Aberrant Wnt/β-catenin signaling following loss of the tumor
suppressor adenomatous polyposis coli (APC) is thought to
initiate colon adenoma formation. Considerable evidence for this
model has come from mouse models of Apc truncation where
nuclear β-catenin is detectable soon after loss of Apc. However,
examination of tumors from familial adenomatous polyposis
coli (FAP) patients has failed to confirm the presence of nuclear
β-catenin in early lesions following APC loss despite robust
staining in later lesions. This observation presents the possibility
that colon adenomas arise through a β-catenin-independent
function of APC. Additionally, there is a well established role for
inflammation and specifically COX-2 and prostaglandin E2 in
the progression of colorectal cancer. Here we review the current
literature regarding the functions of APC in regulating WNT/β-
catenin signaling as well as its control of intestinal cell fate and
differentiation. Further, we provide a brief commentary on our
current understanding of the role that inflammation plays in
colorectal tumorigenesis and how it fits in with APC dysfunc-
tion. Though there are currently contrasting models to explain
colon tumorigenesis, our goal is to begin to reconcile data from
multiple different model systems and provide a functional view
into the initiation and progression of colon cancer.
The Origin of Colorectal Cancer: Loss of APC Function
Colon cancer is an important unmet medical need that is
common world wide. Fortunately, surgical excision of early, nonin-
vasive adenomas is essentially curative. In contrast, there are few
effective treatment options for patients suffering from advanced
forms of the disease, and the prognosis is often very poor. Despite
a prolonged latency phase, too few lesions are identified at a stage
where they can be surgically excised. Thus, colorectal cancer is
currently the third most common cause of cancer death in the
US.1-6 Poor treatment outcomes highlight the need to better
understand the underlying mechanisms that account for tumor
initiation, progression and spreading. Nearly 20 years ago, muta-
tions in the tumor suppressor adenomatous polyposis coli (APC)
gene were identified as the causative lesion in autosomal dominant
colorectal cancer or familial adenomatous polyposis coli (FAP).7 In
addition, ~85% of sporadic colorectal cancers have been reported
to harbor APC truncating mutations.8 Since its identification,
many labs have studied the functions of APC in both normal and
Emerging Non-Canonical Functions of APC
The precise mechanisms that link loss of APC function to
colorectal carcinogenesis remain the subject of active research as a
number of un-answered questions arise when data from multiple
models and species are considered. APC loss is considered a key
initiating step in a model of genetic and epigenetic events that
result in the initiation and progression of colorectal cancer.9
Loss of p53 function or activation of KRAS and genome-wide
hypomethylation accompanied by promoter-specific hyperm-
ethylation are examples of additional events contributing to tumor
progression. A key aspect of this model often views loss of APC as
synonymous with increased activation of the Wnt signaling axis
through the stabilization and nuclear localization of the proto-
oncogene β-catenin.10,11 This sequence of events places aberrant
Wnt signaling as a key initiating event in carcinogenesis, owing to
its direct mechanistic link to deregulated APC function. In support
of this model, the levels of β-catenin and putative β-catenin target
genes such as c-myc12 and CyclinD1,13 are elevated in adenomas
from familial adenomatous polyposis (FAP) patients compared
to healthy tissues. Also, stabilizing mutations in β-catenin in the
absence of mutations in APC have been described in approximately
7% of sporadic human colon carcinomas and provide additional
genetic evidence linking APC loss and β-catenin activation.14-18
Further, transgenic mice carrying stabilized, mutant β-catenin
develop numerous intestinal adenomas.19 Taken together, these
studies offer dysregulation of β-catenin as a key oncogenic event
that follows loss of APC.
Although studies in genetically engineered mice that harbor
truncated forms of Apc have provided considerable support for
this model,20-22 other data raise questions about the ordering of
*Correspondence to: David A. Jones; Huntsman Cancer Institute; University of
Utah; 2000 Circle of Hope; Salt Lake City, UT 84112 USA; Tel.: 801.585.6107;
Submitted: 05/18/09; Accepted: 06/16/09
Previously published online as a Cell Cycle E-publication:
New perspectives on APC control of cell fate and proliferation in
Reid A. Phelps,1,2 Talmage J. Broadbent,1,2 Diana M. Stafforini1,3 and David A. Jones1-4,*
1Huntsman Cancer Institute; and the 2Departments of Oncological Sciences; 3Internal Medicine; and 4Medicinal Chemistry; University of Utah; Salt Lake City, UT USA
Key words: APC, COX-2, β-catenin, KRAS, differentiation, colon cancer
[Cell Cycle 8:16, 2549-2556; 15 August 2009]; ©2009 Landes Bioscience
events following loss of APC.
For example, evidence gener-
ated from studies using tissue
samples from FAP patients raise
questions about the sequence
of events following APC loss.
A recent study evaluated 10
early adenomas with mild
dysplasia and reported elevated
levels of total, but not nuclear,
β-catenin.23 In contrast, the
authors convincingly docu-
β-catenin levels in late adenomas
with severe dysplasia.23 Similarly,
Anderson et al. demonstrated
that microadenomas and early
adenomas from FAP patients
do not express detectable levels
of nuclear β-catenin.24 Finally,
tumors carrying mutation in
distinct from those harboring
mutated APC.25 Taken together, these findings suggest that
additional, Wnt-independent functions of APC contribute to the
initiation and development of colorectal cancer.
Consistent with this possibility, examination of APC protein
function over the past two decades has uncovered a number of
unexpected, non-canonical functions. For example, a number of
groups have demonstrated roles for APC in cytoskeletal organiza-
tion,26,27 chromosomal segregation28 and cell adhesion29 that have
been reviewed elsewhere.30 In addition, APC appears to control
components of chromatin remodeling and the production and
response of tissues to retinoic acid biosynthesis.31-35 These diverse
functions highlight the multi-faceted character of APC and under-
score the necessity to closely examine all the data in light of these
alternate functions. This reviews attempts to integrate new data
regarding APC function with traditional views established from
studies in a variety of model systems.
Key Structural Features of APC
The APC protein product is relatively large and harbors
several functional domains (Fig. 1). The protein includes a total
of ten amino acid repeats, including three 15-amino acid repeats
followed by seven 20-amino acid repeats that bind β-catenin. In
addition, three SAMP (Serine-Alanine-Methionine-Proline) motifs
interspersed within the 20-amino acid repeats are essential for
the ability of APC to bind Axin.36 These independent domains
are important for the function of APC as a scaffold protein for
the β-catenin-destruction complex. Composed of APC, AXIN1,
GSK-3β and Casein Kinase, (CK1), the destruction complex facil-
itates sequential phosphorylation of β-catenin thereby targeting it
for ubiquitination and proteasomal degradation.10,11,37 In addition
to a well described function for APC in ordering the assembly of
the destruction complex, multiple groups have identified a role for
APC in the nuclear shuttling38-40 and sequestration of β-catenin.41
The vast majority of human APC mutations occur in a region
known as the mutation cluster region (mcr) that starts around
codon 1300 (Fig. 1). MCR truncations generally contain all three
15-amino acid repeats and at least one 20-amino acid repeat. For
example, the human colon cancer cell line DLD-1 harbors a trun-
cation in APC at codon 1427 and the encoded protein harbors two
20-amino acid repeat domains (Fig. 1). Similarly, in SW-480 cells
APC is truncated at codon 1337 and the encoded protein harbors
one 20-amino acid repeat.
The relationship between APC structure and function and
it regulation is complex. Early work by Rubinfield and Polakis
demonstrated that the middle domain of APC was phosphory-
lated by GSK-3β and that this phosphorylation was necessary for
recruitment of β-catenin to the destruction complex.42 Additional
studies examined the importance of the APC/Axin interaction and
demonstrated that the SAMP motifs were necessary for this inter-
action.36 Recent studies have also demonstrated similar regulation
of distinct signaling pathways. Specifically, multiple studies have
demonstrated that APC can bind to the transcriptional corepressor
CtBP1 and direct its function and degradation in a potentially
β-catenin-independent manner.31,32,35 These data have added
significantly to our understanding of the functions of APC in both
normal and tumorigenic settings.
Murine Models of Apc Inactivation: Utility and Limitations
To investigate the molecular consequences associated with Apc
inactivation, a number of genetically engineered models have been
developed over the years. Studies in these animals have resulted in
valuable information and they form the foundation for much of
what is known about the relationship between loss of Apc function
and Wnt activation. The most widely used mouse models are the
Apcmin/+, Apcfl/fl and Apc2lox14.
A model for colon tumorigenesis following APC loss
2550Cell Cycle 2009; Vol. 8 Issue 16
Figure 1. APC domain structure. The central region of APC is composed of three 15 amino acid repeat domains,
seven 20 amino acid repeat domains and three Ser-Ala-Met-Pro (SAMP) motifs. Shown are the different APC
fragments present in the most common mouse and rat models of Apc truncation as well as two representative
human colon cancer cell lines and the apc mutant zebrafish. MCR, mutation cluster region.
A model for colon tumorigenesis following APC loss
www.landesbioscience.com Cell Cycle2551
detectable in the absence of mutational activation of KRAS or loss
of p53. A key contribution of this study is that it provides the
opportunity to study colonic lesions that progress to carcinoma
in mice, thus enabling studies focused on the characterization of
events that take place during disease progression. As an impor-
tant note, Apc2lox14, like Apcmin/+ mice, express truncated forms
of Apc shorter than those typical of human colorectal tumors.
Finally, recent developments include mice known as Apc1322T
which express a 1,322-amino acid Apc truncation product. This
protein retains the first 20-amino acid repeat and is similar to
products detected in human colon tumors. Interestingly, these
mice developed multiple adenomas, primarily in the small intes-
tine. However, the level of nuclear β-catenin was much lower than
that observed in adenomas from Apcmin/+ mice.
In summary, the models of Apc truncation or deletion
described above are valuable tools that can be utilized to
elucidate the role of Apc in normal intestinal homeostasis. In
addition, these animals provide the opportunity to study how
impaired Apc function combined with other genetic alterations
affect intestinal homeostasis and/or the initiation and progres-
sion of colorectal cancer. Indeed, these models are widely and
effectively utilized in cancer biology studies. However, there is
the need to consider data from other model systems to form a
more complete view of APC dysfunction in human colorectal
An important feature that may have key mechanistic implica-
tions is that APC mutations in FAP are restricted to the MCR
domain. Examination of 133 adenomas from six different FAP
patients led Albuquerque et al. to propose the ‘just right’ model
for APC truncation. These investigators found that if the germ
line truncation did not contain any of the 20-amino acid repeats,
the second hit generally retained at least one 20-amino acid
repeat. On the other hand, if the inherited allele retained at least
one 20-amino acid repeat, the second hit was usually loss of the
entire allele.48 These findings strongly suggest that expression of
at least one 20-amino acid repeat is necessary for cellular survival.
Importantly, these observations may explain the presence of early
nuclear β-catenin in Apcfl/fl, Apcmin/+ and Apc2lox14 mice, as these
animals lack expression of Apc (Apcfl/fl) or express protein products
that lack β-catenin binding sites (Fig. 1).
Rat Models of Apc Truncation
To more closely recapitulate features of human colorectal
cancer, Amos-Landgraff et al. recently reported a new rat model
carrying Apc truncations within the MCR. These rats are known
as “Pirc” (polyps in the rat colon) and develop adenomas in the
colon—a feature that is absent in most murine models of Apc loss,
except those with targeted, colonic deletion of Apc. In addition,
these animals develop microadenomas that recapitulate histologic
and molecular features similar to those observed in humans.
Importantly, homozygous loss of Apc was insufficient for nuclear
localization of β-catenin in early lesions. However, in agreement
with studies in humans, advanced adenomas displayed robust
nuclear β-catenin staining.49
Of these, the best characterized is the Apcmin/+ model where Apc
is truncated at codon 850 (Fig. 1). Homozygous deletion of Apc
is embryonically lethal, but animals that harbor one copy of the
wild-type allele are viable and develop hundreds of adenomatous
polyps in the small intestine.43 Incompletely characterized mecha-
nisms, some of which possibly arise as a consequence of intestinal
lesion formation, lead to malnourishment, anemia and death
prior to carcinoma formation in the intestine. Kongkanuntn et al.
reported elevated levels of β-catenin in intestinal lesions ranging
from a single dysplastic crypt (group I) to advanced adenoma
(group IV). The location of β-catenin was reportedly nuclear in
at least a subset of cells in each of the categories.21 While these
observations are consistent with the canonical model of APC
function, the results differ from findings in adenomas from FAP
patients, which have been reported not to express β-catenin in
the nucleus. Therefore, while the Apcmin/+ mouse is an important
experimental model in evaluating the biological consequences of
Apc loss, it has a number of limitations. Among these is that the
mutation present in these animals leads to the production of a
much shorter protein product compared to those that are typical
of human colorectal cancer (see below). In addition, the intestinal
lesions do not fully recapitulate key features observed in advanced
A second genetically altered mouse was developed with the goal
of completely deleting Apc in the intestine. This was accomplished
by crossing Apcfl/fl mice to inducible Cyp1A-Cre mice. Upon cre
induction with β-napthoflavone, Apc is rapidly knocked out of
the intestinal epithelial cells.22,44-46 One of the advantages of this
model, unlike the Apcmin/+ approach, is that it can be utilized to
evaluate the consequences of early loss of Apc. Studies by Sansom
and Clarke have been highly informative from this standpoint.
Interestingly, these investigators reported robust expression of
nuclear β-catenin as early as five days following Cre induction in
the majority of intestinal cells.22 Additional reports have pointed
at genetic interactions with Myc45 and K-ras.44 These combined
studies contributed new insight into the in vivo functions of Apc,
and its relationship to other signaling pathways. However, the
model has limitations for studies aimed at characterizing molecular
events relevant to colon cancer in humans. Again, in humans APC
mutations result in the generation of truncated products that
may retain some essential functions of the wild-type protein (see
below); these potential functions are lost in the Apcfl/fl mice as they
express no Apc product.
In contrast to the Apcfl/fl mouse, the Apc2lox14 mouse produces
a truncated Apc product that harbors amino acids 1–580.47 The
phenotype of Fabpl-Cre:Apc2lox14 animals in which deletion of
Apc occurs in all tissues is characterized by the development of
adenomas in the small intestine, in a fashion similar to that of
Apcmin/+ mice. However, an interesting study investigated the
consequences of altered Apc expression in the colon by crossing
Apc2lox14 animals to Cdx2-Cre mice.20 This analysis revealed that
lesions in Cdx2-Apc2lox14 mice develop in the colon and prog-
ress to invasive adenocarcinomas, thus recapitulating important
features of human colon cancer. These animals, like Apcmin/+ and
Apcfl/fl mice, develop lesions in which nuclear β-catenin is readily
A model for colon tumorigenesis following APC loss
2552Cell Cycle2009; Vol. 8 Issue 16
Control of cEBP and COX-2 expression. Clinical observations
have long supported a link between chronic inflammation
and the development of colorectal cancer. A strong association
between ulcerative colitis (UC) and colon cancer formation was
appreciated as early as the 1940s.55,56 Prostaglandins are major
inflammatory products that have been shown to play an essential
role in both normal intestinal homeostasis as well as tumorigenesis.
Cyclooxygenase enzymes (COX-1,-2) catalyze the rate-limiting step
in the conversion of arachidonic acid to prostaglandins. COX-1 is
constitutively expressed throughout the intestinal tract and is
necessary for maintaining the integrity of the gastric mucosa.57-59
In contrast, COX-2 is expressed at low levels chronically, but can
be rapidly induced by inflammatory stimuli.60,61 Additionally,
COX-2 is overexpressed in up to 40% of colon adenomas and
85% of colon adenocarcinomas compared to matched normal
tissue.62-65 In recent years, compelling epidemiologic, genetic and
pharmacologic evidence revealed a central role for inflammation in
the progression of colorectal tumorigenesis.
In a randomized trial of 600,000 individuals, it was observed
that chronic use of aspirin (>16 times/month) reduced the relative
risk of colon cancer development to 0.60.66 This observation led
to the use of nonsteroidal anti-inflammatory drugs (NSAIDs),
such as sulindac, as chemopreventive agents in patients with FAP.
In fact, administration of NSAIDs caused a reduction in polyp
number and size and was associated with adenoma regression in
FAP patients in multiple independent trials.67-70 In addition to
these drug trials in humans, numerous genetic and pharmacologic
studies in rodents have demonstrated that COX-2 is an amenable
and effective drug target. Specifically, treatment of either Apcmin/+
mice71,72 or azoxymethane (AOM)-treated rats73-75 with a COX-2-
specific inhibitor resulted in significantly decreased tumor burden.
Additionally, crossing prostaglandin-endoperoxide synthase-2-/-
(Ptgs-2; Cox-2) mice to ApcΔ716 animals resulted in decreased
number of tumors.76 These data demonstrate that COX-2 plays
an important role in tumorigenesis; however, they do not provide
a mechanism for this reduction.
Because of its early role and the assumption that Wnt is an
initiating signal in colorectal tumorigenesis, many postulated
that expression of COX-2 is regulated by β-catenin. One line of
evidence suggested that, in the presence of active KRAS, β-catenin
could upregulate the transcription of COX-2.77 However, more
recently, in vivo data have challenged this view. Using the
zebrafish as a model, Eisinger et al. demonstrated that APC, via
its control of RA, regulates COX-2 in a β-catenin-independent
manner. Specifically, RA downregulates the transcription factor
C-EBPβ. When APC is truncated and RA biosynthesis is attenu-
ated, C-EBPβ accumulates and translocates to the nucleus where
it directly activates the transcription of COX-2. By controlling
the accumulation of C-EBPβ, APC and RA control the level of
COX-2.78 These data support a model wherein APC regulation
of COX-2 is β-catenin independent. Further, recent studies have
uncovered an intricate regulatory pathway that positions β-catenin
downstream of COX-2.79-81
In 2005, two different groups demonstrated that COX-2 and its
product prostaglandin E2 are upstream of β-catenin.79,80 In their
Genetic, Molecular and Pharmacological Manipulation
of Zebrafish: A Novel Approach to Study Intestinal
Development and Signaling
The development of apc mutant zebrafish has provided an
alternative model system to examine the immediate consequences
of apc loss. apc mutant zebrafish harbor a truncation at codon
1318 (Fig. 1). This is important because that truncation is within
MCR and encodes a protein product similar to those observed
in human colorectal cancers.50 In addition to the availability of
an apc mutant, zebrafish also offer some important advantages in
examination of intestinal development and tumorigenesis. Namely,
morpholino technology, which allows for rapid knockdown of
most genes, large clutch numbers for genetic experiments, high
permeability to pharmacologic agents, cross-reactivity with most
human antibodies and a short developmental window during
which the intestinal tube forms. Multiple groups have utilized this
model to further our understanding of both canonical and non-
canonical functions of apc.
Mutation of apc in Zebrafish Reveals Novel Functions for
this Tumor Suppressor
Control of retinoic acid biosynthesis. Based on the canonical
function of APC in controlling Wnt/β-catenin signaling, one
might expect to find a “Wnt” phenotype upon mutation of apc
in zebrafish embryos. Specifically, work in Xenopus demonstrated
that overexpression of Wnt ligand resulted in axis duplication with
a majority of the embryos developing two heads.51 Surprisingly,
the apcmcr zebrafish do not display axis duplication defects.
Rather, the phenotype of apcmcr embryos resembles that of neckless
mutants, which are characterized by being deficient in retinoic acid
(RA) biosynthesis.52 Briefly these animals lack pectoral fins and
jaws; they display retinal defects and lack intestinal differentiation,
endocrine and exocrine pancreas.33 Though this function of APC
is not accounted for by the traditional view of APC function, Jette
et al. demonstrated that this finding is consistent with molecular
features that characterize early human disease. Microarray and
rt-PCR data comparing FAP adenoma to normal tissue revealed
the downregulation of RA biosynthetic enzymes and suggested
that APC plays a role in the regulation of RA biosynthesis in
human colonocytes.53 Further, these data suggest that this function
of APC is lost in the earliest stages of tumorigenesis.
In a series of papers, Nadauld et al.32-34,54 demonstrated that,
in zebrafish, apc controls the expression of a class of enzymes called
the retinol dehydrogenases (rdh1, rdh1l). Further, the production
of RA controls zebrafish intestinal differentiation such that lack of
RA prevents the differentiation of enterocytes and maintains the
intestinal tube in a primordial state. This failure to differentiate can
be rescued by administration of exogenous all-trans retinoic acid
(ATRA). Mechanistically, apc regulates transcription of the rdh
enzymes by directing proteasome-dependent degradation of the
transcriptional repressor C-terminal Binding Protein-1 (CtBP1),
thus relieving repression. These findings were confirmed in human
colon cancer cell lines and demonstrated an important, nonca-
nonical function of APC in the regulation of RA.
A model for colon tumorigenesis following APC loss
www.landesbioscience.com Cell Cycle2553
activation of PKCδ. Rac1 subsequently activates JNK2 and causes
phosphorylation of Serine-191 and Serine-605, but not Serine-246
(a JNK2 consensus site).85 It appears, therefore, that post-trans-
lational modifications of β-catenin are necessary for its nuclear
accumulation in response to multiple stimuli.
Consistent with this possibility, our recent studies in zebrafish
revealed a surprising decrease in the number of epithelia cells
within the intestines apcmcr embryos (~20) in comparison to wild-
type (~45) at 72 hour post fertilization (hpf). Further, although
apcmcr intestinal cells express higher levels of total β-catenin
compared to wild-type zebrafish, the protein was confined to the
cytoplasm.86 Additional functional studies using the TOPdGFP
β-catenin reporter fish demonstrated lack of β-catenin activation
in the intestine of apc mutant zebrafish embryos. These observa-
tions strongly suggested that truncation of apc and activation
of Wnt/β-catenin signaling are not biologically equivalent. It
indicates that additional components and signaling events are
necessary before migration of β-catenin to the nucleus and activa-
tion of Wnt target genes can occur.
To investigate this issue, we considered other factors known
to contribute to the progression of colon cancer. KRAS is a small
GTPases that transmits a signal from the epidermal growth factor
receptor to the MAPK cascade. Importantly, oncogenic mutation
of KRAS has been reported in a large portion of colon cancers.
In the canonical MAPK cascade, EGFR activates KRAS, which
activates RAF, which activates MEK, which activates ERK. ERK
then translocates to the nucleus and transmits the signal by acti-
vating ETS transcription factors (reviewed in ref. 87). Based on
its role in the normal transmission of mitogenic signals, KRAS is
ideally suited to transmit a tumorigenic signal when it is aberrantly
activated. In fact, multiple groups have reported that Kras activa-
tion in the setting of Apc loss resulted in larger, more aggressive
lesions.44,88,89 In these studies however, it was concluded that
Kras only exacerbated already aberrant Wnt signaling. In our
studies, we demonstrated that expression of active kras in apcmcr
zebrafish embryos resulted in nuclear translocation and activation
of β-catenin. Further, we noted robust intestinal cell proliferation
as measured by an increase in intestinal cell number (~90 per cross
section) and expression of pcna.86 Further, through comparative
studies in human cell lines we delineated a novel pathway wherein
KRAS activates RAC1 through RAF1, not MEK1. Activation of
RAC1 was a necessary step in initiating the nuclear localization
of β-catenin following loss of APC. Taken together, our data
demonstrate that, although loss of APC is sufficient to stabilize
β-catenin in vivo in zebrafish intestinal cells and in vitro in human
cell lines, the nuclear localization of β-catenin requires the activity
of RAS/RAF and RAC. Following the nuclear localization of
β-catenin, cellular proliferation ensues. Though these observations
challenge the current model that equates APC loss with WNT
activation, our results provide mechanistic insight into a growing
body of literature in multiple different model systems that fails to
detect nuclear β-catenin in the earliest lesions following APC loss.
Specifically, although a number of studies have examined early
lesions for mutational activation of Kras, they have not accounted
for aberrant EGF receptor activation that has been noted in a
model, PGE2-mediated activation of the EP2 receptor activated
PI3K and AKT ultimately leading to the phosphorylation of
GSK-3β. This resulted in the destabilization of the β-catenin
destruction complex. Further, in the DLD-1 colon cancer cell
line, treatment with PGE2 resulted in the nuclear translocation of
β-catenin.79 These studies convincingly place COX-2 upstream
of β-catenin and yield important implications about the initia-
tion and progression of colorectal cancer. Following these seminal
studies, Eisinger et al. demonstrated an important and unrecog-
nized connection between RA, COX-2 and β-catenin stability.
Treatment of apcmcr embryos that retain a 1,318-amino acid
fragment of apc with a COX-2-specific inhibitor led to decreased
β-catenin levels. This decrease in β-catenin was sensitive to
treatment with MG132 and, therefore, dependent upon the prote-
osome. In contrast, treatment of apc morphant embryos, which do
not retain any translated apc protein, maintained elevated levels
of β-catenin.81 These results demonstrated that truncated apc
protein, once considered an inert gene product, is able to direct
the degradation of β-catenin in the face of low levels of PGE2.
These data have far-reaching implications. First, the truncations
that are seen in human disease retain certain functions of APC
when COX-2 is inhibited and PGE2 is maintained at a low level.
Second, the mouse models of Apc inactivation where the trunca-
tion is far removed from the mutation cluster region may provide
inaccurate assumptions about the function or lack of function of
truncated Apc. Finally, these observations provide an important
experimental tool in that blockade of COX-2 in an apcmcr embryo
functionally abolishes accumulated β-catenin. Therefore, even
though knockout of β-catenin is lethal to the embryos because of
its function at cell-cell junctions, interrogation of the other func-
tions of β-catenin in a nonlethal manner following loss of apc is
Effects on differentiation and cellular proliferation. Taken
together, the current data indicate that the functions of APC are
diverse and suggest that homozygous loss of APC alone leads to the
stabilization of β-catenin but not necessarily nuclear accumulation.
Though this model presents a departure from the canonical view of
the APC/β-catenin relationship, it is consistent with an increasing
body of literature that suggests specific post-translational modi-
fications of β-catenin are necessary to direct nuclear localization
in response to Wnt ligand stimulation. In two different papers in
2007, Kajiguchi et al. demonstrated that tyrosine phosphoryla-
tion of β-catenin resulted in its nuclear translocation in human
leukemia cells. In their studies, receptor tyrosine kinases such as
c-Kit were mutationally activated and resulted in a shift of the
β-catenin pool from the cytoplasm to the nucleus.82,83 Additionally,
multiple groups have demonstrated that serine phosphorylation of
β-catenin can increase transcriptional activity84 and nuclear local-
ization.85 In studies examining the PKA pathway, it was shown
that PKA-mediated phosphorylation of β-catenin on Serine-675 is
necessary to increase transcription of the TOPFLASH reporter.84
More recently, Wu et al. demonstrated that phosphorylation by
JNK2 is necessary to achieve nuclear localization of β-catenin
in response to Wnt ligand stimulation. Specifically, they demon-
strated that Wnt ligand activates Rac1 through non-canonical
A model for colon tumorigenesis following APC loss
2554Cell Cycle2009; Vol. 8 Issue 16
β-catenin. Activating KRAS mutations, or exacerbated signaling
following growth factor receptor stimulation, may serve to target
stabilized β-catenin to the nucleus through the actions of RAF,
RAC and JNK. This model represents a departure from previous
thinking about colon tumor initiation and progression where APC
loss and WNT activation were considered functionally equivalent.
However, the view may provide mechanistic insight into a number
of studies suggesting inconsistent detection of nuclear β-catenin in
the earliest lesions following APC loss in humans.
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number of different studies following loss of APC.
Furthermore, genetic and pharmacologic evidence
has shown that the combination of a COX-2
inhibitor with an EGFR specific inhibitor is a viable
treatment option.90-92 Previous views of these inhib-
itors considered their effects as largely independent.
Our data suggest that the efficacy of this combinato-
rial approach results from the targeting of β-catenin
signaling through distinct mechanisms. It is possible
that inhibition of COX-2 promotes downregulation
of β-catenin while inhibition of EGFR prevents its
In addition to dysregulated intestinal cell prolif-
eration, colon cancer is characterized by defects in
cell fating and differentiation. A number of studies
have demonstrated important contributions of
β-catenin-independent pathways in APC-mediated
intestinal cell differentiation; however, there remains
a debate about whether these differentiation defects
are mediated by dysregulated WNT signaling as
multiple reports have attributed intestinal differen-
tiation defects to aberrant β-catenin activation. In a
study published by Haramis et al. it was demonstrated that apcmcr/+
adult zebrafish are susceptible to intestinal tumors.93 Further,
these tumors harbor differentiation defects as demonstrated by a
lack of i-fabp expression. In these same lesions, there was detect-
able nuclear β-catenin thereby providing a descriptive relationship
between dysregulated WNT signaling and failed intestinal differ-
entiation. Further, a recent report by Faro et al. demonstrated that
downregulation of tcf4, a primary binding partner for β-catenin
in the nucleus, rescued intestinal differentiation defects in apcmcr/
mcr zebrafish.94 Although these studies have convincingly demon-
strated aberrant intestinal cell differentiation and fating in the
context of apc loss, they do not directly address a role for β-catenin
or consider β-catenin-independent functions of apc or tcf4.
To address the problem of intestinal cell fating, we exploited
an indirect mechanism to downregulate β-catenin in the intestines
of apcmcr embryos. As demonstrated by Eisinger et al. treat-
ment of apcmcr embryos with a COX-2 inhibitor results in the
downregulation of β-catenin.81 Consistent with a model wherein
differentiation defects are mediated by a β-catenin-independent
function of apc, downregulation of β-catenin does not rescue
defects in intestinal cell fate or differentiation. Rather, these
defects are rescued by knockdown of CtBP1.86 These data support
a model wherein intestinal cell fate and differentiation defects
following loss of APC represent the initiating event in colon
tumorigenesis, are mediated by dysregulation of CtBP1 and are
β-catenin-independent (Fig. 2).
Summary and Perspectives
The molecular mechanisms underlying colon tumorigenesis
remain complex. Our current model of adenoma initiation and
progression posits that APC loss underlies cell fating and differen-
tiation defects through dysregulation of CtBP1. Further, APC loss
poises the cells for a proliferative response by stabilizing cytoplasmic
Figure 2. Model of APC function. (A) Wildtype APC functions to regulate the intracellular
levels of β-catenin and control cell fate through the downregulation of CtBP1. (B) APC trun-
cation leads to an increase in cytoplasmic levels of β-catenin and dysregulation of retinoic
acid and cell fate. Free, cytoplasmic β-catenin is subsequently directed to the nucleus fol-
lowing RAS-dependent phosphorylation of β-catenin.
A model for colon tumorigenesis following APC loss
www.landesbioscience.com Cell Cycle2555
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