res.
61, 818822 (2001).
46. Cahill, D. P., Kinzler, K. W., Vogelstein, B. & Lengauer, C.
Genetic instability and Darwinian selection in tumours.
Trends Cell Biol. 9, M57M60 (1999).
47. Stoler, D. L. et al. The onset and extent of genomic
instability in sporadic colorectal tumor progression. Proc.
Natl Acad. Sci. USA 96, 1512115126 (1999).
REVIEWS
48. Groden, J. et al. Identification and characterization of the
familial adenomatous polyposis coli gene. Cell 66,
589600 (1991).
49. Kinzler, K. W. et al. Identification of FAP locus genes from
chromosome 5q21. Science 253, 661665 (1991).
References 48 and 49 are the first reports showing
the molecular cloning of the APC gene.
50. Nagase, H. & Nakamura, Y. Mutations of the APC
(adenomatous polyposis coli) gene. Hum. Mutat. 2,
425434 (1993).
51. Su, L. K., Vogelstein, B. & Kinzler, K. W. Association of the
APC tumor suppressor protein with catenins. Science
262, 17341737 (1993).
52. Rubinfeld, B. et al. Association of the APC gene product
with -catenin. Science 262, 17311734 (1993).
References 51 and 52 show how two cellular
proteins were found to associate with APC, and that
these were identified as the E-cadherin-associated
proteins - and catenin. A 15-amino-acid motif
that is repeated three times in APC was shown to be
sufficient for interaction with the catenins.
53. Hart, M. J., de los Santos, R., Albert, I. N., Rubinfeld, B. &
Polakis, P. Downregulation of -catenin by human axin and
its association with the APC tumor suppressor, -catenin
and GSK3 . Curr. Biol. 8, 573581 (1998).
54. Behrens, J. et al. Functional interaction of an axin
homolog, conductin, with -catenin, APC, and GSK3.
Science 280, 596599 (1998).
55. Fagotto, F. et al. Domains of axin involved in
proteinprotein interactions, Wnt pathway inhibition, and
intracellular localization. J. Cell Biol. 145, 741756
(1999).
56. Kishida, M. et al. Axin prevents Wnt-3a-induced
accumulation of -catenin. Oncogene 18, 979985
(1999).
57. Ikeda, S., Kishida, M., Matsuura, Y., Usui, H. & Kikuchi, A.
GSK-3-dependent phosphorylation of adenomatous
polyposis coli gene product can be modulated by catenin and protein phosphatase 2A complexed with Axin.
Oncogene 19, 537545 (2000).
58. Jiang, J. & Struhl, G. Regulation of the Hedgehog and
Wingless signalling pathways by the F-box/WD40-repeat
protein Slimb. Nature 391, 493496 (1998).
59. Marikawa, Y. & Elinson, R. P. -TrCP is a negative regulator
of Wnt/-catenin signaling pathway and dorsal axis
formation in Xenopus embryos. Mech. Dev. 77, 7580
(1998).
60. Bhanot, P. et al. A new member of the Frizzled family from
Drosophila functions as a Wingless receptor. Nature 382,
225230 (1996).
61. Behrens, J. et al. Functional interaction of -catenin with
the transcription factor LEF-1. Nature 382, 638642
(1996).
62. Molenaar, M. et al. XTcf-3 transcription factor mediates catenin-induced axis formation in Xenopus embryos. Cell
86, 391399 (1996).
63. Roose, J. et al. The Xenopus Wnt effector XTcf-3 interacts
with Groucho-related transcriptional repressors. Nature
395, 608612 (1998).
64. Cavallo, R. A. et al. Drosophila Tcf and Groucho interact to
repress Wingless signalling activity. Nature 395, 604608
(1998).
65. Rosin-Arbesfeld, R., Townsley, F. & Bienz, M. The APC
tumour suppressor has a nuclear export function. Nature
406, 10091012 (2000).
66. Henderson, B. R. Nuclear-cytoplasmic shuttling of APC
regulates -catenin subcellular localization and turnover.
Nature Cell Biol. 2, 653660 (2000).
67. Eleftheriou, A., Yoshida, M. & Henderson, B. R. Nuclear
export of human beta-catenin can occur independent of
CRM1 and the adenomatous polyposis coli tumor
suppressor. J. Biol. Chem. 276, 2588325888 (2001).
68. Ben-Zeev, A. & Geiger, B. Differential molecular
interactions of -catenin and plakoglobin in adhesion,
signaling and cancer. Curr. Opin. Cell Biol. 10, 629639
(1998).
69. Munemitsu, S. et al. The APC gene product associates
with microtubules in vivo and promotes their assembly in
vitro. Cancer Res. 54, 36763681 (1994).
70. Smith, K. J. et al. Wild-type but not mutant APC
associates with the microtubule cytoskeleton. Cancer Res.
54, 36723675 (1994).
71. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. Adenomatous
polyposis coli (APC) protein moves along microtubules and
concentrates at their growing ends in epithelial cells. J. Cell
Biol. 148, 505518 (2000).
72. Mimori-Kiyosue, Y., Shiina, N. & Tsukita, S. The
dynamic behavior of the APC-binding protein EB1 on
the distal ends of microtubules. Curr. Biol. 10, 865868
(2000).
66
73. Nathke, I. S., Adams, C. L., Polakis, P., Sellin, J. H. &

Nelson, W. J. The adenomatous polyposis coli tumor
suppressor protein localizes to plasma membrane sites
involved in active cell migration. J. Cell Biol. 134, 165179
(1996).
74. Su, L. K. et al. APC binds to the novel protein EB1. Cancer
Res. 55, 29722977 (1995).
75. Deka, J., Kuhlmann, J. & Muller, O. A domain within the
tumor suppressor protein APC shows very similar
biochemical properties as the microtubule-associated
protein tau. Eur. J. Biochem. 253, 591597 (1998).
76. Askham, J. M., Moncur, P., Markham, A. F. & Morrison, E. E.
Regulation and function of the interaction between the APC
tumour suppressor protein and EB1. Oncogene 19,
19501958 (2000).
77. Tirnauer, J. S. & Bierer, B. E. EB1 proteins regulate
microtubule dynamics, cell polarity, and chromosome
stability. J. Cell Biol. 149, 761766 (2000).
78. Korinek, V. et al. Constitutive transcriptional activation by a
-catenin-Tcf complex in APC/ colon carcinoma. Science
275, 17841787 (1997).
TCF4 transactivates transcription only when
associated with -catenin. Nuclei of APC/ colon
carcinoma cells were found to contain a stable and
constitutively active -cateninTCF4 complex.
Reintroduction of APC removed -catenin from TCF4
and abrogated the transcriptional transactivation.
79. Morin, P. J. et al. Activation of -catenin-TCF signaling in
colon cancer by mutations in -catenin or APC. Science
275, 17871790 (1997).
Colorectal tumours with intact APC were found to
contain activating -catenin mutations that alter
functionally significant phosphorylation sites. These
results indicate that regulation of -catenin is crucial
to APCs tumour-suppressing function and that loss
of this function can be achieved by mutations in
either APC or catenin.
80. Smits, R. et al. Apc1638T: a mouse model delineating critical
domains of the adenomatous polyposis coli protein
involved in tumorigenesis and development. Genes Dev.
13, 13091321 (1999).
Genetic demonstration that the carboxyl terminus of
APC does not encompass its tumour-suppressing
function. A mouse model carrying a targeted
mutation in Apc codon 1638, Apc1638T, is viable and
tumour free. The truncated Apc protein
encompasses three of seven 20-amino-acid repeats
and one SAMP motif. The results indicate that the
association with DLG, EB1, and microtubulin is not
as crucial for tumour suppression as the proper
catenin regulation by APC.
81. Sparks, A. B., Morin, P. J., Vogelstein, B. & Kinzler, K.
W. Mutational analysis of the APC/-catenin/TCF
pathway in colorectal cancer. Cancer Res. 58,
11301134 (1998).
82. Liu, W. et al. Mutations in AXIN2 cause colorectal cancer
with defective mismatch repair by activating catenin/TCF signalling. Nature Genet. 26, 146147
(2000).
83. Satoh, S. et al. AXIN1 mutations in hepatocellular
carcinomas, and growth suppression in cancer cells by
virus-mediated transfer of AXIN1. Nature Genet. 24,
245250 (2000).
84. Clevers, H. Axin and hepatocellular carcinomas. Nature
Genet. 24, 206208 (2000).
85. Miyoshi, Y. et al. Somatic mutations of the APC gene in
colorectal tumors: mutation cluster region in the APC
gene. Hum. Mol. Genet. 1, 229233 (1992).
86. Miyaki, M. et al. Characteristics of somatic mutation of the
adenomatous polyposis coli gene in colorectal tumors.
Cancer Res. 54, 30113020 (1994).
87. Ichii, S. et al. Detailed analysis of genetic alterations in
colorectal tumors from patients with and without familial
adenomatous polyposis (FAP). Oncogene 8, 23992405
(1993).
88. Lamlum, H. et al. The type of somatic mutation at APC in
familial adenomatous polyposis is determined by the site
of the germline mutation: a new facet to Knudsons twohit hypothesis. Nature Med. 5, 10711075 (1999).
Familial adenomatous polyposis patients with germline APC mutations within a small region around
codon 1300 mainly show allelic loss in their
colorectal adenomas, in contrast to others, the
second hits of which tend to occur by truncating
mutations in the mutation cluster region. The results
indicate that different APC mutations provide cells
with different selective advantages, with mutations
close to codon 1,300 providing the greatest
advantage. Allelic loss is selected strongly in cells
with one mutation near codon 1300.
89. Rowan, A. J. et al. APC mutations in sporadic colorectal

tumors: a mutational hotspot and interdependence of the
two hits. Proc. Natl Acad. Sci. USA 97, 33523357
(2000).
90. Smits, R. et al. Somatic Apc mutations are selected upon
their capacity to inactivate the -catenin downregulating
activity. Genes Chromosom. Cancer 29, 229239 (2000).
91. Kim, K., Pang, K. M., Evans, M. & Hay, E. D.
Overexpression of -catenin induces apoptosis
independent of its transactivation function with LEF-1 or
the involvement of major G1 cell cycle regulators. Mol.
Biol. Cell 11, 35093523 (2000).
92. He, T. C. et al. Identification of c-MYC as a target of the
APC pathway. Science 281, 15091512 (1998).
93. Tetsu, O. & McCormick, F. -catenin regulates expression
of cyclin D1 in colon carcinoma cells. Nature 398,
422426 (1999).
94. Shtutman, M. et al. The cyclin D1 gene is a target of the catenin/LEF-1 pathway. Proc. Natl Acad. Sci. USA 96,
55225527 (1999).
References 9294 are the first reports on the
identification of downstream targets of the APC/catenin pathway. MYC was shown to be repressed
by wild-type APC and activated by -catenin, and
through TCF4 binding sites in its promoter. -catenin
also activates cyclin D1 transcription through
promoter sequences to the consensus TCF/LEFbinding sites. The oncoprotein p21ras further
activates transcription of the cyclin D1 gene,
through sites within the promoter that bind the
transcriptional regulators ETS or CREB.
95. Risio, M., Coverlizza, S., Ferrari, A., Candelaresi, G. L. &
Rossini, F. P. Immunohistochemical study of epithelial cell
proliferation in hyperplastic polyps, adenomas, and
adenocarcinomas of the large bowel. Gastroenterology
94, 899906 (1988).
96. Kikuchi, Y., Dinjens, W. N. & Bosman, F. T. Proliferation and
apoptosis in proliferative lesions of the colon and rectum.
Virchows Arch. 431, 111117 (1997).
97. Bostick, R. M. et al. Colorectal epithelial cell proliferative
kinetics and risk factors for colon cancer in sporadic
adenoma patients. Cancer Epidemiol. Biomarkers Prev. 6,
10111019 (1997).
98. Johnston, P. G., OBrien, M. J., Dervan, P. A. & Carney, D. N.
Immunohistochemical analysis of cell kinetic parameters in
colonic adenocarcinomas, adenomas, and normal mucosa.
Hum. Pathol. 20, 696700 (1989).
99. Crawford, H. C. et al. The metalloproteinase matrilysin is a
target of -catenin transactivation in intestinal tumors.
Oncogene 18, 28832891 (1999).
100. Brabletz, T., Jung, A., Dag, S., Hlubek, F. & Kirchner, T.
-catenin regulates the expression of the matrix
metalloproteinase-7 in human colorectal cancer. Am. J.
Pathol. 155, 10331038 (1999).
101. Wielenga, V. J. et al. Expression of CD44 in Apc and Tcf
mutant mice implies regulation by the WNT pathway.
Am. J. Pathol. 154, 515523 (1999).
102. Brabletz, T., Herrmann, K., Jung, A., Faller, G. & Kirchner, T.
Expression of nuclear -catenin and c-myc is correlated
with tumor size but not with proliferative activity of
colorectal adenomas. Am. J. Pathol. 156, 865870 (2000).
103. Mann, B. et al. Target genes of -catenin-T cellfactor/lymphoid-enhancer-factor signaling in human
colorectal carcinomas. Proc. Natl Acad. Sci. USA 96,
16031608 (1999).
104. Smith, K. J. et al. The APC gene product in normal and
tumor cells. Proc. Natl Acad. Sci. USA 90, 28462850
(1993).
105. Midgley, C. A. et al. APC expression in normal human
tissues. J. Pathol. 181, 426433 (1997).
106. Korinek, V. et al. Depletion of epithelial stem-cell
compartments in the small intestine of mice lacking Tcf-4.
Nature Genet. 19, 379383 (1998).
107. Barker, N., Huls, G., Korinek, V. & Clevers, H. Restricted
high level expression of Tcf-4 protein in intestinal and
mammary gland epithelium. Am. J. Pathol. 154, 2935
(1999).
108. Melhem, M. F. et al. Distribution of cells expressing myc
proteins in human colorectal epithelium, polyps, and
malignant tumors. Cancer Res. 52, 58535864 (1992).
109. Yuwono, M., Rossi, T. M., Fisher, J. E. & Tjota, A.
Oncogene expression in patients with familial polyposis
coli/Gardners syndrome. Int. Arch. Allergy Immunol. 111,
8995 (1996).
110. Roose, J. et al. Synergy between tumor suppressor APC
and the -catenin-Tcf4 target TCF1. Science 285,
19231926 (1999).
111. Shih, I. M. et al. Top-down morphogenesis of colorectal
tumors. Proc. Natl Acad. Sci. USA 98, 26402645
(2001).
REVIEWS
112. Moser, A. R., Dove, W. F., Roth, K. A. & Gordon, J. I. The
Min (multiple intestinal neoplasia) mutation: its effect on gut
epithelial cell differentiation and interaction with a modifier
system. J. Cell Biol. 116, 15171526 (1992).
113. Harada, N. et al. Intestinal polyposis in mice with a
dominant stable mutation of the -catenin gene. EMBO J.
18, 59315942 (1999).
114. Doxsey, S. The centrosome a tiny organelle with big
potential. Nature Genet. 20, 104106 (1998).
115. Aladjem, M. I. et al. ES cells do not activate p53dependent stress responses and undergo p53independent apoptosis in response to DNA damage. Curr.
Biol. 8, 145155 (1998).
116. Hao, X., Tomlinson, I., Ilyas, M., Palazzo, J. P. & Talbot, I. C.
Reciprocity between membranous and nuclear expression
of -catenin in colorectal tumours. Virchows Arch. 431,
167172 (1997).
117. Brabletz, T. et al. Nuclear overexpression of the
oncoprotein -catenin in colorectal cancer is localized
predominantly at the invasion front. Pathol. Res. Pract.
194, 701704 (1998).
118. Smits, R. et al. E-cadherin and adenomatous polyposis
coli mutations are synergistic in intestinal tumor initiation in
mice. Gastroenterology 119, 10451053 (2000).
119. Gottardi, C. J., Wong, E. & Gumbiner, B. M. E-cadherin
suppresses cellular transformation by inhibiting -catenin
signaling in an adhesion-independent manner. J. Cell Biol.
153, 10491060 (2001).
120. Giaretti, W. et al. K-ras-2 GC and GT transversions
correlate with DNA aneuploidy in colorectal adenomas.
Gastroenterology 108, 10401047 (1995).
121. Nigro, S., Geido, E., Infusini, E., Orecchia, R. & Giaretti, W.
Transfection of human mutated K-ras in mouse NIH-3T3
cells is associated with increased cloning efficiency and
DNA aneuploidization. Int. J. Cancer 67, 871875 (1996).
122. Giaretti, W. et al. Intratumor heterogeneity of K-ras2
mutations in colorectal adenocarcinomas: association with
degree of DNA aneuploidy. Am. J. Pathol. 149, 237245
(1996).
123. Giaretti, W. et al. Specific K-ras2 mutations in human
sporadic colorectal adenomas are associated with DNA
near-diploid aneuploidy and inhibition of proliferation.
Am. J. Pathol. 153, 12011209 (1998).
124. Orecchia, R. et al. K-ras activation in vitro affects G1 and
G2M cell-cycle transit times and apoptosis. J. Pathol. 190,
423429 (2000).
125. Saavedra, H. I. et al. The RAS oncogene induces genomic
instability in thyroid PCCL3 cells via the MAPK pathway.
Oncogene 19, 39483954 (2000).
126. Ries, S. et al. Opposing effects of Ras on p53:
transcriptional activation of Mdm2 and induction of
p19ARF. Cell 103, 321330 (2000).
127. Fukasawa, K., Wiener, F., Vande Woude, G. F. & Mai, S.
Genomic instability and apoptosis are frequent in p53
deficient young mice. Oncogene 15, 12951302 (1997).
128. Glick, A. et al. Defects in transforming growth factor-
signaling cooperate with a Ras oncogene to cause rapid
aneuploidy and malignant transformation of mouse

keratinocytes. Proc. Natl Acad. Sci. USA 96,
1494914954 (1999).
129. Yin, X. Y., Grove, L., Datta, N. S., Long, M. W. &
Prochownik, E. V. C-myc overexpression and p53 loss
cooperate to promote genomic instability. Oncogene 18,
11771184 (1999).
130. Li, Q. & Dang, C. V. c-Myc overexpression uncouples DNA
replication from mitosis. Mol. Cell Biol. 19, 53395351
(1999).
131. Shao, C. et al. Chromosome instability contributes to loss
of heterozygosity in mice lacking p53. Proc. Natl Acad.
Sci. USA 97, 74057410 (2000).
132. Samowitz, W. S. et al. -catenin mutations are more
frequent in small colorectal adenomas than in larger
adenomas and invasive carcinomas. Cancer Res. 59,
14421444 (1999).
133. Lynch, H. T. & Lynch, J. Lynch syndrome: genetics, natural
history, genetic counseling, and prevention. J. Clin. Oncol.
18, 19S31S (2000).
134. Zhang, H. et al. Apoptosis induced by overexpression
of hMSH2 or hMLH1. Cancer Res. 59, 30213027
(1999).
135. Hickman, M. J. & Samson, L. D. Role of DNA mismatch
repair and p53 in signaling induction of apoptosis by
alkylating agents. Proc. Natl Acad. Sci. USA 96,
1076410769 (1999).
136. Fishel, R. Mismatch repair, molecular switches, and signal
transduction. Genes Dev. 12, 20962101 (1998).
137. Fishel, R. Signaling mismatch repair in cancer. Nature
Med. 5, 12391241 (1999).
138. Wang, Y. et al. BASC, a super complex of BRCA1associated proteins involved in the recognition and repair
of aberrant DNA structures. Genes Dev. 14, 927939
(2000).
139. Wang, Q., Zhang, H., Fishel, R. & Greene, M. I.
BRCA1 and cell signaling. Oncogene 19, 61526158
(2000).
140. Eshleman, J. R. et al. Chromosome number and structure
both are markedly stable in RER colorectal cancers and
are not destabilized by mutation of p53. Oncogene 17,
719725 (1998).
141. Kinzler, K. W. & Vogelstein, B. Cancer-susceptibility genes.
Gatekeepers and caretakers. Nature 386, 761763
(1997).
142. Cotran, R. Collins, K. V. & Robbins, T. Pathologic Basis of
Disease (W. B. Saunders Company, Philadelphia, USA,
1999).
143. Bird, R. P. Role of aberrant crypt foci in understanding the
pathogenesis of colon cancer. Cancer Lett. 93, 5571
(1995).
144. Takayama, T. et al. Aberrant crypt foci of the colon as
precursors of adenoma and cancer. N. Engl. J. Med. 339,
12771284 (1998).
145. Howe, J. R. et al. Mutations in the SMAD4/DPC4
gene in juvenile polyposis. Science 280, 10861088
(1998).
146. Howe, J. R. et al. Germline mutations of the gene

encoding bone morphogenetic protein receptor 1A in
juvenile polyposis. Nature Genet. 28, 184187 (2001).
147. Zhu, Y., Richardson, J. A., Parada, L. F. & Graff, J. M.
Smad3 mutant mice develop metastatic colorectal cancer.
Cell 94, 703714 (1998).
148. Takaku, K. et al. Gastric and duodenal polyps in Smad4
(Dpc4) knockout mice. Cancer Res. 59, 61136117 (1999).
149. Xu, X. et al. Haploid loss of the tumor suppressor
Smad4/Dpc4 initiates gastric polyposis and cancer in
mice. Oncogene 19, 18681874 (2000).
150. Takaku, K. et al. Intestinal tumorigenesis in compound
mutant mice of both Dpc4 (Smad4) and Apc genes. Cell
92, 645656 (1998).
151. Jass, J. R. Serrated route to colorectal cancer: back street
or super highway? J. Pathol. 193, 283285 (2001).
152. Markowitz, S. et al. Inactivation of the type II TGF-
receptor in colon cancer cells with microsatellite instability.
Science 268, 13361338 (1995).
153. Massagu, J. How cells read TGF- signals. Nature Rev.
Mol. Cell Biol. 1, 169178 (2000).
Online links
DATABASES
The following terms in this article are linked online to:
Flybase: http://flybase.bio.indiana.edu/
Armadillo
InterPro: www.ebi.ac.uk/interpro/
F-box
LocusLink: www.ncbi.nlm.nih.gov/LocusLink/
APC | Apc | axin | BRCA1 | BUB1 | cadherin | E-cadherin |
-catenin | CD44 | conductin | cyclin D1 | Dishevelled | EB/RP |
EB1 | Frizzled | GSK3 | Groucho | KRAS | LRP6 | MAPK |
matrilysin | MDM2 | MLH1 | MSH2 | MSH6 | MYC | PMS2 |
SMAD2 | SMAD4 | TCF4 | Tcf4 | TGF- | TP53 | TrCP |
urokinase-type plasminogen activator receptor | WNT
OMIM:http://www.ncbi.nlm.nih.gov/Omim/
FAP | HNPCC
Saccharomyces Genome Database: http://genomewww.stanford.edu/Saccharomyces
Bim1 | Mal3
FURTHER INFORMATION
APC Mutation Database:
http://perso.curie.fr/Thierry.Soussi/APC.html
The Hopkins hereditary colon cancer web site:
http://www.hopkinscoloncancer.org/subspecialties/heredicolor_cancer/overview.
htm
IARC Unit of Descriptive Epidemiology information:
http://www-dep.iarc.fr
The Internet Pathology Laboratory: http://wwwmedlib.med.utah.edu/WebPath/webpath.html
Roel Nusses Wnt web site:
http://www.stanford.edu/~rnusse/pathways/targets.html

Alsnlasnf

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Alsnlasnf

Uploaded by

Copyright:

Available Formats

REVIEWS

APC, SIGNAL TRANSDUCTION

The adenomatous polyposis coli (APC) gene can be

Western Europe, and one of the leading causes of

NATURE REVIEWS | C ANCER

VOLUME 1 | OCTOBER 2001 | 5 5

2001 Macmillan Magazines Ltd

Box 1 | Histopathology of colorectal cancer

Aberrant crypt focus

Nuclear -catenin levels and chromosomal instabilty

Disordered growth: that is,

(LOH). In cells that carry a

gene mutations are found in most tumours, whereas

polyps12,19. Apparently, the synergistic action of the

| OCTOBER 2001 | VOLUME 1

2001 Macmillan Magazines Ltd

Box 2 | The TGF- signal transduction pathway in colorectal cancer

Proto-oncogenes become active

mutations in the remaining TP53 allele, in line with

SMAD4, also known as DPC4 (deleted in pancreatic

NATURE REVIEWS | C ANCER

VOLUME 1 | OCTOBER 2001 | 5 7

2001 Macmillan Magazines Ltd

Box 3 | APC mouse models

Structure of gene product

Tumour burden Extra-intestinal

A system to repair base-pair

This gene encodes one of the

different types of genetic instability contribute to

understood at the molecular level. CIN tumours have a

| OCTOBER 2001 | VOLUME 1

2001 Macmillan Magazines Ltd

Mutation cluster region

-catenin binding (15-amino-acid repeat)

Nuclear export signal

-catenin downregulaion (20-amino-acid repeat)

Nuclear import signal

SAMP (axin/conductin binding)

CDK consensus phosphorylation

SCF (FOR SKP/CULLIN/F-BOX)

In these so-called E3-ubiquitin

Transcription factors of the

Cahill et al.46 have proposed a model to reconcile

APC was identified by positional cloning of the FAP

NATURE REVIEWS | C ANCER

VOLUME 1 | OCTOBER 2001 | 5 9

2001 Macmillan Magazines Ltd

turn, binds actin and actin-associated proteins68. APC

Selective advantage and the second hit. APC, then,

Moreover, although most colorectal tumours carry

| OCTOBER 2001 | VOLUME 1

2001 Macmillan Magazines Ltd

-catenin binding (15-amino-acid repeat)

-catenin downregulation (20-amino-acid repeat)

nuclear -catenin and activation of the downstream

NATURE REVIEWS | C ANCER

VOLUME 1 | OCTOBER 2001 | 6 1

2001 Macmillan Magazines Ltd

Figure 4 | Models of colorectal tumorigenesis. a | The

| OCTOBER 2001 | VOLUME 1

2001 Macmillan Magazines Ltd

A general picture is emerging from the analysis of the

Figure 5 | The mitotic spindle in relation to APC and CIN.