Download Caught up in a Wnt storm: Wnt signaling in cancer

Biochimica et Biophysica Acta 1653 (2003) 1 – 24
www.bba-direct.com
Review
Caught up in a Wnt storm: Wnt signaling in cancer
Rachel H. Giles 1, Johan H. van Es, Hans Clevers *
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
Received 25 October 2002; accepted 14 January 2003
Abstract
The Wnt signaling pathway, named for its most upstream ligands, the Wnts, is involved in various differentiation events during embryonic
development and leads to tumor formation when aberrantly activated. Molecular studies have pinpointed activating mutations of the Wnt
signaling pathway as the cause of approximately 90% of colorectal cancer (CRC), and somewhat less frequently in cancers at other sites, such
as hepatocellular carcinoma (HCC). Ironically, Wnts themselves are only rarely involved in the activation of the pathway during
carcinogenesis. Mutations mimicking Wnt stimulation—generally inactivating APC mutations or activating h-catenin mutations—result in
nuclear accumulation of h-catenin which subsequently complexes with T-cell factor/lymphoid enhancing factor (TCF/LEF) transcription
factors to activate gene transcription. Recent data identifying target genes has revealed a genetic program regulated by h-catenin/TCF
controlling the transcription of a suite of genes promoting cellular proliferation and repressing differentiation during embryogenesis,
carcinogenesis, and in the post-embryonic regulation of cell positioning in the intestinal crypts. This review considers the spectra of tumors
arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting
uncontrolled cell growth following induction of Wnt signaling.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Wnt signaling pathway; Carcinogenesis; Colorectal cancer
1. Brief overview of the Wnt signaling pathway
The American Cancer Society estimates that colorectal
cancer (CRC) will kill over 50,000 people in the US this
year alone [1]. The remarkable thing about CRCs—the most
common cause of non-smoking-related cancer deaths in the
western world—is that the molecular mechanisms underlying virtually all of these cases are uniform. Greater than
90% of all CRCs will have an activating mutation of the
canonical Wnt signaling pathway, ultimately leading to the
stabilization and accumulation of h-catenin in the nucleus of
a cell. Nuclear h-catenin is the hallmark of an active
canonical Wnt pathway; the presence of nuclear h-catenin
is evident in even the smallest detectable lesions resulting
from Wnt mutations [2]. The consistency of mutations at
some level of the Wnt signaling pathway in CRC makes this
cancer an attractive model for molecular intervention.
* Corresponding author. Tel.: +31-30-212-1800; fax: +31-30-2516464.
E-mail addresses: [email protected] (R.H. Giles),
[email protected] (J.H. van Es), [email protected] (H. Clevers).
1
To whom proofs should be sent.
In the absence of Wnt signal, unstimulated cells regulate
h-catenin levels by a multiprotein complex which phosphorylates h-catenin marking it for subsequent ubiquitination
and degradation [3]. This h-catenin degradation complex
consists of the adenomatous polyposis coli (APC) tumor
suppressor protein, axin, and the glycogen synthase kinase,
GSK3h. Upon docking of the Wnt ligand to its Frizzled (Fz)
receptor, a cascade of events is relayed that destabilizes the
degradation complex, allowing unphosphorylated h-catenin
levels to accumulate and translocate to the nucleus where
h-catenin functions as a cofactor for transcription factors of
the T-cell factor/lymphoid enhancing factor (TCF/LEF)
family (Fig. 1).
The genetic program initiated by h-catenin and TCF/LEF
transcription factors specifies the transcription of a specific
subset of genes, mainly determining cell fate and regulating
proliferation. Signaling through this pathway is present
during embryogenesis, where it has been shown to regulate
many developmental patterning events in organisms ranging
from worm to man (Table 1). In the developing vertebrate
embryo, the formation of the dorsal – ventral axis depends on
the activity of the Wnt signaling pathway. Misregulation of
Wnt signaling causes developmental defects. Like other
0304-419X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0304-419X(03)00005-2
2
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
Fig. 1. The Wnt signaling cascade, simplified. Left: in the absence of Wnt ligand, h-catenin levels are efficiently regulated by a complex containing APC, Axin,
and GSK3h. Transcription of TCF target genes is repressed by the presence of Grg co-repressors. Right: Wnt ligand destabilizes the h-catenin degradation
complex, allowing transportation to the nucleus. Once there, h-catenin recruits Lgs/BCL9 and pygo and activates TCF target genes. Known oncogenes are
lettered in pink; known tumor suppressors are lettered in blue.
major pathways regulating morphogenesis in early embryogenesis, studies showing a high frequency of mutations
constitutively activating Wnt/h-catenin signaling in specific
human tumors have implicated this pathway in the genesis of
cancer (reviewed in Ref. [4]). In this review, we will present
a synopsis of current research with particular attention paid
to how the deregulation of Wnt signaling leads to cancer in
certain anatomical venues such as the intestinal crypt.
2. Components of Wnt signaling
2.1. At the cell membrane
The term ‘‘Wnt’’ (pronounced wint) was introduced 20
years ago and fused the names of two orthologous genes:
Wingless (Wg), a Drosophila segment polarity gene [5], and
Int-1, a mouse protooncogene [6,7]. Wnts are a large family
of secreted glycoproteins with at least 19 known human
members that are expressed in species ranging from Drosophila to man (Table 1), and which play key roles in cell
fate specification, CNS patterning, and control of asymmetric cell division [8]. Transcription of Wnt family genes
appears to be developmentally regulated in a precise temporal and spatial manner.
Wnt signaling is initiated following Wnt ligand binding
to a member of the Fz family of seven-span transmembrane
receptors [9– 12] together with the co-receptors LRP-5 or
LRP-6, members of the low-density lipoprotein receptorrelated protein family (LRP) [13]. Canonical Wnt signaling
is only mediated when both Fz and LRP are complexed with
Wnt [13 – 15]. Most Wnt proteins can bind to multiple Fzs
and vice versa, suggesting redundancy in vivo. Soluble
forms of Fz – FzBs are thought to act as antagonists,
squelching Wnt before it can bind to membrane-bound Fz
[16 – 18]. Like FzBs, Dickkopf (Dkk) proteins are secreted
inhibitors, structurally distinct from Wnts [19,20]. They act
as potent inhibitors of Wnt signaling and are involved in
head induction in Xenopus embryogenesis. Dkk blocks Wnt
signaling by binding LRP-6 in such a way that it sterically
hinders Wnt binding [21]. A transmembrane protein called
Kremen2 then forms a ternary complex with Dkk and LRP6, inducing rapid endocytosis and removal of the Wnt
receptor LRP-6 from the plasma membrane [22].
2.2. In the cytoplasm
Upon binding of Wnt, the scaffold protein axin translocates to the membrane where it interacts with the intracellular tail of LRP-5 [23]. LRP-5 mutants lacking the
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
Table 1
Gene nomenclature of the key Wnt components
Protein
Human
Mouse
Drosophila
C. elegans
Wnt
WNT (19)
Int/Wnt
(19)
Wingless,
Wg, dwnt (7)
Frizzled, Fz
FZD (10)
Fzd (9)
Frizzled, Fz,
Dfz (5)
LRP
LRP5,
LRP6 (2)
DKK (4)
Lrp (2)
Arrow (1)
mom-1,
mom-2,
lin-44,
egl-20,
cwn-1,
cwn-2 (6)
mom-5,
lin-17,
mig-1,
cfz-2 (4)
lrp-1 (1)
Dkk (1)
Dickkopf (1)
?
DVL (3)
Dvl (3)
Disheveled,
Dsh (1)
Axin,
conductin/
axil
GSK3h
AXIN1,
AXIN2
(2)
GSK3B
(1)
Axin,
Conductin/
Axil (2)
Gsk3b (1)
dAxin (1)
mig-5,
dsh-1,
dsh-2 (3)
pry-1 (1)
APC, APC2
APC,
APC2 or
APCL (2)
CTNNB1
JUP (2)
Apc,
Apc2 (2)
TCF, LEF
(4)
TLE (5)
BCL9 (1)
PYGO (2)
Dickkopf,
Dkk
Dishevelled,
Dsh
h-Catenin,
g-catenin/
plakoglobin
TCF/LEF
Groucho
Legless
Pygopus
Shaggy, sgg,
or Zeste white
3, zw3 (1)
dApc, E-Apc
(2)
sgg-1 (1)
Catnb,
Jup (2)
Armadillo (1)
Tcf/Lef (4)
dTcf/pangolin
(1)
Groucho (1)
Legless, lgl (1)
Pygopus,
pygo (1)
bar-1,
wrm-1, and
hmp-2 (3)
pop-1 (1)
Grg (5)
Lgl (1)
Pygo (2)
apr-1 (1)
unc-37 (1)
?
?
Values in parentheses are the number of known homologues.
extracellular domain function behave as constitutively
active forms that bind and destabilize axin [23]. By destabilizing axin, h-catenin becomes refractory to degradation in
the cytoplasm and eventually translocates to the nucleus by
an unknown mechanism where it activates TCF/LEF target
genes [23]. In parallel but by an unknown mechanism,
Wnt binding to Fz results in hyperphosphorylation of
Dishevelled (Dsh), which inhibits the activity of GSK3h
[24].
Dsh is an intersection for Wnt signaling traffic. While this
review focuses on Wnt signaling through h-catenin and TCF,
dubbed the canonical Wnt pathway, two other pathways
branch off at the level of Dsh. These non-canonical Wnt
pathways transduce Wnt signal to either the JNK (c-Jun Nterminal kinase) pathway or to the Ca2 +-releasing pathway
through Fz receptors (reviewed in Refs. [25,26]). Signaling
through JNK, the planar cell polarity (PCP) pathway is a
morphogenic process in Drosophila that orients parallel
arrays where cell polarity dictates the plane of the epithelium, perpendicular to the cells’ apical – basal axes. During
vertebrate gastrulation, a non-canonical Fz- and Wnt-mediated signal drives a critical morphogenic movement, termed
3
convergent extension, which is thought to be orthologous to
PCP [27]. Proteins that bind Dsh decide its participation in
either canonical or non-canonical Wnt signaling. For example, casein kinase I (CKI) can phosphorylate Dsh and
actively promotes Dsh function in canonical Wnt signaling,
but inhibits its activity in convergent extension [28,29].
Conversely, Naked cuticle (Nkd) is a feedback inhibitor that
blocks Dsh function in canonical Wnt signaling but can also
activate PCP-like convergent extension signal in vertebrates
[30]. Strabismus (Stbm, also known as van Gogh/vang) is a
tetra-membrane-spanning protein that acts to suppress canonical Wnt signaling and potentiate the PCP signal [31].
Lastly, Dapper is also a negative inhibitor of h-catenin
signaling, working at the level of Dsh [32]. In Xenopus,
protein kinase C (PKC), activated by the Wnt/Ca2 + pathway,
blocks the Wnt/h-catenin pathway by phosphorylating Dsh
[26]. Calmodulin-dependent protein kinase II (CamKII), also
activated by the Wnt/Ca2 + pathway, inhibits the Wnt/hcatenin signaling cascade downstream of h-catenin [26].
Thus, five proteins (Stbm, Nkd, CKI, Dapper, and PKC)
promote activity of one pathway and inhibit the other
through their interaction with Dsh. Interestingly, gene
expression profiling recently correlated overexpression of
Wnt5a and concomitant increase in PKC activity with human
melanoma progression [33]. Nuclear h-catenin was not
observed, providing the first indication that the Wnt/Ca2 +
pathway is directly involved in cancer [33]. In addition,
mutations in Fz4 that render it unable to activate the Wnt/
Ca2 + pathway have recently been implicated in familial
exudative vitreoretinopathy (FEVR), a hereditary ocular
disorder characterized by a failure of peripheral retinal
vascularization [34].
In the absence of canonical Wnt signaling, GSK3h
phosphorylates h-catenin at four amino-terminal residues
[35 – 37], targeting it for binding to the F-box protein
h-TrCP and subsequent ubiquitination and degradation by
the proteasome [38 – 40]. Unlike most protein kinases
involved in signaling, GSK3h is active in unstimulated,
resting cells and is a key component in many signaling
pathways including Wnt. Its activity is abrogated during
cellular responses and its substrates therefore tend to be
dephosphorylated. GSK3h substrates are generally functionally inhibited by GSK3h, as is the case with h-catenin.
Before phosphorylation of h-catenin by GSK3h can occur,
however, CKI phosphorylates h-catenin at Ser-45 creating a
priming site which is necessary and sufficient for GSK3h to
subsequently phosphorylate the remaining sites of Thr-41,
Ser-37, and Ser-33, respectively [41 – 43]. Very recently, a
protein called Diversin has been shown to recruit CKI to the
degradation complex and facilitates phosphorylation of
h-catenin [44]. Morpholino-based gene ablation in zebrafish
shows that Diversin is crucial for axis formation, which
depends on h-catenin signaling. Diversin is also involved in
JNK activation and convergent extension in zebrafish [44].
Similar to Stbm, Nkd, and Dapper, Diversin suppresses Wnt
signals mediated by the canonical h-catenin pathway and
4
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
stimulates signaling via JNK, but unlike these proteins it
does not exert this function through Dsh.
Exactly how GSK3h is regulated by Dsh is still unknown.
Dsh has been shown to interact with axin and GSK3h
inhibitory protein Frat1, while GSK3h directly interacts with
Axin and Frat1 but not Dsh [45,46]. A quaternary complex
of Axin, GSK3h, Dsh and Frat1 thus appears to exist in
unstimulated cells. Following Wnt stimulation, Dsh is
recruited to the membrane and phosphorylated which is
postulated to cause a conformational change allowing the
Frat1-mediated disassociation of GSK3h from axin. With
GSK3h no longer bound to axin, phosphorylation of
h-catenin does not occur and the cytosolic levels of
h-catenin accumulate and translocate to the nucleus. An
alternative scenario could be that the recruitment of Dsh to
the membrane triggers dephosphorylation of the multiprotein
complex responsible for h-catenin degradation by protein
phosphatase 2A (PP2A). The catalytic unit of PP2A directly
interacts with axin [47,48], whereas the regulatory subunit
binds the N-terminus of APC [49].
The h-catenin degradation complex consists of GSK3h,
axin, and the tumor suppressor APC. Axin and APC form a
structural scaffold that allows GSK3h to specifically phosphorylate h-catenin, as well as APC and axin. Phosphorylation of axin by GSK3h is important for its stability [50].
Axin was identified as an inhibitor of Wnt signaling when
mutations in murine axin were observed to cause axis
duplication in homozygous mouse embryos [51]. Furthermore, injection of axin mRNA into frog embryos inhibited
dorsal axis formation [51]. Humans have two paralogous
AXIN genes, encoding 900 amino acid polypeptides with
roughly 87% identity to the mouse protein. Axin appears to
serve as a scaffold to bring together GSK3h, CKI, APC,
and h-catenin stimulating phosphorylation and thus regulating cytosolic and nuclear levels of h-catenin. The relevance of axin’s function is illustrated by studies showing
that truncating mutations in AXIN1 leading to nuclear
accumulation of h-catenin are found in hepatocellular
carcinomas (HCCs) [52]. Adenoviral transfer of wild-type
AXIN1 into these cells, as well as HepG2 cells which
express mutant h-catenin, was able to decrease nuclear
accumulation of h-catenin and lower TCF/LEF1-mediated
transcriptional activity [52].
APC is a large (312 kDa) protein that is known to interact
with at least 10 protein partners including h-catenin, axin,
EB-1 and DLG [53]. It can also form homodimers, the
functional relevance of which has not yet been determined
[54 –56]. APC has multiple, diverse functions in cell migration and adhesion, in cell cycle regulation and in chromosome stability [57]. However, its critical role in tumorigenesis appears to lie in the control of cellular levels of
h-catenin, thus acting as a negative regulator of the Wnt
signaling pathway [58]. Although APC, located on chromosome 5q, is mutated in 85% of familial and sporadic CRCs,
it is not absolutely necessary for the proper functioning of
the h-catenin degradation complex. Overexpression of axin
can compensate for the absence of functional APC [59,60].
In addition to its structural role in the h-catenin degradation
complex, APC has been shown to capture and escort nuclear
h-catenin to the cytoplasmic destruction machinery [61 –
63]. Inclusion of APC in the h-catenin degradation complex
likely results in an improved presentation of h-catenin to
GSK3h, leading to more efficient phosphorylation and
subsequent destruction.
Three different structural motifs in APC are responsible
for its h-catenin-regulating function: three 15-amino acid
repeats binding h-catenin and plakoglobin, seven 20-amino
acid repeats involved with both binding and downregulating
these proteins [55,64,65], and three so-called SAMP repeats
facilitating axin and conductin binding [35,59,60,66,67].
Although APC is expressed in the epithelium of the bladder,
small and large intestine, esophagus, stomach, and epidermis, APC expression is restricted to regions where cell
replication has ceased and terminal differentiation is established [68].
A paralogue of APC termed APC2 or APCL was
identified in humans and mouse [69,70]. APC2 localizes
to human chromosome band 19p13.3 and encodes a 300
kDa protein that appears to be ubiquitously expressed in
normal tissues although clear differences in expression
levels are present [69]. APC2 bears significant homology
to APC at the N-terminus and central region, including the
Armadillo (Arm) repeats, but not at the C-terminus. The
dimerization domain is conserved suggesting that APC2 can
also form homo- and/or hetero-dimers with APC itself.
APC2 can bind h-catenin and regulate its concentrations
as efficiently as APC [69,70]. The C-terminus of APC2, but
not APC, has been shown to bind to the p53-binding protein
2, suggesting that distinct functions of these proteins may be
mediated by the C-terminus [71]. All other protein partners
appear to be conserved, however, including members of the
EB family of microtubule binding proteins [72,73]. Given
the similarities between APC and APC2, it seems likely that
APC2 may participate in Wnt signaling and consequently
tumorigenesis. However, no specific mutations in APC2 in
tumors have yet been reported [74], although one recent
report does document allelic imbalance of the APC2 locus in
19/20 ovarian carcinomas [75].
h-Catenin is the mammalian orthologue of the Drosophila Arm protein and is the key mediator of the Wnt signal.
It was originally identified as a component of the adherens
junctions, where it links E-cadherin to a-catenin and consequently, the actin microfilament network of the cytoskeleton [76 – 79]. At about 1000 amino acids, a large part of
this protein is taken up by 12 tandemly arranged imperfect
42 residue repeats, aptly called Arm repeats that mediate
protein – protein interactions with cadherins, APC, axin and
TCF [80,81]. h-Catenin’s arm repeats are sufficient for
docking at the nuclear envelope and for nuclear accumulation of the protein [82]. Because importin-h and transportin also contain arm repeats and function in nuclear
transport, it has been suggested that arm repeats facilitate
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
nuclear localization [83]. Supporting this hypothesis, APC
also contains seven arm repeats and can be found in the
nucleus; a polypeptide containing just these repeats was
sufficient for nuclear localization [62,84,85]. In addition
to being effectors of Wnt signaling, h-catenin and its
paralogue plakoglobin are essential components of the
cadherin and desmosomal-based cell adhesion systems.
Because the cadherins and desmosomal proteins can be
very abundant in certain tissues, they may modulate the
amount of h-catenin/plakoglobin available for signaling.
APC can bind and regulate both h-catenin and plakoglobin
equally well, perhaps being one of the reasons why the
frequency of APC mutations in colon cancer far exceeds that
of h-catenin mutations. Like h-catenin, plakoglobin may act
as an oncogene [86 – 88].
2.3. In the nucleus
The C-terminus of h-catenin contains a transactivation
domain [89]. h-Catenin does not bind DNA itself, but is an
essential cofactor for TCF/LEF transcription factors. Interactions with additional proteins probably modulate h-catenin’s activity at certain promoters. The acetyltransferase
CBP (CREB-binding protein) acetylates h-catenin at lysine
49 and this alteration has been reported to improve transactivation at the c-MYC locus, but not at other target genes
[90]. By recruiting CBP to a specific promoter and allowing
it to acetylate nearby histones, h-catenin promotes transactivation; the transcription machinery gains better access to
promoter sequences when local chromatin is relaxed [91].
Likewise, h-catenin recruits Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodeling complexes, to
TCF target gene promoters, facilitating chromatin remodeling as a prerequisite for transcriptional activation [92].
Nuclear h-catenin/TCF activity was recently shown to be
regulated by legless (Lgs) [93] and pygopus (pygo) [93 – 96].
Lgs was identified in a genetic screen for suppressors of an
eye phenotype in Drosophila generated by overexpression of
Wnt/Wg. Embryos lacking lgs are phenotypically similar to
Wg mutants [93]. Lgs is a nuclear protein orthologous to
BCL9, a known oncogene involved in the development of
non-Hodgkin’s lymphoma [97] and in B cell acute lymphoblastic leukemia (ALL) exhibiting a somatic translocation
t(1;14)(q21;q32) [98]. While Thompson et al. [94] identified
pygo in a genetic screen for suppressors of an activated
h-catenin/Arm phenotype in the fly eye, Kramps et al. [93]
identified pygo as a binding partner of Lgs. Again, flies
mutant for pygo have a phenotype similar to a loss of Wg. A
fusion protein consisting of Lgs/Bcl-9 h-catenin-binding
HD2 domain and the part of pygo not used for Lgs-binding
(the NHD domain) is sufficient for h-catenin/Arm transactivation, suggesting that the primary function of Lgs is to
recruit pygo to h-catenin/Arm. The question that remains to
be answered is: what does the NHD domain of pygo then
bind? Elements involved in chromatin remodeling seem
likely candidates.
5
The human TCF/LEF family of transcription factors
consists of four members: TCF1, LEF1, TCF3, and TCF4.
T-cell factor 1 (TCF1) and lymphoid enhancing factor 1
(LEF1) were originally identified as lymphoid-specific transcription factors [99 – 101]. These proteins contain a highmobility group (HMG) box which binds DNA in a sequence-specific manner, bending the DNA in the process
[102]. LEF1 is different than the TCFs in that it has a socalled context-dependent activation domain which instigates
transactivation in the presence of the coactivator ALY
[103,104]. TCFs must bind h-catenin for transactivation
[89,105]. In the absence of Wnt signal, TCF/LEF binds a
family of broadly expressed transcriptional repressors,
called Grg or groucho proteins [106,107], which help
actively repress transcription of TCF target genes by recruiting histone deacetylases which subsequently act to condense
chromatin [108].
3. Alterations of the Wnt signaling pathway in colorectal
cancer
3.1. APC
Activating mutations of the Wnt pathway are the only
known genetic alterations present in early premalignant
lesions in the intestine, such as aberrant crypt foci and
small polyps [109]. Human APC has monoallelic inactivating mutations in patients with familial adenomatous polyposis (FAP), an inherited autosomal dominant condition
leading to the development of multiple adenomas in the
colorectum [110,111]. In these patients, polyps also develop
in the upper gastrointestinal tract and malignancies may
occur at other sites including the brain and thyroid. The
remaining wild-type APC allele is mutated in most FAP
patient polyps. Although FAP is a rare disease, CRC is not,
and up to 85% of all sporadic CRCs have mutations in APC.
Over 300 different disease-associated mutations of the APC
gene have been reported [112]. The vast majority of these
changes are insertions, deletions, and nonsense mutations
that lead to frameshifts and/or premature stop codons in the
resulting transcript of the gene. The most common APC
mutation (10% of FAP patients) is a deletion of AAAAG in
codon 1309; no other mutations appear to predominate.
A great deal of clinical variability is seen in FAP patients,
apparently due to the nature and position of the germline
APC mutation. This rough genotype –phenotype correlation
provides insight into APC tumorigenesis potential. The most
severe and the most common human APC mutations are
located between codons 450 and 1578 and result in stable
truncated proteins (reviewed in Ref. [113] and references
therein). Within this group, patients with truncations between codons 1395– 1578 suffer desmoids, osteomas, epidermoid cysts, and polyps of the upper gastrointestinal tract
in addition to their high colorectal polyp counts. On the other
hand, mutations affecting the extreme 5Vend of APC result in
6
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
an attenuated form of FAP, characterized by a later age of
onset and fewer colorectal polyps. These mutations are
believed to allow residual APC expression through alternatively spliced exons. Mutations occurring at the 3Vend of
APC often result in undetectable levels of truncated protein
and also confer an attenuated form of FAP but with an
increased risk for extraintestinal manifestations. Attenuated
FAP caused by mutations at either the 5Vor 3Vends of APC
can be characterized by neoplasia with a serrated glandular
structure, called serrated adenomas [114].
APC is generally considered a classical tumor suppressor: both alleles must be affected for loss of growthsuppressing activity. However, CRCs with APC mutations
do not completely lack APC protein. In the vast majority of
cases, one allele acquires a truncating mutation while the
second undergoes either a loss-of-heterozygosity or a second truncating mutation [53,115]. In particular, mutations
close to codon 1300 are usually associated with allelic loss
of the second allele, whereas tumors with mutations outside
this region tend to harbor a second truncating mutation
[116,117]. This interdependence of mutations suggests a
strong selective process for tumor progression. Upon closer
analysis of the mutation spectra in CRCs, it appears that the
retention of a single truncated APC allele containing one or
two of the total seven h-catenin regulating 20-amino acid
repeats confers the best selective advantage [115,118]. Since
polypeptides containing two or three of the 20 aa motifs but
none of the axin-binding SAMP repeats are still effective at
regulating h-catenin levels [65,119– 121], these truncated
APC products are presumably capable of residual h-catenin
regulating activity. A recent hypothesis dubbed the ‘‘justright signaling model’’ suggests that APC function must
be impaired sufficiently to allow the accumulation of hcatenin, but not above a certain limit [118]. Interestingly,
extracolonic lesions such as desmoid tumors and gastric
polyps in FAP patients are characterized by the retention of
a single APC allele with two or three 20 aa repeats,
suggesting that these tissues require a higher level of
residual h-catenin regulation than intestinal tumors [122 –
124]. Notably, mice do not share this selective pressure to
maintain a truncated APC allele, and allelic loss is the most
common somatic mutation mechanism.
Pathologies caused by a mutation in a single allele that
results in a 50% protein decrease are said to be haploinsufficient for that protein. There has been much discussion
about whether FAP and thus CRCs are caused by haploinsufficiency for APC. The very first mapping of the gene
responsible for FAP utilized a patient with an interstitial
deletion on chromosome 5 [125]. Although western blots
demonstrating the presence of only 50% APC were never
performed, it seems unlikely that the remaining APC allele
would be upregulated. More than a decade later, Laken et al.
[126] examined APC levels in FAP patients lacking obvious
mutations and found that seven of the nine studied had
significantly reduced expression, although the reduction was
not quantitated. Recently, Yan et al. [127] made use of a
quantitative PCR technology termed Digital-SNP to revisit
one of the patients described by Laken et al. in addition to
other FAP patients. Intriguingly, this analysis yielded data
supporting the notion that even modestly reduced levels of
APC expression from one allele are associated with a
pronounced predisposition to hereditary colorectal tumor.
Another group also developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions [128].
Analyzing 60 classic polyposis and 143 multiple adenoma
patients with no apparent APC germline mutation, deletions
were found exclusively in individuals with classic polyposis
(7 of 60, 12%). Fine mapping of the region suggested that
most (6 of 7) of these deletions encompassed the entire APC
locus, confirming that haploinsufficiency can result in a
classic polyposis phenotype.
Alternatively, because truncated APC retains its N-terminal coiled-coil domain responsible for dimerization, it is
possible that it might interfere with the remaining wild-type
APC function in a dominant negative manner. Looking at
the preneoplastic small intestinal epithelium of the Min
mouse (where both the 312 kDa full-length and the 95
kDa truncated Apc proteins are stably expressed) Mahmoud
et al. [129] found that truncated Apc is associated with
changes in the growth characteristics of preneoplastic tissue,
increased h-catenin expression, and an extended proliferative compartment. In a follow-up study, Min mice were
compared to mice expressing extremely low levels of Apc
truncated at residue 1638 and to wild-type littermates;
significant differences were seen in enterocyte migration,
in addition to the previously reported differences in proliferation and apoptosis [130]. These observations suggested
that a dominant-negative effect altering cell migration was
exerted by the truncated Apc protein present in the Min
mouse. Further experimental evidence arguing for a dominant-negative effect of truncated APC gene products was
supplied when wild-type APC activity in h-catenin/TCFmediated transcription was strongly inhibited by a mutant
APC truncated at codon 1309 [119]. In contrast, mutant
APC gene products that are associated with attenuated
polyposis, such as those involving codon 386 or 1465,
interfered only weakly with wild-type APC activity.
3.2. b-Catenin
Mutations in the gene encoding b-catenin (CTNNB1) are
present in approximately 10% of the remaining CRC tumors.
APC and CTNNB1 mutations are mutually exclusive, consistent with the notion that mutation of either gene has more
or less the same effect on h-catenin stability and TCF
transactivation. However, small adenomas with h-catenin
mutations do not appear to be as likely to progress to larger
adenomas and invasive carcinomas nor are h-catenin mutations present in 10% of invasive cancers. Thus, APC and
h-catenin mutations do not appear to be functionally identical [131]. Most h-catenin mutations occur in or around
exon 3 of the CTNNB1 gene, affecting the putative phos-
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
phorylation sites for GSK3h, making it refractory to degradation [132,133]. Inactivating mutations in axin have also
been found in a few CRC cell lines [4,134]. Taken together,
it appears that any mutation leading to stabilized nuclear
h-catenin is sufficient for neoplastic transformation in
colonic mucosa.
4. Wnt signaling is active in many cancers
Activating mutations of the Wnt pathway are not restricted to CRC (Table 2). A large number of sporadic tumor
types have been described with mutations in APC or
CTNNB1, and occasionally mutations in AXIN or other
components have been described. Upper gastrointestinal
tumors, for example, seemingly exhibit a high number of
h-catenin mutations. Approximately 20% (33 of 187) of the
gastric carcinomas screened had activating mutations in
CTNNB1 [135 – 137], as opposed to the large majority
(79%) of the gastric fundic gland polyps [138]. Gastric
adenomas featured somatic APC gene mutations in 76% (59
of 78) of adenomas or flat dysplasias without associated
adenocarcinoma, but in only 3% (1 of 30) of adenomas/
dysplasias associated with adenocarcinoma, and in only 4%
(3 of 69) of adenocarcinomas [139]. Also occurring in FAP
patients, 12 of 16 (75%) sporadic juvenile nasopharyngeal
angiofibromas had mutations in CTNNB1. Interestingly,
immunohistochemistry recognized nuclear h-catenin in all
16 tumors, but in the stromal cells and not in the endothelial
cells [140]. Tumors of the esophagus and esophagogastric
junction were also examined; activating Wnt mutations were
found to be rare (2 of 178) [141,142].
Human melanoma samples were one of the first tumor
types to be screened for CTNNB1 and APC mutations [143].
Initial results indicated a prominent role for Wnt signaling in
the etiology of malignant melanoma; unusual messenger
RNA splicing and missense mutations in the CTNNB1 gene
that result in stabilization of the protein were identified in 6
of the 26 cell lines (22%), and the APC gene was altered or
missing in 2 others (7%). Furthermore, in the APC-deficient
cells, ectopic expression of wild-type APC eliminated
excess h-catenin [143]. Screening an additional 195 cell
lines and primary tumors, however, delivered just five
additional CTNNB1 mutations [144 – 147]. Analyzing the
data collectively suggests that CTNNB1 and APC mutations
may each be present in approximately 5% of all melanomas
(Table 2). Recent data also support a role for Wnt5a
expression in increased melanoma progression [33].
A consistent role for activating CTNNB1 mutations in the
majority of hair matrix cell tumors, called pilomatricomas,
has been established. Mutations were initially identified in
12 of 16 (75%) human pilomatricomas [148]. All 40 of the
pilomatricomas evaluated in another study showed intense
nuclear h-catenin staining, and upon sequencing exon 3 of
CTNNB1, 3 of 11 (26%) harbored mutations affecting
GSK3h phosphorylation sites [149]. A third study identified
7
stabilizing h-catenin mutations in 100% of the tumors
studied [150]. In an effort to understand the mechanisms
underlying this tumor type, transgenic mice expressing
h-catenin controlled by an epidermal promoter were generated [151]. The mice undergo a process resembling de
novo hair morphogenesis and develop human-like epithelioid cysts and trichofolliculomas. Older transgenic mice
develop pilomatricomas. These findings suggest that transient h-catenin stabilization regulates the epidermal signal
leading to hair development and tumorigenesis.
HCC accounts for 75% of all liver cancer [1]. Understanding the etiology of this carcinoma is therefore of great
interest to the medical community. A number of papers have
described HCCs with mutations in h-catenin, pointing a
finger at Wnt signaling (Table 2). Collectively, these groups
analyzed 497 HCCs, of which 91 (18%) had activating
CTNNB1 mutations [52,152 –157]. Huang et al. [158] show
that h-catenin mutations are much more common (41%) in
HCC associated with hepatitis C infection. Among tumors
lacking a h-catenin mutation, no APC mutation was
detected in a subset of 30 cases tested [154]. Two studies
used immunostaining to detect nuclear h-catenin in a total of
25 of 95 (26%) HCCs [155,156]. Nhieu et al. [155] found a
significant correlation between the presence of nuclear hcatenin and patient clinical outcome, whereas Wong et al.
[156] found that the 63% of HCCs studied with non-nuclear
h-catenin localization had a poorer clinicopatholigical outcome. Legoix et al. [154] looked at h-catenin mutation
status and chromosome instability in a set of 48 hyperploid
HCC tumors. A significant association was observed; like
CRC, HCC with h-catenin mutations do not exhibit chromosome instability [154]. Satoh et al. [52] performed
mutation analysis of the CTNNB1 and AXIN1 genes on
106 HCCs; 15 had CTNNB1 mutations affecting phosphorylation sites and 8 of 91 remaining HCCs harbored inactivating mutations in AXIN1. This study provided the first
evidence that Axin functions as a tumor suppressor protein.
The female and male reproductive organs are also prone
to cancers exhibiting active Wnt signaling, particularly
endometrioid-type ovarian carcinomas (Table 2). Approximately one-third (65 of 208, 31%) of endometrioid-type
ovarian carcinoma exhibit nuclear h-catenin [159 – 164].
Sequence analysis of exon 3 of CTNNB1 identified 44
mutations in 156 primary tumors (28%) [159 – 163,165].
Tumors exhibiting either microsatellite instability or chromosome instability almost never had h-catenin mutations,
suggestive of two distinct mechanisms [163,164]. Two
studies linked CTNNB1 mutations with a favorable prognosis for patients with endometrioid-type ovarian carcinomas [161,164]. Mutations in APC, AXIN1, and AXIN2 have
also been described in these tumors [162]. Endometrial
cancer showed nuclear localization of h-catenin in 14 of
the 25 (56.0%) endometrial hyperplasia samples, 12
(60.0%) endometrial cancers, and 11 (55.0%) associated
hyperplasias of the 20 endometrial cancers associated with
hyperplasia [166]. CTNNB1 mutation analyses has thus far
8
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
Table 2
Overview of mutations of the Wnt signaling pathway in selected human
cancers
Table 2 (continued )
Cancer type
Gene
Mutation frequency
References
Breast fibromatoses
CTNNB1
APC
CTNNB1
CTNNB1
CTNNB1
APC
hTRCP
CTNNB1
N/A
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
APC
APC
AXIN1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
AXIN1
AXIN1
AXIN2
CTNNB1
45% (15/33)
33% (11/33)
5% (5/104)
5% (7/138)
9% (2/22)
14% (3/22)
9% (2/22)
46% (26/57)
67% (52/78), IHC
48% (25/52)
33% (1/3)
89% (8/9)
67% (12/18)
13% (4/30)
75% (12/16)
65% (44/68)
70% (19/27)
69% (9/13)
0% (0/68)
7% (2/27)
26% (8/31)
19% (14/73)
18% (21/119)
34% (12/35)
14% (15/106)
12% (7/60)
19% (14/73)
8% (8/106)
10% (7/73)
3% (2/73)
41% (9/22)
[322]
[322]
[171]
[172]
[173]
[173]
[173]
[323]
[324]
[177]
[174]
[175]
[178]
[179]
[325]
[176]
[157]
[180]
[176]
[157]
[152]
[153]
[154]
[155]
[52]
[156]
[157]
[52]
[157]
[157]
[158]
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
APC
APC
AXIN1
AXIN1
CTNNB1
CTNNB1
CTNNB1
4% (3/67)
9% (4/46)
18% (9/51)
5% (4/97)
4% (1/23)
4% (2/46)
2% (2/97)
12% (13/97)
4% (1/23)
58% (7/13)
52% (22/42)
Novel case
25% (3/12)
50% (3/6)
21% (9/42)
15% (6/40)
14% (21/153)
83% (15/18)
90% (18/20)
[183]
[184]
[181]
[185]
[182]
[184]
[185]
[186]
[182]
[188]
[189]
[326]
[190]
[327]
[189]
[328]
[329]
[330]
[331]
CTNNB1
APC
6% (1/21)
18% (3/21)
[332]
[332]
CTNNB1
CTNNB1
CTNNB1
APC
55% (5/9)
8% (4/49)
0% (0/15)
8% (4/49)
[187]
[333]
[334]
[335]
Cancer type
Gene
Mutation frequency
References
Colorectal cancer
APC
80%
Prostate
CTNNB1
N/A
10%
48% (10/21), IHC
[110,111,
315]
[132,316]
[317]
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
91% (52/57)
64% (29/45)
8% (7/88)
26% (19/73)
27% (7/26)
[138]
[318]
[137]
[135]
[136]
Thyroid carcinoma
APC
76% (59/78)
[139]
CTNNB1
38% (27/72)
[319]
CTNNB1
APC and
CTNNB1
CTNNB1
0% (0/69)
2% (2/109)
[142]
[141]
75% (12/16)
[140]
Hepatocellular
carcinoma
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
APC
APC
CTNNB1
CTNNB1
CTNNB1
CTNNB1
22% (6/26)
2% (1/65)
3% (1/31)
2% (1/62)
5% (2/37)
7% (2/26)
3% (1/37)
75% (12/16)
26% (3/11)
100%
3% (3/93)
[143]
[144]
[145]
[146]
[147]
[143]
[147]
[148]
[149]
[150]
[320]
Hepatocellular
carcinoma
associated with
hepatitis C
Medulloblastoma
CTNNB1
50% (3/6)
endometrioid-type
33% (4/12)
endometrioid-type
54% (7/13)
endometrioid-type
16% (10/63)
endometrioid-type
31% (15/47)
endometrioid-type
38% (8/21)
endometrioid-type
14% (1/7)
mucinous-type
2% (1/47)
endometrioid-type
4% (2/47)
endometrioid-type
2% (1/47)
endometrioid-type
37% (12/32), IHC
13% (10/76)
22% (2/9)
45% (13/29)
11% (5/44)
25% (1/4)
10% (2/20)
[159]
Small intestinal
adenocarcinoma
Fundic gland
polyps (gastric)
Gastric carcinoma
Gastric,
intestinal-like
Gastric adenoma
(without associated
adenocarcinoma)
Gastrointestinal
carcinoid tumor
Esophageal
adenocarcinoma
Juvenile
nasopharyngeal
angiofibromas
Melanoma
Pilomatricomas
Lung
adenocarcinomas
Ovarian carcinoma
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
APC
AXIN1
AXIN2
Uterine cervix
Uterine endometrial
N/A
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
CTNNB1
Hepatoblastoma
[160]
[161]
[165]
Desmoid tumor
[162]
[149]
APC
[160]
[162]
[162]
[162]
[321]
[167]
[168]
[169]
[170]
[163]
[166]
Wilm’s tumor
(kidney)
Pancreatic:
non-ductal solid
pseudopapillary
Pancreatic:
non-ductal
acinar cell
carcinomas
Pancreatoblastoma
Synovial sarcoma
CTNNB1
N/A, not applicable; IHC, immunohistochemistry staining showed nuclear
h-catenin.
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
identified 33 activating mutations in the 182 endometrial
tumors (18%) examined [163,166 – 170]. A single study has
reported accumulation of h-catenin as seen by immunohistochemistry in 12 of 32 (37%) tumors of the uterine cervix.
Lastly, three groups recognized a low (5%) but consistent
level of CTNNB1 mutations in a total of 264 prostate tumors
[171 –173]. Interestingly, Gerstein et al. [173] also identified
two tumors in their sample set of 22 with inactivating
mutations in hTRCP, a protein involved in h-catenin degradation never previously implicated as a tumor suppressor.
These data suggest a role for Wnt signaling in the etiology
of reproductive cancers.
It is remarkable that CTNNB1 mutations are so prevalent
in childhood cancers. Hepatoblastoma represents the most
frequent malignant liver tumor in childhood, occurring in
children at an average age of 2– 3 years. Although most
cases are sporadic, the incidence is highly elevated in
patients with FAP. To determine whether Wnt signaling is
involved in the pathogenesis of sporadic hepatoblastomas,
several groups have looked at sporadic hepatoblastomas for
mutations in the APC and CTNNB1 genes (Table 2).
Virtually all (25 of 27) tested hepatoblastomas exhibited
nuclear h-catenin detected by immunohistochemistry [174 –
176]. CTNNB1 mutations affecting exon 3 which ablated
GSK3h target phosphorylation sites were found in 113 of
223 (51%) samples [157,174 – 179]. Less frequent somatic
mutations of APC and AXIN1 have also been described in
hepatoblastomas [157,180]. Thus, h-catenin accumulation
may play a role in the development of hepatoblastoma and
heterozygous activating mutations of the CTNNB1 gene
may substitute for biallelic APC inactivation in this tumor
type. Like hepatoblastomas, medulloblastomas (MB)—the
most frequent malignant brain tumors in childhood generally occurring in the cerebellum—also occur in FAP
patients. Two studies used immunostaining for h-catenin
to establish Wnt involvement; in total, 14 of 74 (19%) MB
samples showed strong nuclear h-catenin staining
[181,182]. They and others also looked at MB samples for
the presence of mutations resulting in APC inactivation or
h-catenin activation [181 – 185]. These data collectively
report CTNNB1 mutations in 21 of 284 (7%), and APC
mutations in 4 of 143 (3%) MBs. These mutation frequencies were unexpectedly low considering the relatively common phenomenon of nuclear h-catenin. Mutation analyses
of the AXIN1 gene filled the discrepancy: deletions and
point mutations were reported in 14 of 120 (12%) of
examined MB samples [182,186]. Only one study has
performed mutation analysis on pancreatoblastomas, but it
too showed five of nine tumors to have activating h-catenin
mutations [187].
Because desmoid tumors commonly occur in FAP
patients, sporadic cases were examined for molecular
changes affecting stabilization of h-catenin. The combined
results of three studies identified CTNNB1 mutations in 32
of 67 (48%) desmoid tumors [188 – 190]. Using real-time
PCR, Saito et al. [190] were able to quantitate h-catenin
9
RNA expression and confirmed that tumors with somatic
CTNNB1 mutations had increased levels of h-catenin as
compared to samples with wild-type h-catenin. Because
real-time PCR is fast, sensitive, and reliable, this technology
could offer a good alternative to immunohistochemical
staining of h-catenin.
Several lines of research point to a role of Wnt signaling
in mammary development and carcinogenesis. Animals
ectopically expressing Wnt-1 develop mammary and salivary adenocarcinomas [191]. The Wnt-2 gene is amplified in
mammary tumors from mice [192]. Interestingly, a proportion of human breast carcinomas also express high levels of
WNT-2, WNT-5A, and WNT-7B [193 – 196]. Transgenic
mice overexpressing axin exhibit problems in mammary
and lymphoid development [197]. Similarly, mice harboring
mammary-specific mutations in Apc show markedly delayed
development of the mammary ductal network, and during
lactation, these mice develop multiple benign growths
[198]. Mice lacking Lef1 do not develop mammary glands
[199], while mice lacking Tcf1 eventually develop adenomas in the intestine and mammary glands [200]. These
results require follow-up studies to ascertain how Wnt
signaling affects mammary carcinogenesis.
5. Model organisms
5.1. Lower organisms
Most components of the Wnt pathway were identified in
genetic experiments in lower organisms. While useful for
delineating the pathway itself and its role in axis and
mesoderm formation, initiation of organ development and
cell polarity, Drosophila and Xenopus make poor models for
cancer research. The canonical pathways in these organisms
are strikingly similar to the vertebrate pathway, and are
reviewed elsewhere [201,202]. New to the Wnt field, the
zebrafish is becoming a popular model organism for cancers
[203]. A homozygous mutation in zebrafish axin abolishing
its ability to bind GSK3h results in fish without eyes or
telencephalon, a phenotype called masterblind [204,205].
The signaling and adhesion functions of h-catenin are
represented by three distinct orthologues in the nematode
C. elegans: WRM-1, BAR-1 and HMP-2 [206]. WRM-1
was originally identified as a h-catenin orthologue, although
initial studies showed that it did not transactivate with the
TCF orthologue POP-1 [207,208]. The notion that canonical
Wnt signaling in C. elegans significantly deviated from
other organisms was further perpetrated when APR, a
distant relative of APC, and SGG/GSK3h were found to
be positive, not negative, regulators of Wnt signal [209].
However, Korswagen et al. [206] demonstrated that BAR-1
is the h-catenin homologue that interacts with TCF/POP-1
and is the effector of canonical Wnt signaling. The third
homologue, HMP-2, mediates cell – cell adhesion by binding to the a-catenin/HMP-1 and interacting with the
10
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
E-cadherin/HMR-1. A conserved canonical Wnt pathway in
C. elegans is further supported by the recent studies of a
distant relative of axin, PRY-1, which participates in a
functional degradation complex regulating BAR-1 levels
[210]. PRY-1 is able to rescue the zebrafish masterblind
phenotype, demonstrating that despite its evolutionary
divergence, PRY-1 is a functional axin protein [210].
Another recent report revisits APR-1 and places it in a more
APC-like light; loss of APR-1 mimics the phenotype seen
when BAR-1 h-catenin protein is constitutively activated,
suggesting that APR-1 may actually play a negative role in
C. elegans Wnt signaling, as one would expect [211].
5.2. Apc mouse models
For the purpose of understanding the role of Wnt signaling in cancer, the generation of mouse models has provided
the best tools for dissecting the individual functions of Wnt
pathway components (see Table 3). Chemical mutagenesis
produced a mouse model for FAP carrying a nonsense
mutation at codon 850, and stably expressing truncated
Apc. Mice heterozygous for this mutation develop multiple
intestinal neoplasia (Min) and seldom live longer than 3
months [212,213]. The multiplicity and size of the tumors
varies with genetic background suggesting the presence of
genetic modifying components. Of note is the fact that while
the Min adenomas resemble the human disease quite well,
they arise in the small intestine, not the colon. Adenomas
harvested from Min mice harbor somatic mutations of the
wild-type Apc allele [214]. Female Min mice that live long
enough may also develop mammary tumors. Transplantation of mammary cells from Min/+ or +/+ donors into +/+
hosts demonstrated that the propensity to develop mammary
tumors is intrinsic to the Min/+ mammary cells [215]. Mice
homozygous for the Min mutation die in utero, about 8 days
postcoitus (dpc) [216].
Min mice are frequently used in studies ascertaining
environmental and genetic factors involved in tumorigenesis
[217]. For example, when crossed with mice mutant for p53,
Min/+ p53 / mice exhibited a shift in phenotype, consistently resulting in pancreatic neoplasia in addition to the
same number and size of intestinal adenomas seen in Min
mice with p53 [218]. A different group revisited the
interaction between Apc and p53 in congenic mouse strains
Table 3
Murine models of Wnt component function in carcinogenesis
Gene
Type of mutation
Mouse phenotype
+/
ApcMIN
ApcD716
ApcD580
Apc1638N
Apc1638T
ApcD716
h-Catenin
(Catnb)
truncated at aa 850
truncated at aa 716
intestine-specific
CRE-mediated deletion
of exon 14
(truncated at aa 580)
expresses about 2%
Apc truncated at aa 1638
truncated at aa 1638
Tg, constitutively active 32
knock-out
Tg, constitutively active
inducible Tg, mesencymal
Tg, renal or
intestinal epithelial
Tg, hepatocyte
Tg, hepatocyte,
adenoviral transfer
Tg, secretory epithelia
References
/
200 – 500 intestinal adenomas
200 – 500 intestinal adenomas
N/A
embryonic lethal 8 dpc
embryonic lethal 8 dpc
after induction of mutation
by CRE infection, adenomas
develop within 4 weeks
[212,213,216]
[227]
[228]
5 – 6 upper gastrointestinal
adenomas, desmoid tumors,
cutaneous cysts
normal
normal
normal
embryonic lethal
[229,231]
nearly normal, no cancer
N/A
embryonic lethal 7 dpc
[236]
[353]
[246]
200 – 500 intestinal adenomas
hyperplastic gastrointestinal
polyps, desmoid tumors
polycystic kidney disease
N/A
N/A
[238,239]
[240]
N/A
[239,241]
N/A
N/A
[243]
[244]
N/A
[245]
adenomas in the gut and
mammary glands
increased tumor load
[200,248]
Tcf-1
HR knock-out
severe hepatomegaly
severe hepatomegaly,
no neoplastic foci observed
extensive squamous metaplasia
and keratinization, high-grade
prostate intraepithelial neoplasias
reminiscent of early
human prostate cancer
normal
Tcf1 / ,
Apc Min/ +
Tcf-4
N/A
N/A
HR knock-out
normal
small intestine stem cell
compartment depleted
aa, amino acid; dpc, days postcoitus; N/A, not applicable; HR, homologous recombination; Tg, transgenic.
[200]
[253]
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
in order to minimize the influence of polymorphic modifiers. Deficiency of p53 enhanced the number and size of
intestinal adenomas in Min/+ mice [219]. Conversely, crossing Min mice with mice hemizygous for DNA methyltransferase that were then treated with 5-aza-deoxycytidine
dramatically reduced the average number of polyps from
103 to 2 [220]. Studies into environmental factors affecting
colon cancer incidence worldwide led to the hypothesis that
this variation can be explained largely by dietary influences.
Using the Min mouse, Wasan et al. [221] demonstrated that
higher dietary fat results in an increase in tumor counts in
both the large and the small intestine. A positive correlation
could be made with increased dietary fat and polyp size. As
discussed later, Min mice provide useful reagents for testing
therapeutic agents targeting the Wnt pathway.
Min mice have also been used to identify modifier loci of
tumor development. Just months after the first report of the
Min phenotype, Dietrich et al. [222] reported the genetic
mapping of a locus, Mom1 (modifier of Min 1), that
modifies tumor number and size by about 50% in Min/+
animals. Mom1 was localized to a region syntenic to human
chromosome 1p36– p35, a region of frequent somatic loss of
heterozygosity in a variety of human tumors, including
colon tumors. A transgene overexpressing the protein encoded by Mom1, secretory phospholipase Pla2g2a, drastically decreased the number of tumors in Min mice [223]. A
second spontaneous modifier was recently described,
Mom2, which can suppress up to 95% of tumor formation
in Min mice [224]. The gene encoding Mom2 is currently
being cloned.
Although perhaps most widely used, Min mice are not
the only model generated to study Apc. Conventional gene
targeting has also generated mice with truncated alleles at
codons 474 and 1309, respectively [225,226]. Both mice
share phenotypes with the Min mouse. Oshima et al. [227]
used homologous recombination to generate mice expressing Apc truncated at residue 716. Like Min mice, the
ApcD716 heterozygotes developed numerous adenomas
throughout the intestinal tract, mostly in the small intestine.
Genotyping even the smallest tumors revealed that all had
lost their wild-type Apc allele. This study was pivotal in two
respects: it determined that loss of Apc is an early, if not
initiating event in tumorigenesis, and it established that
these microadenomas stem from single crypts by forming
abnormal outpockets protruding into the neighboring villus
[227]. Shibata et al. [228] created a conditional Apc model
in which Apc exon 14 is deleted upon Cre recombinase
expression in the colon, resulting in Apc truncated at codon
580. The mice developed adenomas within 4 weeks, again
implying that inactivation of Apc is sufficient to drive polyp
formation.
Two Apc mouse models make use of an introduced
termination mutation at residue 1638. The first, called
Apc1638N, introduces the PGK-neomycin gene at residue
1638 in the opposite transcriptional orientation of Apc,
resulting in greatly reduced levels of the truncated polypep-
11
tide [229]. Although undetectable by western blot, the allele
is leaky and approximately 2% of the 182 kDa Apc1638N is
present and functional [229,230]. In the same genetic background, Apc1638N/+ mice develop considerably fewer intestinal tumors than Min mice and live considerably longer, but
their tumors tend to develop higher up in the gastrointestinal
tract, at the transition from stomach to small intestine
(periampullary region) [231,232]. Interestingly, Apc1638N/+
mice also develop high numbers of extracolonic lesions,
particularly cutaneous follicular cysts and benign desmoid
growths. Human FAP patients with APC mutations located
between codons 1445 and 1578 are also often associated
with severe desmoids as well as osteomas, epidermoid cysts,
and polyps of the upper gastrointestinal tract [233], making
this mouse model indispensable for studying these particular
extraintestinal manifestations. Furthermore, gastric tumors
occur in Apc1638N/+ mice [234,235]. Homozygosity for the
Apc1638N mutation is not compatible with adult life [229,
230].
When the same PGK-neomycin gene at residue 1638 is
introduced in alignment with the transcriptional orientation
of Apc, it results in a truncated allele, called 1638T. The
truncated protein lacks the C-terminal domains binding
to tubulin, EB/RP proteins, and DLG, but can regulate
h-catenin levels efficiently. Heterozygous as well as homozygous Apc1638T mice are not only viable and fertile, but
they remain tumor-free [236]. Because the C-terminus of
Apc binds microtubules, EB-family proteins, and DLG,
Apc1638T mice provide an excellent model for delineating
the critical domains of Apc involved in tumorigenesis and
development. The targeted deletion of the last remaining
SAMP repeat, resulting in a truncation at codon 1572,
eliminated h-catenin regulatory function [236]. Embryonic
stem cells homozygous for APC1638T exhibit chromosome
instability underscoring the role of the C-terminus in chromosome segregation [237]. Evidently, the predisposition to
chromosomal instability is not sufficient to drive tumor
formation in these mice.
5.3. b-catenin mouse models
In approximately 5 –10% of human CRCs, mutations in
h-catenin make it refractory to degradation and are oncogenic [132,133]. Likewise, transgenic mouse models inducibly expressing constitutively active h-catenin suffer
intestinal tumors indistinguishable from Min mice
[238,239]. Thus, constitutive h-catenin signaling is sufficient for intestinal tumor induction. Furthermore, stabilized
h-catenin expressed in mesenchymal cells under control of a
tetracycline-regulated promoter produced transgenic mice
exhibiting increased fibroblast proliferation, motility, and
invasiveness and induced tumors after induction of the
transgene when grafted into nude mice. After 3 months of
transgene induction, mice developed aggressive fibromatoses (desmoids) and hyperplastic gastrointestinal polyps
[240].
12
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
Transgenic mice overproducing an oncogenic form of
h-catenin have also been generated to study other tumor
types. Mice expressing stable h-catenin controlled by an
epidermal promoter develop human-like epithelioid cysts,
trichofolliculomas, and eventually pilomatricomas [151].
Targeted to the epithelial cells of either the intestine or
kidney, dominant stable h-catenin caused severe polycystic
lesions soon after birth [239,241]. Because autosomal dominant polycystic kidney disease is one of the most commonly inherited human diseases and results in renal failure
at a young age [242], this mouse model has generated a
considerable amount of interest. Alternatively, when selectively targeted to hepatocytes in transgenic mice, oncogenic
h-catenin caused dramatic hepatomegaly, producing hyperplastic livers three to four times heavier than those of
littermates [243]. In a different study, adenovirus-mediated
expression of dominant stable h-catenin again caused hepatomegaly, although a thorough examination detected no
neoplastic foci [244]. These results suggest that, in contrast
to intestinal polyposis, the Wnt pathway activation by
stabilized h-catenin is not sufficient for hepatocarcinogenesis. Likewise, stabilization of h-catenin in secretory epithelia including salivary, preputial, harderian, and mammary
glands induced extensive squamous metaplasia and keratinization associated with terminal differentiation of the target
cells, but failed to cause neoplastic transformation [245].
However, stabilized h-catenin was able to induce high-grade
prostate intraepithelial neoplasias reminiscent of early
human prostate cancer [245].
h-Catenin knock-out mice suffer severe gastrulation
defects and die 7 dpc [246]. A conditional knock-out
targeted to the epidermis and hair follicles demonstrated
that without h-catenin, stem cells fail to differentiate into
follicular keratinocytes, but instead adopt an epidermal fate
[247].
5.4. Other cancer mouse models for Wnt pathway
components
A large number of mutations affecting Wnt signaling
components have been introduced into the mouse. Most do
not produce a cancer phenotype. Only the few that contribute relevant information concerning tumorigenesis will be
discussed here. Transgenic mice overexpressing ectopic
Wnt1 RNA in mammary and salivary glands of male and
female mice were generated long before the Wnt signaling
pathway was defined [191]. Mammary and salivary adenocarcinomas developed in these animals, but not in their
nontransgenic littermates. A somewhat similar phenotype
was recapitulated in the Tcf1 knock-out mice. Human TCF1
is expressed in T-lymphocytes. Mice nullizygous for Tcf1
are impaired in the generation of T cells [248]. The thymus
in these mice is reduced in size and show up to 100-fold
reduction in T cells as compared to their heterozygous
littermates. Despite this defect, initial examination of the
Tcf1 /
mice showed them to be fully immunocompe-
tent, and they enjoy normal longevity and fertility [249].
Follow-up studies found adenomas in the intestine and
mammary glands [200]. Crossing these mice with Min mice
increased the number of tumors, suggesting that Tcf1 is a
target of Tcf4/h-catenin in the intestine. Because the predominant isoform of Tcf1 expressed in intestinal epithelium
is unable to bind h-catenin yet retains its ability to bind
members of the groucho repressors, expression of Tcf-1
regulated by Wnt signaling would create a negative regulatory feedback loop.
Lef1 is expressed in pre-B and T lymphocytes of adult
mice, and in the neural crest, mesencephalon, tooth germs,
whisker follicles, and other sites during mouse embryogenesis. Knock-out mice lacking Lef1 protein caused postnatal lethality [199]. The mutant mice lacked teeth,
mammary glands, whiskers, and hair, although they developed rudimentary hair follicles. The mutant mice showed no
obvious defects in lymphoid cell populations at birth [199].
To test whether LEF1 patterning might be functionally
important for hair patterning and morphogenesis, Zhou et
al. [250] overexpressed human LEF1 in the surface ectoderm of mice. Striking abnormalities arose in the positioning and orientation of hair follicles, and elevated levels of
LEF1 in the lip furrow epithelium of developing transgenic
mice triggered these cells to invaginate, sometimes leading
to the inappropriate adoption of hair follicle and tooth cell
fates. Another Lef1 transgenic model introduced a form of
Lef1 lacking the h-catenin binding site under the control of
a keratin promoter. No skin abnormalities were detected
before the first postnatal hair cycle. However, from 6 weeks
of age, mice underwent progressive hair loss which correlated with the development of dermal cysts. Adult mice
developed spontaneous skin tumors, most of which
exhibited sebaceous differentiation, which could be indicative of an origin in the upper part of the hair follicle. The
transgene continued to be expressed in the tumors and
h-catenin signaling was still inhibited, as evidenced by the
absence of cyclin D1 expression [251]. This phenotype
shares some phenotypic overlap with the targeted deletion
of h-catenin in the epidermis [247]. Niemann et al. [251]
conclude that the level of h-catenin signaling determines
whether keratinocytes differentiate into hair or interfollicular
epidermis, and that perturbation of the pathway can lead to
skin tumor formation.
In the intestinal epithelium TCF4 is the most prominently
expressed TCF family member [252]. Gene disruption of the
murine germline has revealed that Tcf4 is required to
establish the proliferative progenitors of the prospective
crypts in the small intestine during embryonic development
[253]. APC loss and subsequent h-catenin stabilization
results in a size increase of the proliferative compartment
of the crypt. Because Tcf4 is required for crypt maintenance,
it is likely that inappropriate Tcf4/h-catenin signaling is
responsible for this phenomenon. Cells that would normally
differentiate while migrating up the crypt – villus axis are
now kept in a proliferative and non-differentiated state. This
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
is supported by the observation that h-catenin suppresses
differentiation [230,238,254].
6. How does Wnt signaling cause colorectal tumor
initiation?
Activation of the Wnt signaling pathway can lead to a
number of outcomes including effects on transcription, cell
migration, and cell polarity [57]. The spectrum of target
genes controlled by h-catenin/TCF is expected to hold the
key to understanding the primary transformation of intestinal and other cells. Once a growth advantage is provided by
the activation of certain critical target genes, a number of
inactivating mutations must be acquired in tumor suppressor
genes. In CRC cells, this is most often accomplished by
chromosome instability. Architectural changes coupled with
suppression of cellular differentiation in the intestinal crypt
may provide a venue for these additional mutations to take
place. Changes in cell adhesion and migration contribute to
the invasiveness of a particular tumor. All of these requirements are met by the activation of Wnt signaling and are
summarized as follows.
6.1. Target genes
Several individual TCF/LEF target genes have been
identified, some of which have important implications in
understanding the role of Wnt signaling in cancer. Among
the most prevalent, Wnt signaling promotes the expression
of c-MYC [255]. Table 4 provides a select list of target
genes; a more detailed list can be found at the Wnt home
page hosted by R. Nusse (www.stanford.edu/frnusse/
wntwindow.html). The observation that loss of Tcf4 funcTable 4
Selected human h-catenin/TCF target genes
Gene
References
CD44
BMP4
claudin-1
ENC1
cyclin D1
fra-1
PPARd
c-MYC
Nr-Cam
ITF-2
IL-8
PKD1
Gastrin
AVIN2
MRD1
Matrilysin
MMP7
Osteopontin
TLE/Groucho, MSX1, MSX2, CBP/p300, REST/NRSF
EPHB2, EPHB3, c-MYB, ETS-2, BMP4
[336]
[337]
[338]
[339]
[340,341]
[342]
[301]
[255]
[343]
[344]
[345]
[346]
[347]
[348]
[349]
[350]
[351]
[352]
[257]
[254]
See also www.stanford.edu/frnusse/wntwindow.html.
13
tion in mouse leads to the depletion of intestinal stem cells is
indicative of a role of the Wnt signaling pathway in epithelial stem cell maintenance [253], and computer modeling
studies imply that tumor initiation in the colon is caused by
crypt stem cell overproduction [256].
Recently, two large-scale analyses using DNA microarray
technology have begun to unravel the downstream genetic
program activated by h-catenin/TCF in embryonic carcinoma cells and CRC cells, respectively [254,257]. In the first
study, Wnt-3A conditioned or control medium was applied
to human teratocarcinoma cells for various lengths of time
before RNA was isolated and the expression profiles compared by microarray hybridization. Several genes were
identified in this and in earlier studies that interfere with
the differentiation of cells, e.g. the ID, MSX, and REST/
NRSF family of genes [254,257,258]. Expression of these
genes was amplified even further upon exposure to BMP-4,
suggesting either an additive or synergistic effect with Wnt
[257]. Consistent with these results, van de Wetering et al.
[254] confirmed the generalization that h-catenin/TCF targets often work to repress differentiation. Microarray expression profiling was performed using CRC cell lines carrying
doxycycline-inducible expression plasmids encoding dominant-negative TCF (dnTCF) proteins. Upon induction with
doxycycline, these dnTCF proteins act as potent inhibitors of
the endogenous h-catenin/TCF complexes present in CRC
cells, imposing a robust cell cycle and proliferation arrest.
Genes downregulated by the induction of dnTCF are thus
putative targets of h-catenin/TCF and include the genes
encoding c-MYC, c-MYB, ETS, BMP4, and ephrin receptors EPHB2 and EPHB3 [254]. Reintroduction of c-MYC
rescued the cell cycle arrest by blocking the expression of
cyclin-dependent kinase inhibitor p21CIP/WAF1. Furthermore,
upon expression of dnTCF and subsequent G1 arrest, these
cells recapitulate the physiological differentiation program of
normal intestinal cells in a p21CIP1/WAF1-dependent manner
[254]. Many of the genes upregulated by dnTCF represent
differentiation markers of mucososecretory and/or absorptive intestinal cells. Thus h-catenin/TCF4 activity constitutes
the master switch controlling proliferation versus differentiation in the intestinal epithelium and imposes a crypt
progenitor phenotype in CRC cells.
LEF1 is a target gene ectopically activated in colon
cancer [259]. The pattern of this ectopic expression is
unusual because it derives from selective activation of a
promoter for a full-length LEF1 isoform that binds hcatenin, but not a second, intronic promoter that drives
expression of a dominant-negative isoform. h-Catenin/TCF
complexes can activate the promoter for full-length LEF1,
indicating that in cancer, high levels of these complexes
misregulate transcription to favor a positive feedback loop
for Wnt signaling by inducing selective expression of fulllength, h-catenin-sensitive forms of LEF/TCFs [259]. LEF1
anchors h-catenin in the nucleus by blocking APC-mediated
nuclear export. LEF1 is a key regulator of h-catenin nuclear
localization and stability suggesting that overexpression of
14
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
LEF1 in colon cancer and melanoma cells may contribute to
the accumulation of oncogenic h-catenin in the nucleus
[260]. In the mouse, Tcf1 has been shown to be a target
gene of h-catenin/Tcf4 [200]. Crossing Min mice with
Tcf1 /
mice substantially increased the multiplicity
and speed of tumor development, indicating that Tcf1
negatively regulates h-catenin/Tcf4 and functions as a tumor
suppressor [200].
6.2. Genetic instability
A common feature of many tumors is genetic instability,
often manifested as aneuploidy or hyperploidy, supernumery centrosomes and abnormal mitotic spindle assembly
[261 –265]. These phenomena lead to multipolar cell divisions, incomplete or abnormal chromosome segregation,
and occasionally chromosomal breakage [266]. While
genetic instability may provide genetic diversity, stochastic
mutations are more likely to be deleterious than growth
promoting. A normal 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
at other genes aiding tumor progression and malignant
transformation. Most tumors resulting from activated Wnt
signaling fulfill the first requirement by transactivating TCF
target genes such as c-MYC.
Fulfillment of the second requirement has been best
studied in the intestine. CRC develops through a series of
genetic alterations involving the accumulation of mutations
in a number of genes and progression through the
adenoma – carcinoma sequence [267] (Fig. 2). The underlying hypothesis is that the tumor microenvironment is an
additional driving force of tumor progression. This tumor
progression model was deduced from comparison of genetic
alterations seen in normal colon epithelium, adenomas of
progressively larger size, and malignancies [268]. There are
at least two mechanisms inducing the molecular events that
lead to CRC. About 85% of CRCs exhibit chromosomal
instability (CIN), whereas the remaining 15% are due to
events that result in microsatellite instability (MIN) [269 –
271]. A strong correlation between APC mutation in CRC
and the presence of CIN, coupled with the association of
CTNNB1 mutations occurring with MIN led to the hypothesis that mutations in APC not only confer a growth
advantage but also promote CIN in CRCs. Mouse ES cells
expressing truncated APC show CIN, aberrant spindle
formation and supernumery centrosomes [237,272]. Moreover, 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 [272]. Based on
these observations, the hypothesis has been put forward that
a truncating mutation in APC initiates CIN, thus accelerating the loss of the second allele which ultimately leads to
deregulated cellular proliferation.
Although genetic instability is global, cells with specific
losses are selected for clonal growth. Generally, CRCs
Fig. 2. The adenoma – carcinoma sequence for colorectal cancer. A mutation in APC or h-catenin results in the activation of the Wnt signaling pathway,
triggering tumor formation. Subsequent progression towards malignancy is accompanied by sequential mutations in KRAS, deletion of chromosome 18q
affecting genes encoding SMAD2 and SMAD4, p53, and genes involved in tumor invasiveness such as E-cadherin. Tumor suppressor proteins are represented
above the adenoma – carcinoma sequence, whereas oncogenes are depicted below. Increasing levels of nuclear h-catenin accompany tumor progression.
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
manifesting CIN acquire sequential chromosome losses
(Fig. 2) at the loci for APC (chromosome 5q) [273], DCC/
DPC/JV18 (chromosome 18q), and p53 (chromosome 17p)
[268]. These chromosome losses perpetrate further instability at the molecular and chromosomal level [269]. The
paradigm of sequential genetic alterations driving tumor
progression is not totally inflexible; not every CRC tumor
needs to acquire every mutation, nor do the mutations
always need to occur in a specific order. However, the type
of mutation may influence the rate or type of pathologic
change in the tumors. As opposed to CIN, tumors manifesting MIN are characterized by a largely intact chromosome
complement, but they have acquired defects in DNA repair.
Thus mutations that may occur in important cancer-associated genes are allowed to persist. These types of cancers are
detectable at the molecular level by alterations in repeating
units of DNA that occur normally throughout the genome,
known as a DNA microsatellite. Many of the same genes are
affected in MIN tumors as in CIN tumors.
6.3. APC and b-catenin in cytoskeletal dynamics, cell
adhesion and cell migration
Although the Arm repeats in APC show high conservation
to those in h-catenin, they do not bind the same proteins
[64,86,87]. Remarkably little is known about the function of
APC’s Arm repeats [67,274,275]. However, two recent
studies report the association of APC’s Arm repeats to
proteins involved in cytoskeletal dynamics. Suggesting a role
for APC in cell morphology and migration through modifications of the actin cytoskeleton, the Rac-specific guanine
nucleotide exchange factor Asef binds the arm repeats of
APC. This interaction stimulated cell flattening, membrane
ruffling, and lamellipodia formation in MDCK cells induced
by exogenous expression of Asef [276]. Microtubules are
essential for cellular processes including migration, organelle
transport, and chromosome segregation during cell division
by formation of the mitotic spindle. APC has been localized
to clusters near the distal ends of microtubules at the edges of
migrating epithelial cells [277], and other studies have shown
APC to bind microtubules both directly and indirectly [278 –
281]. The C-terminus of Apc is important for the stabilization
of microtubule structures; mice heterozygous for Apc demonstrate a significant decrease in apico-basal arrays of microtubule bundles [282]. Because APC associates with
microtubules and accumulates at their growing tips even in
the absence of its C-terminal sequences (including both EBand microtubule-binding domains) [283], it has been suggested that another domain at the N-terminus targets APC to
the microtubular cytoskeleton [284,285]. Jimbo et al. [286]
demonstrated an interaction between the arm repeats of APC
and the kinesin superfamily-associated protein 3 (KAP3), an
adaptor for the kinesin superfamily KIF3A/3B plus-end
directed microtubule motor proteins.
The observations that APC and h-catenin bind actin and
microtubular cytoskeletal systems are suggestive of a more
15
fundamental role for these proteins in cell migration [277].
Both proteins bind to E-cadherin at adherens junctions, and
g-catenin/plakoglobin participates in desmosomes as well.
Overexpression of Apc in mouse intestine produces a disordered migratory phenotype [287]. Furthermore, APC
mutations result in increased localization of h- and g-catenin
at the membrane, increasing adhesion and consequently
decreasing migration [288,289]. Two recent reports suggest
that mutations in h-catenin also affect cell adhesion properties. E-APC in Drosophila can alter adhesion by acting
through the Drosophila orthologue of h-catenin [290].
Furthermore, mutated h-catenin does not bind as well to Ecadherin [291]. An earlier study also implied that CTNNB1
mutations might involve E-cadherin in neoplasia when it was
observed that deletion of residues 28 – 134 of h-catenin
abrogated E-cadherin cell –cell adhesion in a gastric cancer
cell line [292].
6.4. Why are certain cell types seemingly more vulnerable
to Wnt-mediated cancer than others?
For convenience’s sake, researchers generally discuss the
Wnt pathway as being either ‘‘on’’ or ‘‘off.’’ While this
simplistic view helps delineate extreme situations, it does
not reflect the in vivo scenario. Cells in the intervillus
pockets of the developing intestine, the prospective crypts,
express Tcf4 and exhibit nuclear h-catenin [293]. DNA
microarray experiments have demonstrated that patterns of
gene expression in polyp cells resemble that of proliferating
crypt cells [254], suggesting that the Wnt cascade could be
active in normal adult colonic epithelium. Accordingly,
h-catenin was recently shown to reside in the nuclei of
cells in the bottom third of normal adult intestinal crypts
[254]. Yet—considering each human intestine produces 100
billion epithelial cells per day [294]—neoplastic transformation is a very rare event. Immunohistochemical staining
of nuclear h-catenin suggests that TCF/h-catenin signaling
in normal adult tissue is considerably less than seen in
intestinal or colorectal polyps [254]. Therefore, a certain
amount of h-catenin signaling is permitted before some
threshold is crossed. Therefore, if active Wnt signaling is
already present in certain tissues, alterations at these venues
which simply increase the signal may be sufficient to
instigate uncontrolled cellular growth.
Cancer is often initiated by a block in differentiation
allowing a pool of undifferentiated cells to accumulate in
which an environment is fostered for further transforming
mutations. Mesenchymal and epithelial differentiation is
known to be a process mediated by Wnt signaling [295].
Using a panel of truncated Apc alleles (Min, 1638N, 1638T),
Kielman et al. [230] assayed embryonic stem (ES) cells for
their differentiation potential by subcutaneously injecting
them into recipient animals and allowing them to form
teratomas, a benign tumor containing a broad spectrum of
tissue types. ES cells derived from wild-type and Apc1638T
mice were able to differentiate into cell types derived from
16
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
all embryonic germ layers. Teratomas generated from ES
cells harboring alleles defective in h-catenin regulation,
however, showed a diminished differentiation capacity
which became increasingly more severe as more certain
combinations of Apc alleles resulted in more nuclear hcatenin. ES cells from homozygous Min mice were not even
able to form teratomas [230]. It thus follows that in the
intestinal crypt, increased h-catenin levels interfere with
differentiation and encourage conditions beneficial to tumor
progression.
6.5. Architectural changes in the intestinal crypt
Wnt signaling appears to regulate the complex balance of
proliferation, migration and differentiation which is essential to normal functioning of the rapidly proliferating intestinal epithelium. Any perturbation of this balance appears to
disrupt normal intestinal homeostasis leading to tumor
development. The colorectal mucosa contains large numbers
of invaginations termed the crypts of Lieberkühn. Epithelial
cells are constantly being renewed in these crypts in a
coordinated series of events involving proliferation, differentiation and cell migration towards the intestinal lumen
(Fig. 3). Pluripotent stem cells are believed to reside at the
bottom positions of the crypt. From these stem cells,
progenitors are generated that occupy the lower third of
the crypt, the amplification compartment. Cells in this
compartment divide approximately every 12 h until their
migration brings them to the mid-crypt region. Here they
cease proliferating and differentiate into one of the functional cell types of the colon. At the surface epithelium, cells
undergo apoptosis and/or extrusion into the lumen. Apc
mutations result in an extended proliferative compartment
and reduced cellular turnover of the normal intestinal
epithelium of the Min mouse [129,130] perhaps providing
an early mechanism for disease progression: an increased
number of cells in the crypt – villus compartment would
allow for opportunities for a second hit.
In the gut, TCF/h-catenin inversely control the expression of the EphB2/EphB3 receptors and their ligand, ephrinB1 along the crypt –villus axis [254,296]. The interactions
between Eph receptors and ephrin ligands involve direct
cell-to-cell interactions and frequently result in repulsion.
Eph-ephrin signaling provides repulsive cues in a wide
range of developmental phenomena including axon , the
migration of neural crest cells to their target tissues and
boundary formation between adjacent cell populations in
segmented structures such as rhombomeres [297]. It has
been strongly suggested that Eph receptors and ephrin
ligands transduce repulsive signals and that this bi-direc-
Fig. 3. Schematic representation of a colon crypt and proposed model for polyp formation. At the bottom third of the crypt, the progenitor proliferating cells
accumulate nuclear h-catenin. Consequently, they express h-catenin/TCF target genes. An uncharacterized source of WNT factors likely resides in the
mesenchymal cells surrounding the bottom of the crypt, depicted in red. As the cells reach the mid-crypt region, h-catenin/TCF activity is downregulated and
this results in cell cycle arrest and differentiation. Cells undergoing mutation in APC or b-catenin become independent of the physiological signals controlling
h-catenin/TCF activity. As a consequence, they continue to behave as crypt progenitor cells in the surface epithelium giving rise to aberrant crypt foci.
Modified from Ref. [254].
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
tional signaling is required to maintain boundaries at the
interface of adjacent cell compartments [298]. Polyps in the
small intestine of Min mice develop at the crypt –villus
junction and form pockets that migrate inside the normal
epithelium of the villus [299]. These cells proliferate inside
the mucosa as a disorganized mass that will eventually give
rise to a tumor (Fig. 3). This abnormal migratory behavior is
likely to be the outcome of the h-catenin/Tcf target gene
program autonomously activated in the Apc mutant cells.
Intriguingly, this initial outpocketing arises in the crypt –
villus junction where that APC mutant cells the overexpress
EphB receptors encounter the maximum threshold of ephrin-mediated repulsion. These observations suggest that the
initial founding polyp cells expressing high levels of EphB
receptors likely initiate an abnormal migration inside the
villus to avoid the cells expressing high levels of ephrin-B.
Future analysis of the Min phenotype in an EphB-deficient
background will determine the role of the ephrin/EPH
system in architectural changes associated with neoplasm.
7. Molecular medicine
Despite recent advances in clinical treatment, CRC is still
a major public health threat. Current clinical treatment of
CRC usually involves removal of the colon and rectum.
This rather crude intervention is moderately successful, but
certainly interferes with the patients’ quality of life. Understanding the molecular basis of tumors generated from
activated Wnt signaling suggests a plethora of Wnt-specific
possibilities. For example, small molecule inhibitors that
physiologically interfere with h-catenin binding to TCF or
Lgs, are being hotly pursued by a number of commercial
concerns.
Nonsteroidal anti-inflammatory drugs (NSAIDs) have
been reported to suppress colon carcinogenesis, probably
through the inhibition of cyclooxygenase-2 (COX-2), that
catalyzes the rate-limiting step in prostaglandin synthesis
from arachidonic acid. Compelling evidence from genetic
and clinical studies indicates that COX-2 upregulation is a
key step in carcinogenesis. A model has developed whereby
Wnt-mediated tumorigenesis activates COX-2-derived prostaglandin production, which promotes cellular viability
through PPARy, the nuclear receptor peroxisome proliferator receptor (PPAR) delta [300]. The gene encoding PPARy
is a h-catenin/TCF target [301]. Epidemiologic studies
indicate a 40– 50% reduction in mortality due to CRC in
individuals taking NSAIDs (e.g. aspirin). Inhibition of
COX-2 promises to be an effective approach in the prevention and treatment of cancer, especially CRC.
In addition, inhibitors of COX have shown a great deal of
promise in vitro and in animal models as potential antitumor
therapies. Oshima et al. [302] bred mice carrying an Apc
mutation with mice lacking COX-2. All of the animals were
heterozygous at the Apc locus; if homozygous for wild-type
COX-2, they developed an average of 652 polyps at 10
17
weeks, while heterozygotes had 224 polyps and homozygously deficient mice had only 93 polyps. This experiment
provided definitive genetic evidence that induction of COX2 is an early, rate-limiting step for adenoma formation. As
supporting evidence, treating ApcD716 mice with a novel
COX-2 inhibitor reduced the polyp number more significantly than with sulindac, which inhibits both COX-1 and
COX-2 [302]. Although the general COX inhibitor sulindac
has been shown to reduce the number of Min polyps
[303,304], a specific COX-2 inhibitor (MF-tricyclic) was
observed to be more effective in preventing polyps in Min
mice [305]. The findings suggested that drugs inhibiting
COX-2 should be broadly effective in chemoprevention of
colon cancer [306].
In response to these studies in mice, the COX inhibitor
sulindac was tested as a chemopreventive and therapeutic
agent in humans at risk for colorectal neoplasia. Despite the
relatively disappointing results with respect to the ability of
sulindac to prevent adenomas in patients with FAP [307 –
309], NSAIDs and COX-2 inhibitors still hold hope for the
primary prevention or treatment of established CRC
[310,311]. Another group studied the effect of celecoxib, a
selective COX-2 inhibitor, on colorectal polyps in patients
with FAP [312]. In a double-blind, placebo-controlled study
of 77 patients, they found that 6 months of twice-daily
treatment with 400 mg of the agent significantly reduced
the number of colorectal polyps. Certainly more studies are
warranted on the effect of specific COX-2 inhibitors on
developing CRC, but the preliminary results look promising.
Perhaps somewhat less specific, the pharmacological
agents piroxicam and difluoromethylornithine each reduced
intestinal adenoma multiplicity in Min/+ mice deficient for
p53 [219]. In an attempt to explore immunotherapeutic
options, Min/+ mice were crossed with mice transgenic
for the human carcinoembryonic antigen (CEA), a cellsurface glycoprotein that is expressed on normal human
intestinal epithelium and that is overexpressed in intestinal
tumors. Intestinal adenomas in the resultant offspring overexpressed CEA, which could be successfully targeted by a
specific antibody retained in tumors at levels higher than in
areas of normal gut [313].
8. Final comments
As discussed in this review, a large body of literature is
beginning to sketch out the mechanisms by which active Wnt
signaling causes cancer. Nuclear h-catenin transactivates
TCF/LEF target genes, simultaneously promoting cellular
growth and repressing differentiation programs. Once a
growth advantage has been provided, architectural changes
provide a harbor for tumor progression. By controlling the
EPH/ephrin system, cells with an activated Wnt signal might
form outpockets allowing pools of undifferentiated cells to
foster conditions beneficial for the acquisition of additional
mutations [296]. Genetic instability, such as that incurred in
18
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
the presence of APC mutations, destabilize the mitotic
spindle and promote loss of tumor suppressor genes
[237,272]. Changes in cell adhesion and migration, brought
on by APC or CTNNB1 mutations, contribute to the invasiveness of a particular tumor [314].
Mutations in several components of the Wnt signaling
cascade cause cancer in a large number of anatomical
venues, yet—despite all the variables—they all manifest a
single molecular defect: nuclear h-catenin. The inventory of
tumors testifies to the robustness of clonal selection. The
presence of nuclear h-catenin in normal adult intestinal
crypts was difficult to detect and has just now been
published. It is interesting to speculate that other sites prone
to tumors due to activated Wnt signaling, e.g. the liver,
might also maintain low levels of this signal in normal adult
tissue. Furthermore, identifying nuclear h-catenin clinically
will aid molecular profiling of tumor types. For example,
the presence of nuclear h-catenin is correlated with poor
prognosis in patients with hepatoblastoma [179].
In summary, while significant progress has been made in
our understanding of Wnt signaling through model systems,
genetics, biochemistry, it is just the beginning of the story.
Effectively regulating Wnt signaling under physiological
circumstances is considerably more difficult. Yet, with Wnt
signaling such a prevalent cause of human tumors, the
research drive to harness this pathway is strong.
Acknowledgements
We are grateful to M. van de Wetering, E. Sancho, and E.
Batlle for help preparing the figures.
References
[1] The American Cancer Society. www.cancer.org.
[2] R. Kongkanuntn, V.J. Bubb, O.J. Sansom, A.H. Wyllie, D.J. Harrison, A.R. Clarke, Oncogene 18 (1999) 7219 – 7225.
[3] K. Orford, C. Crockett, J.P. Jensen, A.M. Weissman, S.W. Byers,
J. Biol. Chem. 272 (1997) 24735 – 24738.
[4] P. Polakis, Genes Dev. 14 (2000) 1837 – 1851.
[5] R.P. Sharma, V.L. Chopra, Dev. Biol. 48 (1976) 461 – 465.
[6] R. Nusse, H.E. Varmus, Cell 31 (1982) 99 – 109.
[7] F. Rijsewijk, M. Schuermann, E. Wagenaar, P. Parren, D. Weigel, R.
Nusse, Cell 50 (1987) 649 – 657.
[8] J.R. Miller, Genome Biol. 3 (2002) 3001 (Reviews).
[9] P. Bhanot, M. Brink, C.H. Samos, J.C. Hsieh, Y. Wang, J.P. Macke,
D. Andrew, J. Nathans, R. Nusse, Nature 382 (1996) 225 – 230.
[10] Y. Wang, J.P. Macke, B.S. Abella, K. Andreasson, P. Worley, D.J.
Gilbert, N.G. Copeland, N.A. Jenkins, J. Nathans, J. Biol. Chem.
271 (1996) 4468 – 4476.
[11] X. He, J.P. Saint-Jeannet, Y. Wang, J. Nathans, I. Dawid, H. Varmus,
Science 275 (1997) 1652 – 1654.
[12] T.C. Dale, Biochem. J. 329 (1998) 209 – 223.
[13] M. Wehrli, S.T. Dougan, K. Caldwell, L. O’Keefe, S. Schwartz, D.
Vaizel-Ohayon, E. Schejter, A. Tomlinson, S. DiNardo, Nature 407
(2000) 527 – 530.
[14] K. Tamai, M. Semenov, Y. Kato, R. Spokony, C. Liu, Y. Katsuyama,
F. Hess, J.P. Saint-Jeannet, X. He, Nature 407 (2000) 530 – 535.
[15] K.I. Pinson, J. Brennan, S. Monkley, B.J. Avery, W.C. Skarnes,
Nature 407 (2000) 535 – 538.
[16] L. Leyns, T. Bouwmeester, S.H. Kim, S. Piccolo, E.M. De Robertis,
Cell 88 (1997) 747 – 756.
[17] S. Wang, M. Krinks, M. Moos Jr., Biochem. Biophys. Res. Commun. 236 (1997) 502 – 504.
[18] P.W. Finch, X. He, M.J. Kelley, A. Uren, R.P. Schaudies, N.C.
Popescu, S. Rudikoff, S.A. Aaronson, H.E. Varmus, J.S. Rubin,
Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 6770 – 6775.
[19] P. Fedi, A. Bafico, A. Nieto Soria, W.H. Burgess, T. Miki, D.P.
Bottaro, M.H. Kraus, S.A. Aaronson, J. Biol. Chem. 274 (1999)
19465 – 19472.
[20] A. Glinka, W. Wu, H. Delius, A.P. Monaghan, C. Blumenstock, C.
Niehrs, Nature 391 (1998) 357 – 362.
[21] B. Mao, W. Wu, Y. Li, D. Hoppe, P. Stannek, A. Glinka, C. Niehrs,
Nature 411 (2001) 321 – 325.
[22] B. Mao, W. Wu, G. Davidson, J. Marhold, M. Li, B.M. Mechler, H.
Delius, D. Hoppe, P. Stannek, C. Walter, A. Glinka, C. Niehrs,
Nature 417 (2002) 664 – 667.
[23] J. Mao, J. Wang, B. Liu, W. Pan, G.H. Farr III, C. Flynn, H. Yuan, S.
Takada, D. Kimelman, L. Li, D. Wu, Mol. Cell 7 (2001) 801 – 809.
[24] S. Yanagawa, F. van Leeuwen, A. Wodarz, J. Klingensmith, R.
Nusse, Genes Dev. 9 (1995) 1087 – 1097.
[25] A. Wodarz, Curr. Biol. 11 (2001) R975 – R978.
[26] M. Kuhl, K. Geis, L.C. Sheldahl, T. Pukrop, R.T. Moon, D. Wedlich,
Mech. Dev. 106 (2001) 61 – 76.
[27] J.B. Wallingford, S.E. Fraser, R.M. Harland, Dev. Cell 2 (2002)
695 – 706.
[28] J.M. Peters, R.M. McKay, J.P. McKay, J.M. Graff, Nature 401
(1999) 345 – 350.
[29] R.M. McKay, J.M. Peters, J.M. Graff, Dev. Biol. 235 (2001)
388 – 396.
[30] D. Yan, M. Wiesmann, M. Rohan, V. Chan, A.B. Jefferson, L. Guo,
D. Sakamoto, R.H. Caothien, J.H. Fuller, C. Reinhard, P.D. Garcia,
F.M. Randazzo, J. Escobedo, W.J. Fantl, L.T. Williams, Proc. Natl.
Acad. Sci. U. S. A. 98 (2001) 14973 – 14978.
[31] M. Park, R.T. Moon, Nat. Cell Biol. 4 (2002) 20 – 25.
[32] B.N. Cheyette, J.S. Waxman, J.R. Miller, K. Takemaru, L.C. Sheldahl, N. Khlebtsova, E.P. Fox, T. Earnest, R.T. Moon, Dev. Cell 2
(2002) 449 – 461.
[33] A.T. Weeraratna, Y. Jiang, G. Hostetter, K. Rosenblatt, P. Duray, M.
Bittner, J.M. Trent, Cancer Cell 1 (2002) 279 – 288.
[34] J. Robitaille, M.L. MacDonald, A. Kaykas, L.C. Sheldahl, J. Zeisler,
M.P. Dube, L.H. Zhang, R.R. Singaraja, D.L. Guernsey, B. Zheng,
L.F. Siebert, A. Hoskin-Mott, M.T. Trese, S.N. Pimstone, B.S. Shastry, R.T. Moon, M.R. Hayden, Y.P. Goldberg, M.E. Samuels, Nat.
Genet. 12 (2002) 12.
[35] J. Behrens, B.A. Jerchow, M. Wurtele, J. Grimm, C. Asbrand, R.
Wirtz, M. Kuhl, D. Wedlich, W. Birchmeier, Science 280 (1998)
596 – 599.
[36] S. Kishida, H. Yamamoto, S. Ikeda, M. Kishida, I. Sakamoto, S.
Koyama, A. Kikuchi, J. Biol. Chem. 273 (1998) 10823 – 10826.
[37] M. van Noort, M. van de Wetering, H. Clevers, Exp. Cell Res. 276
(2002) 264 – 272.
[38] M. Hart, J.P. Concordet, I. Lassot, I. Albert, R. del los Santos, H.
Durand, C. Perret, B. Rubinfeld, F. Margottin, R. Benarous, P. Polakis, Curr. Biol. 9 (1999) 207 – 210.
[39] M. Kitagawa, S. Hatakeyama, M. Shirane, M. Matsumoto, N. Ishida,
K. Hattori, I. Nakamichi, A. Kikuchi, K. Nakayama, EMBO J. 18
(1999) 2401 – 2410.
[40] C. Liu, Y. Kato, Z. Zhang, V.M. Do, B.A. Yankner, X. He, Proc.
Natl. Acad. Sci. U. S. A. 96 (1999) 6273 – 6278.
[41] S. Amit, A. Hatzubai, Y. Birman, J.S. Andersen, E. Ben-Shushan,
M. Mann, Y. Ben-Neriah, I. Alkalay, Genes Dev. 16 (2002)
1066 – 1076.
[42] C. Liu, Y. Li, M. Semenov, C. Han, G.H. Baeg, Y. Tan, Z. Zhang,
X. Lin, X. He, Cell 108 (2002) 837 – 847.
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[43] S. Yanagawa, Y. Matsuda, J.S. Lee, H. Matsubayashi, S. Sese, T.
Kadowaki, A. Ishimoto, EMBO J. 21 (2002) 1733 – 1742.
[44] T. Schwarz-Romond, C. Asbrand, J. Bakkers, M. Kuhl, H.J.
Schaeffer, J. Huelsken, J. Behrens, M. Hammerschmidt, W. Birchmeier, Genes Dev. 16 (2002) 2073 – 2084.
[45] L. Li, H. Yuan, C.D. Weaver, J. Mao, G.H. Farr III, D.J. Sussman,
J. Jonkers, D. Kimelman, D. Wu, EMBO J. 18 (1999) 4233 – 4240.
[46] E. Fraser, N. Young, R. Dajani, J. Franca-Koh, J. Ryves, R.S.
Williams, M. Yeo, M.T. Webster, C. Richardson, M.J. Smalley,
L.H. Pearl, A. Harwood, T.C. Dale, J. Biol. Chem. 277 (2002)
2176 – 2185.
[47] W. Hsu, L. Zeng, F. Costantini, J. Biol. Chem. 274 (1999)
3439 – 3445.
[48] S. Ikeda, M. Kishida, Y. Matsuura, H. Usui, A. Kikuchi, Oncogene
19 (2000) 537 – 545.
[49] J.M. Seeling, J.R. Miller, R. Gil, R.T. Moon, R. White, D.M. Virshup,
Science 283 (1999) 2089 – 2091.
[50] H. Yamamoto, S. Kishida, M. Kishida, S. Ikeda, S. Takada, A.
Kikuchi, J. Biol. Chem. 274 (1999) 10681 – 10684.
[51] L. Zeng, F. Fagotto, T. Zhang, W. Hsu, T.J. Vasicek, W.L. Perry III,
J.J. Lee, S.M. Tilghman, B.M. Gumbiner, F. Costantini, Cell 90
(1997) 181 – 192.
[52] S. Satoh, Y. Daigo, Y. Furukawa, T. Kato, N. Miwa, T. Nishiwaki,
T. Kawasoe, H. Ishiguro, M. Fujita, T. Tokino, Y. Sasaki, S. Imaoka,
M. Murata, T. Shimano, Y. Yamaoka, Y. Nakamura, Nat. Genet. 24
(2000) 245 – 250.
[53] N.S. Fearnhead, M.P. Britton, W.F. Bodmer, Hum. Mol. Genet. 10
(2001) 721 – 733.
[54] R.B. Pyles, I.M. Santoro, J. Groden, L.M. Parysek, Oncogene 16
(1998) 77 – 82.
[55] L.K. Su, K.A. Johnson, K.J. Smith, D.E. Hill, B. Vogelstein, K.W.
Kinzler, Cancer Res. 53 (1993) 2728 – 2731.
[56] G. Joslyn, D.S. Richardson, R. White, T. Alber, Proc. Natl. Acad.
Sci. U. S. A. 90 (1993) 11109 – 11113.
[57] M. Peifer, P. Polakis, Science 287 (2000) 1606 – 1609.
[58] R. Fodde, Eur. J. Cancer 38 (2002) 867 – 871.
[59] T. Nakamura, F. Hamada, T. Ishidate, K. Anai, K. Kawahara, K.
Toyoshima, T. Akiyama, Genes Cells 3 (1998) 395 – 403.
[60] M.J. Hart, R. de los Santos, I.N. Albert, B. Rubinfeld, P. Polakis,
Curr. Biol. 8 (1998) 573 – 581.
[61] B.R. Henderson, Nat. Cell Biol. 2 (2000) 653 – 660.
[62] R. Rosin-Arbesfeld, F. Townsley, M. Bienz, Nature 406 (2000)
1009 – 1012.
[63] K.L. Neufeld, F. Zhang, B.R. Cullen, R.L. White, EMBO Rep. 1
(2000) 519 – 523.
[64] B. Rubinfeld, B. Souza, I. Albert, O. Muller, S.H. Chamberlain,
F.R. Masiarz, S. Munemitsu, P. Polakis, Science 262 (1993)
1731 – 1734.
[65] S. Munemitsu, I. Albert, B. Souza, B. Rubinfeld, P. Polakis, Proc.
Natl. Acad. Sci. U. S. A. 92 (1995) 3046 – 3050.
[66] M. Peifer, S. Berg, A.B. Reynolds, Cell 76 (1994) 789 – 791.
[67] F. Hamada, Y. Murata, A. Nishida, F. Fujita, Y. Tomoyasu, M.
Nakamura, K. Toyoshima, T. Tabata, N. Ueno, T. Akiyama, Genes
Cells 4 (1999) 465 – 474.
[68] C.A. Midgley, S. White, R. Howitt, V. Save, M.G. Dunlop, P.A.
Hall, D.P. Lane, A.H. Wyllie, V.J. Bubb, J. Pathol. 181 (1997)
426 – 433.
[69] J.H. van Es, C. Kirkpatrick, M. van de Wetering, M. Molenaar, A.
Miles, J. Kuipers, O. Destree, M. Peifer, H. Clevers, Curr. Biol. 9
(1999) 105 – 108.
[70] H. Nakagawa, Y. Murata, K. Koyama, A. Fujiyama, Y. Miyoshi,
M. Monden, T. Akiyama, Y. Nakamura, Cancer Res. 58 (1998)
5176 – 5181.
[71] H. Nakagawa, K. Koyama, Y. Murata, M. Morito, T. Akiyama, Y.
Nakamura, Cancer Res. 60 (2000) 101 – 105.
[72] J.H. van Es, R.H. Giles, H.C. Clevers, Exp. Cell Res. 264 (2001)
126 – 134.
19
[73] H. Nakagawa, K. Koyama, Y. Murata, M. Morito, T. Akiyama, Y.
Nakamura, Oncogene 19 (2000) 210 – 216.
[74] H. Nakagawa, K. Koyama, M. Monden, Y. Nakamura, Jpn. J. Cancer Res. 90 (1999) 982 – 986.
[75] C.R. Jarrett, J. Blancato, T. Cao, D.S. Bressette, M. Cepeda, P.E.
Young, C.R. King, S.W. Byers, Cancer Res. 61 (2001) 7978 – 7984.
[76] A. Nagafuchi, M. Takeichi, Cell Regul. 1 (1989) 37 – 44.
[77] M. Ozawa, H. Baribault, R. Kemler, EMBO J. 8 (1989) 1711 – 1717.
[78] B.M. Gumbiner, Neuron 11 (1993) 551 – 564.
[79] P. Cowin, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 10759 – 10761.
[80] A.H. Huber, W.J. Nelson, W.I. Weis, Cell 90 (1997) 871 – 882.
[81] J.P. von Kries, G. Winbeck, C. Asbrand, T. Schwarz-Romond, N.
Sochnikova, A. Dell’Oro, J. Behrens, W. Birchmeier, Nat. Struct.
Biol. 7 (2000) 800 – 807.
[82] N. Funayama, F. Fagotto, P. McCrea, B.M. Gumbiner, J. Cell Biol.
128 (1995) 959 – 968.
[83] F. Fagotto, U. Gluck, B.M. Gumbiner, Curr. Biol. 8 (1998)
181 – 190.
[84] K.L. Neufeld, R.L. White, Proc. Natl. Acad. Sci. U. S. A. 94 (1997)
3034 – 3039.
[85] J.A. Efstathiou, M. Noda, A. Rowan, C. Dixon, R. Chinery, A.
Jawhari, T. Hattori, N.A. Wright, W.F. Bodmer, M. Pignatelli, Proc.
Natl. Acad. Sci. U. S. A. 95 (1998) 3122 – 3127.
[86] J. Hulsken, W. Birchmeier, J. Behrens, J. Cell Biol. 127 (1994)
2061 – 2069.
[87] B. Rubinfeld, B. Souza, I. Albert, S. Munemitsu, P. Polakis, J. Biol.
Chem. 270 (1995) 5549 – 5555.
[88] F.T. Kolligs, B. Kolligs, K.M. Hajra, G. Hu, M. Tani, K.R. Cho, E.R.
Fearon, Genes Dev. 14 (2000) 1319 – 1331.
[89] M. van de Wetering, R. Cavallo, D. Dooijes, M. van Beest, J. van Es,
J. Loureiro, A. Ypma, D. Hursh, T. Jones, A. Bejsovec, M. Peifer,
M. Mortin, H. Clevers, Cell 88 (1997) 789 – 799.
[90] D. Wolf, M. Rodova, E.A. Miska, J.P. Calvet, T. Kouzarides, J. Biol.
Chem. 277 (2002) 25562 – 25567.
[91] K.I. Takemaru, R.T. Moon, J. Cell Biol. 149 (2000) 249 – 254.
[92] N. Barker, A. Hurlstone, H. Musisi, A. Miles, M. Bienz, H. Clevers,
EMBO J. 20 (2001) 4935 – 4943.
[93] T. Kramps, O. Peter, E. Brunner, D. Nellen, B. Froesch, S. Chatterjee, M. Murone, S. Zullig, K. Basler, Cell 109 (2002) 47 – 60.
[94] B. Thompson, F. Townsley, R. Rosin-Arbesfeld, H. Musisi, M. Bienz, Nat. Cell Biol. 4 (2002) 367 – 373.
[95] T.Y. Belenkaya, C. Han, H.J. Standley, X. Lin, D.W. Houston, J.
Heasman, Development 129 (2002) 4089 – 4101.
[96] D.S. Parker, J. Jemison, K.M. Cadigan, Development 129 (2002)
2565 – 2576.
[97] A. Sarris, R. Ford, Curr. Opin. Oncol. 11 (1999) 351 – 363.
[98] T.G. Willis, I.R. Zalcberg, L.J. Coignet, I. Wlodarska, M. Stul, D.M.
Jadayel, C. Bastard, J.G. Treleaven, D. Catovsky, M.L. Silva, M.J.
Dyer, Blood 91 (1998) 1873 – 1881.
[99] A. Travis, A. Amsterdam, C. Belanger, R. Grosschedl, Genes Dev. 5
(1991) 880 – 894.
[100] M. van de Wetering, M. Oosterwegel, D. Dooijes, H. Clevers, EMBO J. 10 (1991) 123 – 132.
[101] M. Oosterwegel, M. van de Wetering, D. Dooijes, L. Klomp, A.
Winoto, K. Georgopoulos, F. Meijlink, H. Clevers, J. Exp. Med.
173 (1991) 1133 – 1142.
[102] K. Giese, J. Cox, R. Grosschedl, Cell 69 (1992) 185 – 195.
[103] P. Carlsson, M.L. Waterman, K.A. Jones, Genes Dev. 7 (1993)
2418 – 2430.
[104] L. Bruhn, A. Munnerlyn, R. Grosschedl, Genes Dev. 11 (1997)
640 – 653.
[105] M. Molenaar, M. van de Wetering, M. Oosterwegel, J. Peterson-Maduro, S. Godsave, V. Korinek, J. Roose, O. Destree, H. Clevers, Cell
86 (1996) 391 – 399.
[106] J. Roose, M. Molenaar, J. Peterson, J. Hurenkamp, H. Brantjes,
P. Moerer, M. van de Wetering, O. Destree, H. Clevers, Nature
395 (1998) 608 – 612.
20
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[107] H. Brantjes, J. Roose, M. van de Wetering, H. Clevers, Nucleic
Acids Res. 29 (2001) 1410 – 1419.
[108] G. Chen, J. Fernandez, S. Mische, A.J. Courey, Genes Dev. 13
(1999) 2218 – 2230.
[109] S.M. Powell, N. Zilz, Y. Beazer-Barclay, T.M. Bryan, S.R. Hamilton,
S.N. Thibodeau, B. Vogelstein, K.W. Kinzler, Nature 359 (1992)
235 – 237.
[110] J. Groden, A. Thliveris, W. Samowitz, M. Carlson, L. Gelbert, H.
Albertsen, G. Joslyn, J. Stevens, L. Spirio, M. Robertson, et al., Cell
66 (1991) 589 – 600.
[111] I. Nishisho, Y. Nakamura, Y. Miyoshi, Y. Miki, H. Ando, A. Horii,
K. Koyama, J. Utsunomiya, S. Baba, P. Hedge, Science 253 (1991)
665 – 669.
[112] P. Laurent-Puig, C. Beroud, T. Soussi, Nucleic Acids Res. 26 (1998)
269 – 270.
[113] R. Fodde, P.M. Khan, Crit. Rev. Oncog. 6 (1995) 291 – 303.
[114] T. Matsumoto, M. Iida, Y. Kobori, M. Mizuno, S. Nakamura, K.
Hizawa, T. Yao, Gut 50 (2002) 402 – 404.
[115] R. Smits, N. Hofland, W. Edelmann, M. Geugien, S. JagmohanChangur, C. Albuquerque, C. Breukel, R. Kucherlapati, M.F. Kielman, R. Fodde, Genes Chromosomes Cancer 29 (2000) 229 – 239.
[116] H. Lamlum, M. Ilyas, A. Rowan, S. Clark, V. Johnson, J. Bell,
I. Frayling, J. Efstathiou, K. Pack, S. Payne, R. Roylance, P. Gorman, D. Sheer, K. Neale, R. Phillips, I. Talbot, W. Bodmer, I. Tomlinson, Nat. Med. 5 (1999) 1071 – 1075.
[117] A.J. Rowan, H. Lamlum, M. Ilyas, J. Wheeler, J. Straub, A. Papadopoulou, D. Bicknell, W.F. Bodmer, I.P. Tomlinson, Proc. Natl.
Acad. Sci. U. S. A. 97 (2000) 3352 – 3357.
[118] C. Albuquerque, C. Breukel, R. van der Luijt, P. Fidalgo, P. Lage,
F.J. Slors, C.N. Leitao, R. Fodde, R. Smits, Hum. Mol. Genet. 11
(2002) 1549 – 1560.
[119] S. Dihlmann, J. Gebert, A. Siermann, C. Herfarth, M. von Knebel
Doeberitz, Cancer Res. 59 (1999) 1857 – 1860.
[120] B. Rubinfeld, I. Albert, E. Porfiri, S. Munemitsu, P. Polakis, Cancer
Res. 57 (1997) 4624 – 4630.
[121] L.K. Su, C.J. Barnes, W. Yao, Y. Qi, P.M. Lynch, G. Steinbach, Am.
J. Hum. Genet. 67 (2000) 582 – 590.
[122] M. Toyooka, M. Konishi, R. Kikuchi-Yanoshita, T. Iwama, M.
Miyaki, Cancer Res. 55 (1995) 3165 – 3170.
[123] S.C. Abraham, B. Nobukawa, F.M. Giardiello, S.R. Hamilton, T.T.
Wu, Am. J. Pathol. 157 (2000) 747 – 754.
[124] C. Groves, H. Lamlum, M. Crabtree, J. Williamson, C. Taylor, S.
Bass, D. Cuthbert-Heavens, S. Hodgson, R. Phillips, I. Tomlinson,
Am. J. Pathol. 160 (2002) 2055 – 2061.
[125] W.F. Bodmer, C.J. Bailey, J. Bodmer, H.J. Bussey, A. Ellis, P. Gorman, F.C. Lucibello, V.A. Murday, S.H. Rider, P. Scambler, et al.,
Nature 328 (1987) 614 – 616.
[126] S.J. Laken, N. Papadopoulos, G.M. Petersen, S.B. Gruber, S.R.
Hamilton, F.M. Giardiello, J.D. Brensinger, B. Vogelstein, K.W.
Kinzler, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 2322 – 2326.
[127] H. Yan, Z. Dobbie, S.B. Gruber, S. Markowitz, K. Romans, F.M.
Giardiello, K.W. Kinzler, B. Vogelstein, Nat. Genet. 30 (2002) 25 – 26.
[128] O.M. Sieber, H. Lamlum, M.D. Crabtree, A.J. Rowan, E. Barclay,
L. Lipton, S. Hodgson, H.J. Thomas, K. Neale, R.K. Phillips, S.M.
Farrington, M.G. Dunlop, H.J. Mueller, M.L. Bisgaard, S. Bulow,
P. Fidalgo, C. Albuquerque, M.I. Scarano, W. Bodmer, I.P. Tomlinson, K. Heinimann, Proc. Natl. Acad. Sci. U. S. A. 99 (2002)
2954 – 2958.
[129] N.N. Mahmoud, S.K. Boolbol, R.T. Bilinski, C. Martucci, A. Chadburn, M.M. Bertagnolli, Cancer Res. 57 (1997) 5045 – 5050.
[130] N.N. Mahmoud, R.T. Bilinski, M.R. Churchill, W. Edelmann, R.
Kucherlapati, M.M. Bertagnolli, Cancer Res. 59 (1999) 353 – 359.
[131] W.S. Samowitz, M.D. Powers, L.N. Spirio, F. Nollet, F. van Roy,
M.L. Slattery, Cancer Res. 59 (1999) 1442 – 1444.
[132] P.J. Morin, A.B. Sparks, V. Korinek, N. Barker, H. Clevers, B.
Vogelstein, K.W. Kinzler, Science 275 (1997) 1787 – 1790.
[133] P. Polakis, Curr. Opin. Genet. Dev. 9 (1999) 15 – 21.
[134] M.T. Webster, M. Rozycka, E. Sara, E. Davis, M. Smalley, N.
Young, T.C. Dale, R. Wooster, Genes Chromosomes Cancer 28
(2000) 443 – 453.
[135] W.M. Clements, J. Wang, A. Sarnaik, O.J. Kim, J. MacDonald, C.
Fenoglio-Preiser, J. Groden, A.M. Lowy, Cancer Res. 62 (2002)
3503 – 3506.
[136] W.S. Park, R.R. Oh, J.Y. Park, S.H. Lee, M.S. Shin, Y.S. Kim, S.Y.
Kim, H.K. Lee, P.J. Kim, S.T. Oh, N.J. Yoo, J.Y. Lee, Cancer Res.
59 (1999) 4257 – 4260.
[137] D.K. Woo, H.S. Kim, H.S. Lee, Y.H. Kang, H.K. Yang, W.H. Kim,
Int. J. Cancer 95 (2001) 108 – 113.
[138] S.C. Abraham, B. Nobukawa, F.M. Giardiello, S.R. Hamilton, T.T.
Wu, Am. J. Pathol. 158 (2001) 1005 – 1010.
[139] J.H. Lee, S.C. Abraham, H.S. Kim, J.H. Nam, C. Choi, M.C. Lee,
C.S. Park, S.W. Juhng, A. Rashid, S.R. Hamilton, T.T. Wu, Am. J.
Pathol. 161 (2002) 611 – 618.
[140] S.C. Abraham, E.A. Montgomery, F.M. Giardiello, T.T. Wu, Am. J.
Pathol. 158 (2001) 1073 – 1078.
[141] Y.W. Choi, E.I. Heath, R. Heitmiller, A.A. Forastiere, T.T. Wu, Mod.
Path. 13 (2000) 1055 – 1059.
[142] B.P. Wijnhoven, F. Nollet, N.J. De Both, H.W. Tilanus, W.N. Dinjens, Int. J. Cancer 86 (2000) 533 – 537.
[143] B. Rubinfeld, P. Robbins, M. El-Gamil, I. Albert, E. Porfiri, P.
Polakis, Science 275 (1997) 1790 – 1792.
[144] D.L. Rimm, K. Caca, G. Hu, F.B. Harrison, E.R. Fearon, Am. J.
Pathol. 154 (1999) 325 – 329.
[145] K. Omholt, A. Platz, U. Ringborg, J. Hansson, Int. J. Cancer 92
(2001) 839 – 842.
[146] P.M. Pollock, N. Hayward, Melanoma Res. 12 (2002) 183 – 186.
[147] J. Reifenberger, C.B. Knobbe, M. Wolter, B. Blaschke, K.W.
Schulte, T. Pietsch, T. Ruzicka, G. Reifenberger, Int. J. Cancer
100 (2002) 549 – 556.
[148] E.F. Chan, U. Gat, J.M. McNiff, E. Fuchs, Nat. Genet. 21 (1999)
410 – 413.
[149] G. Moreno-Bueno, C. Gamallo, L. Perez-Gallego, F. Contreras, J.
Palacios, Br. J. Dermatol. 145 (2001) 576 – 581.
[150] Y. Kajino, A. Yamaguchi, N. Hashimoto, A. Matsuura, N. Sato, K.
Kikuchi, Pathol. Int. 51 (2001) 543 – 548.
[151] U. Gat, R. DasGupta, L. Degenstein, E. Fuchs, Cell 95 (1998)
605 – 614.
[152] A. de La Coste, B. Romagnolo, P. Billuart, C.A. Renard, M.A.
Buendia, O. Soubrane, M. Fabre, J. Chelly, C. Beldjord, A. Kahn,
C. Perret, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 8847 – 8851.
[153] Y. Miyoshi, K. Iwao, Y. Nagasawa, T. Aihara, Y. Sasaki, S. Imaoka, M. Murata, T. Shimano, Y. Nakamura, Cancer Res. 58
(1998) 2524 – 2527.
[154] P. Legoix, O. Bluteau, J. Bayer, C. Perret, C. Balabaud, J. Belghiti,
D. Franco, G. Thomas, P. Laurent-Puig, J. Zucman-Rossi, Oncogene
18 (1999) 4044 – 4046.
[155] J.T. Nhieu, C.A. Renard, Y. Wei, D. Cherqui, E.S. Zafrani, M.A.
Buendia, Am. J. Pathol. 155 (1999) 703 – 710.
[156] C.M. Wong, S.T. Fan, I.O. Ng, Cancer 92 (2001) 136 – 145.
[157] K. Taniguchi, L.R. Roberts, I.N. Aderca, X. Dong, C. Qian, L.M.
Murphy, D.M. Nagorney, L.J. Burgart, P.C. Roche, D.I. Smith, J.A.
Ross, W. Liu, Oncogene 21 (2002) 4863 – 4871.
[158] H. Huang, H. Fujii, A. Sankila, B.M. Mahler-Araujo, M. Matsuda,
G. Cathomas, H. Ohgaki, Am. J. Pathol. 155 (1999) 1795 – 1801.
[159] J. Palacios, C. Gamallo, Cancer Res. 58 (1998) 1344 – 1347.
[160] S. Sagae, K. Kobayashi, Y. Nishioka, M. Sugimura, S. Ishioka,
M. Nagata, K. Terasawa, T. Tokino, R. Kudo, Jpn. J. Cancer Res.
90 (1999) 510 – 515.
[161] C. Gamallo, J. Palacios, G. Moreno, J. Calvo de Mora, A. Suarez, A.
Armas, Am. J. Pathol. 155 (1999) 527 – 536.
[162] R. Wu, Y. Zhai, E.R. Fearon, K.R. Cho, Cancer Res. 61 (2001)
8247 – 8255.
[163] G. Moreno-Bueno, C. Gamallo, L. Perez-Gallego, J.C. de Mora,
A. Suarez, J. Palacios, Diagn. Mol. Pathol. 10 (2001) 116 – 122.
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[164] J. Palacios, L. Catasus, G. Moreno-Bueno, X. Matias-Guiu, J. Prat,
C. Gamallo, Virchows Arch. 438 (2001) 464 – 469.
[165] K. Wright, P. Wilson, S. Morland, I. Campbell, M. Walsh, T. Hurst,
B. Ward, M. Cummings, G. Chenevix-Trench, Int. J. Cancer 82
(1999) 625 – 629.
[166] K. Ashihara, T. Saito, H. Mizumoto, M. Nishimura, R. Tanaka, R.
Kudo, Med. Electron Microsc. 35 (2002) 9 – 15.
[167] T. Fukuchi, M. Sakamoto, H. Tsuda, K. Maruyama, S. Nozawa, S.
Hirohashi, Cancer Res. 58 (1998) 3526 – 3528.
[168] H. Nei, T. Saito, H. Yamasaki, H. Mizumoto, E. Ito, R. Kudo, Mol.
Carcinog. 25 (1999) 207 – 218.
[169] L. Mirabelli-Primdahl, R. Gryfe, H. Kim, A. Millar, C. Luceri, D.
Dale, E. Holowaty, B. Bapat, S. Gallinger, M. Redston, Cancer Res.
59 (1999) 3346 – 3351.
[170] T. Ikeda, K. Yoshinaga, S. Semba, E. Kondo, H. Ohmori, A. Horii,
Oncol. Rep. 7 (2000) 323 – 326.
[171] H.J. Voeller, C.I. Truica, E.P. Gelmann, Cancer Res. 58 (1998)
2520 – 2523.
[172] D.R. Chesire, C.M. Ewing, J. Sauvageot, G.S. Bova, W.B. Isaacs,
Prostate 45 (2000) 323 – 334.
[173] A.V. Gerstein, T.A. Almeida, G. Zhao, E. Chess, M. Shih Ie, K.
Buhler, K. Pienta, M.A. Rubin, R. Vessella, N. Papadopoulos, Genes
Chromosomes Cancer 34 (2002) 9 – 16.
[174] H. Blaker, W.J. Hofmann, R.J. Rieker, R. Penzel, M. Graf, H.F. Otto,
Genes Chromosomes Cancer 25 (1999) 399 – 402.
[175] Y.M. Jeng, M.Z. Wu, T.L. Mao, M.H. Chang, H.C. Hsu, Cancer Lett.
152 (2000) 45 – 51.
[176] H. Takayasu, H. Horie, E. Hiyama, T. Matsunaga, Y. Hayashi, Y.
Watanabe, S. Suita, M. Kaneko, F. Sasaki, K. Hashizume, T. Ozaki,
K. Furuuchi, M. Tada, N. Ohnuma, A. Nakagawara, Clin. Cancer
Res. 7 (2001) 901 – 908.
[177] A. Koch, D. Denkhaus, S. Albrecht, I. Leuschner, D. von Schweinitz,
T. Pietsch, Cancer Res. 59 (1999) 269 – 273.
[178] Y. Wei, M. Fabre, S. Branchereau, F. Gauthier, G. Perilongo, M.A.
Buendia, Oncogene 19 (2000) 498 – 504.
[179] W.S. Park, R.R. Oh, J.Y. Park, P.J. Kim, M.S. Shin, J.H. Lee, H.S.
Kim, S.H. Lee, S.Y. Kim, Y.G. Park, W.G. An, J.J. Jang, N.J. Yoo,
J.Y. Lee, J. Pathol. 193 (2001) 483 – 490.
[180] H. Oda, Y. Imai, Y. Nakatsuru, J. Hata, T. Ishikawa, Cancer Res. 56
(1996) 3320 – 3323.
[181] C.G. Eberhart, T. Tihan, P.C. Burger, J. Neuropathol. Exp. Neurol.
59 (2000) 333 – 337.
[182] N. Yokota, S. Nishizawa, S. Ohta, H. Date, H. Sugimura, H. Namba,
M. Maekawa, Int. J. Cancer 101 (2002) 198 – 201.
[183] R.H. Zurawel, S.A. Chiappa, C. Allen, C. Raffel, Cancer Res. 58
(1998) 896 – 899.
[184] H. Huang, B.M. Mahler-Araujo, A. Sankila, L. Chimelli, Y.
Yonekawa, P. Kleihues, H. Ohgaki, Am. J. Pathol. 156 (2000)
433 – 437.
[185] A. Koch, A. Waha, J.C. Tonn, N. Sorensen, F. Berthold, M. Wolter,
J. Reifenberger, W. Hartmann, W. Friedl, G. Reifenberger, O.D.
Wiestler, T. Pietsch, Int. J. Cancer 93 (2001) 445 – 449.
[186] R.P. Dahmen, A. Koch, D. Denkhaus, J.C. Tonn, N. Sorensen, F.
Berthold, J. Behrens, W. Birchmeier, O.D. Wiestler, T. Pietsch, Cancer Res. 61 (2001) 7039 – 7043.
[187] S.C. Abraham, T.T. Wu, D.S. Klimstra, L.S. Finn, J.H. Lee, C.J.
Yeo, J.L. Cameron, R.H. Hruban, Am. J. Pathol. 159 (2001)
1619 – 1627.
[188] Y. Miyoshi, K. Iwao, G. Nawa, H. Yoshikawa, T. Ochi, Y. Nakamura, Oncol. Res. 10 (1998) 591 – 594.
[189] S. Tejpar, F. Nollet, C. Li, J.S. Wunder, G. Michils, P. dal Cin, E.
Van Cutsem, B. Bapat, F. van Roy, J.J. Cassiman, B.A. Alman,
Oncogene 18 (1999) 6615 – 6620.
[190] T. Saito, Y. Oda, K. Kawaguchi, K. Tanaka, S. Matsuda, S. Tamiya,
Y. Iwamoto, M. Tsuneyoshi, Lab. Invest. 82 (2002) 97 – 103.
[191] A.S. Tsukamoto, R. Grosschedl, R.C. Guzman, T. Parslow, H.E.
Varmus, Cell 55 (1988) 619 – 625.
21
[192] R. Nusse, J. Steroid Biochem. Mol. Biol. 43 (1992) 9 – 12.
[193] T.C. Dale, S.J. Weber-Hall, K. Smith, E.L. Huguet, H. Jayatilake,
B.A. Gusterson, G. Shuttleworth, M. O’Hare, A.L. Harris, Cancer
Res. 56 (1996) 4320 – 4323.
[194] E.L. Huguet, J.A. McMahon, A.P. McMahon, R. Bicknell, A.L.
Harris, Cancer Res. 54 (1994) 2615 – 2621.
[195] R.V. Iozzo, I. Eichstetter, K.G. Danielson, Cancer Res. 55 (1995)
3495 – 3499.
[196] S. Lejeune, E.L. Huguet, A. Hamby, R. Poulsom, A.L. Harris, Clin.
Cancer Res. 1 (1995) 215 – 222.
[197] W. Hsu, R. Shakya, F. Costantini, J. Cell Biol. 155 (2001)
1055 – 1064.
[198] R.C. Gallagher, T. Hay, V. Meniel, C. Naughton, T.J. Anderson, H.
Shibata, M. Ito, H. Clevers, T. Noda, O.J. Sansom, J.O. Mason, A.R.
Clarke, Oncogene 21 (2002) 6446 – 6457.
[199] C. van Genderen, R.M. Okamura, I. Farinas, R.G. Quo, T.G. Parslow, L. Bruhn, R. Grosschedl, Genes Dev. 8 (1994) 2691 – 2703.
[200] J. Roose, G. Huls, M. van Beest, P. Moerer, K. van der Horn, R.
Goldschmeding, T. Logtenberg, H. Clevers, Science 285 (1999)
1923 – 1926.
[201] G. Giudice, Cell Biol. Int. 25 (2001) 1081 – 1090.
[202] H. Dierick, A. Bejsovec, Curr. Top. Dev. Biol. 43 (1999) 153 – 190.
[203] T. Grosser, S. Yusuff, E. Cheskis, M.A. Pack, G.A. FitzGerald, Proc.
Natl. Acad. Sci. U. S. A. 99 (2002) 8418 – 8423.
[204] S. van de Water, M. van de Wetering, J. Joore, J. Esseling, R. Bink,
H. Clevers, D. Zivkovic, Development 128 (2001) 3877 – 3888.
[205] C.P. Heisenberg, C. Houart, M. Take-Uchi, G.J. Rauch, N. Young, P.
Coutinho, I. Masai, L. Caneparo, M.L. Concha, R. Geisler, T.C. Dale,
S.W. Wilson, D.L. Stemple, Genes Dev. 15 (2001) 1427 – 1434.
[206] H.C. Korswagen, M.A. Herman, H.C. Clevers, Nature 406 (2000)
527 – 532.
[207] R. Lin, S. Thompson, J.R. Priess, Cell 83 (1995) 599 – 609.
[208] C.E. Rocheleau, J. Yasuda, T.H. Shin, R. Lin, H. Sawa, H. Okano,
J.R. Priess, R.J. Davis, C.C. Mello, Cell 97 (1999) 717 – 726.
[209] E.F. Hoier, W.A. Mohler, S.K. Kim, A. Hajnal, Genes Dev. 14
(2000) 874 – 886.
[210] H.C. Korswagen, D.Y. Coudreuse, M.C. Betist, S. van de Water,
D. Zivkovic, H.C. Clevers, Genes Dev. 16 (2002) 1291 – 1302.
[211] J.E. Gleason, H.C. Korswagen, D.M. Eisenmann, Genes Dev. 16
(2002) 1281 – 1290.
[212] A.R. Moser, H.C. Pitot, W.F. Dove, Science 247 (1990) 322 – 324.
[213] L.K. Su, K.W. Kinzler, B. Vogelstein, A.C. Preisinger, A.R.
Moser, C. Luongo, K.A. Gould, W.F. Dove, Science 256 (1992)
668 – 670.
[214] C. Luongo, A.R. Moser, S. Gledhill, W.F. Dove, Cancer Res. 54
(1994) 5947 – 5952.
[215] A.R. Moser, E.M. Mattes, W.F. Dove, M.J. Lindstrom, J.D. Haag,
M.N. Gould, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 8977 – 8981.
[216] A.R. Moser, A.R. Shoemaker, C.S. Connelly, L. Clipson, K.A.
Gould, C. Luongo, W.F. Dove, P.H. Siggers, R.L. Gardner, Dev.
Dyn. 203 (1995) 422 – 433.
[217] A.R. Shoemaker, K.A. Gould, C. Luongo, A.R. Moser, W.F. Dove,
Biochim. Biophys. Acta 1332 (1997) F25 – F48.
[218] A.R. Clarke, M.C. Cummings, D.J. Harrison, Oncogene 11 (1995)
1913 – 1920.
[219] R.B. Halberg, D.S. Katzung, P.D. Hoff, A.R. Moser, C.E. Cole, R.A.
Lubet, L.A. Donehower, R.F. Jacoby, W.F. Dove, Proc. Natl. Acad.
Sci. U. S. A. 97 (2000) 3461 – 3466.
[220] P.W. Laird, L. Jackson-Grusby, A. Fazeli, S.L. Dickinson, W.E.
Jung, E. Li, R.A. Weinberg, R. Jaenisch, Cell 81 (1995) 197 – 205.
[221] H.S. Wasan, M. Novelli, J. Bee, W.F. Bodmer, Proc. Natl. Acad. Sci.
U. S. A. 94 (1997) 3308 – 3313.
[222] W.F. Dietrich, E.S. Lander, J.S. Smith, A.R. Moser, K.A. Gould,
C. Luongo, N. Borenstein, W. Dove, Cell 75 (1993) 631 – 639.
[223] R.T. Cormier, K.H. Hong, R.B. Halberg, T.L. Hawkins, P. Richardson, R. Mulherkar, W.F. Dove, E.S. Lander, Nat. Genet. 17 (1997)
88 – 91.
22
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[224] K.A. Silverman, R. Koratkar, L.D. Siracusa, A.M. Buchberg, Genome Res. 12 (2002) 88 – 97.
[225] H. Sasai, M. Masaki, K. Wakitani, Carcinogenesis 21 (2000)
953 – 958.
[226] C.F. Quesada, H. Kimata, M. Mori, M. Nishimura, T. Tsuneyoshi, S.
Baba, Jpn. J. Cancer Res. 89 (1998) 392 – 396.
[227] M. Oshima, H. Oshima, K. Kitagawa, M. Kobayashi, C. Itakura, M.
Taketo, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 4482 – 4486.
[228] H. Shibata, K. Toyama, H. Shioya, M. Ito, M. Hirota, S. Hasegawa,
H. Matsumoto, H. Takano, T. Akiyama, K. Toyoshima, R. Kanamaru, Y. Kanegae, I. Saito, Y. Nakamura, K. Shiba, T. Noda, Science
278 (1997) 120 – 123.
[229] R. Fodde, W. Edelmann, K. Yang, C. van Leeuwen, C. Carlson,
B. Renault, C. Breukel, E. Alt, M. Lipkin, P.M. Khan, et al., Proc.
Natl. Acad. Sci. U. S. A. 91 (1994) 8969 – 8973.
[230] M.F. Kielman, M. Ridanpaa, C. Gaspar, N. van Poppel, C. Breukel,
S. van Leeuwen, M. Taketo, S. Roberts, R. Smits, R. Fodde, Nat.
Genet. 32 (2002) 594 – 605.
[231] C.W. van der Houven van Oordt, R. Smits, S.L. Williamson, A. Luz,
P.M. Khan, R. Fodde, A.J. van der Eb, M.L. Breuer, Carcinogenesis
18 (1997) 2197 – 2203.
[232] R. Smits, W. van der Houven van Oordt, A. Luz, C. Zurcher, S.
Jagmohan-Changur, C. Breukel, P.M. Khan, R. Fodde, Gastroenterology 114 (1998) 275 – 283.
[233] R. Caspari, S. Olschwang, W. Friedl, M. Mandl, C. Boisson, T.
Boker, A. Augustin, M. Kadmon, G. Moslein, G. Thomas, et al.,
Hum. Mol. Genet. 4 (1995) 337 – 340.
[234] J.G. Fox, C.A. Dangler, M.T. Whary, W. Edelman, R. Kucherlapati,
T.C. Wang, Cancer Res. 57 (1997) 3972 – 3978.
[235] R. Smits, P. Ruiz, S. Diaz-Cano, A. Luz, S. Jagmohan-Changur,
C. Breukel, C. Birchmeier, W. Birchmeier, R. Fodde, Gastroenterology 119 (2000) 1045 – 1053.
[236] R. Smits, M.F. Kielman, C. Breukel, C. Zurcher, K. Neufeld, S.
Jagmohan-Changur, N. Hofland, J. van Dijk, R. White, W. Edelmann, R. Kucherlapati, P.M. Khan, R. Fodde, Genes Dev. 13
(1999) 1309 – 1321.
[237] R. Fodde, J. Kuipers, C. Rosenberg, R. Smits, M. Kielman, C.
Gaspar, J.H. van Es, C. Breukel, J. Wiegant, R.H. Giles, H. Clevers,
Nat. Cell Biol. 3 (2001) 433 – 438.
[238] N. Harada, Y. Tamai, T. Ishikawa, B. Sauer, K. Takaku, M. Oshima,
M.M. Taketo, EMBO J. 18 (1999) 5931 – 5942.
[239] B. Romagnolo, D. Berrebi, S. Saadi-Keddoucci, A. Porteu, A.L.
Pichard, M. Peuchmaur, A. Vandewalle, A. Kahn, C. Perret, Cancer
Res. 59 (1999) 3875 – 3879.
[240] S.S. Cheon, A.Y. Cheah, S. Turley, P. Nadesan, R. Poon, H. Clevers,
B.A. Alman, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 6973 – 6978.
[241] S. Saadi-Kheddouci, D. Berrebi, B. Romagnolo, F. Cluzeaud, M.
Peuchmaur, A. Kahn, A. Vandewalle, C. Perret, Oncogene 20
(2001) 5972 – 5981.
[242] D.J. Peters, M.H. Breuning, Lancet 358 (2001) 1439 – 1444.
[243] A. Cadoret, C. Ovejero, S. Saadi-Kheddouci, E. Souil, M. Fabre, B.
Romagnolo, A. Kahn, C. Perret, Cancer Res. 61 (2001) 3245 – 3249.
[244] N. Harada, H. Miyoshi, N. Murai, H. Oshima, Y. Tamai, M. Oshima,
M.M. Taketo, Cancer Res. 62 (2002) 1971 – 1977.
[245] F. Gounari, S. Signoretti, R. Bronson, L. Klein, W.R. Sellers, J.
Kum, A. Siermann, M.M. Taketo, H. von Boehmer, K. Khazaie,
Oncogene 21 (2002) 4099 – 4107.
[246] H. Haegel, L. Larue, M. Ohsugi, L. Fedorov, K. Herrenknecht, R.
Kemler, Development 121 (1995) 3529 – 3537.
[247] J. Huelsken, R. Vogel, B. Erdmann, G. Cotsarelis, W. Birchmeier,
Cell 105 (2001) 533 – 545.
[248] S. Verbeek, D. Izon, F. Hofhuis, E. Robanus-Maandag, H. te Riele,
M. van de Wetering, M. Oosterwegel, A. Wilson, H.R. MacDonald,
H. Clevers, Nature 374 (1995) 70 – 74.
[249] M.W. Schilham, A. Wilson, P. Moerer, B.J. Benaissa-Trouw, A.
Cumano, H.C. Clevers, J. Immunol. 161 (1998) 3984 – 3991.
[250] P. Zhou, C. Byrne, J. Jacobs, E. Fuchs, Genes Dev. 9 (1995) 700 – 713.
[251] C. Niemann, D.M. Owens, J. Hulsken, W. Birchmeier, F.M. Watt,
Development 129 (2002) 95 – 109.
[252] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger,
K.W. Kinzler, B. Vogelstein, H. Clevers, Science 275 (1997)
1784 – 1787.
[253] V. Korinek, N. Barker, P. Moerer, E. van Donselaar, G. Huls,
P.J. Peters, H. Clevers, Nat. Genet. 19 (1998) 379 – 383.
[254] M. van de Wetering, E. Sancho, C. Verweij, W. de Lau, I.M.
Oving, A. Hurlstone, K. van der Horn, E. Batlle, D.Y. Coudreuse,
A.P. Haramis, M. Tjon-Pon-Fong, P. Moerer, M.M.W. van den
Born, G. Soete, S.T. Pals, M. Eilers, R.H. Medema, H. Clevers,
Cell 111 (2002) 241 – 250.
[255] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da
Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Science 281 (1998)
1509 – 1512.
[256] B.M. Boman, J.Z. Fields, O. Bonham-Carter, O.A. Runquist, Cancer
Res. 61 (2001) 8408 – 8411.
[257] J. Willert, M. Epping, J.R. Pollack, P.O. Brown, R. Nusse, BMC
Dev. Biol. 2 (2002) 8.
[258] S.P. Rockman, S.A. Currie, M. Ciavarella, E. Vincan, C. Dow, R.J.
Thomas, W.A. Phillips, J. Biol. Chem. 276 (2001) 45113 – 45119.
[259] K. Hovanes, T.W. Li, J.E. Munguia, T. Truong, T. Milovanovic, J.
Lawrence Marsh, R.F. Holcombe, M.L. Waterman, Nat. Genet. 28
(2001) 53 – 57.
[260] B.R. Henderson, M. Galea, S. Schuechner, L. Leung, J. Biol. Chem.
277 (2002) 24258 – 24264.
[261] C. Lengauer, K.W. Kinzler, B. Vogelstein, Nature 396 (1998)
643 – 649.
[262] G.A. Pihan, A. Purohit, J. Wallace, H. Knecht, B. Woda, P. Quesenberry, S.J. Doxsey, Cancer Res. 58 (1998) 3974 – 3985.
[263] B.M. Ghadimi, D.L. Sackett, M.J. Difilippantonio, E. Schrock, T.
Neumann, A. Jauho, G. Auer, T. Ried, Genes Chromosomes Cancer
27 (2000) 183 – 190.
[264] K.K. Kuo, N. Sato, K. Mizumoto, N. Maehara, H. Yonemasu, C.G.
Ker, P.C. Sheen, M. Tanaka, Hepatology 31 (2000) 59 – 64.
[265] N. Sato, K. Mizumoto, M. Nakamura, N. Maehara, Y.A. Minamishima, S. Nishio, E. Nagai, M. Tanaka, Cancer Genet. Cytogenet. 126
(2001) 13 – 19.
[266] S. Duensing, K. Munger, Biochim. Biophys. Acta 2 (2001)
M81 – M88.
[267] K.W. Kinzler, B. Vogelstein, Cell 87 (1996) 159 – 170.
[268] B. Vogelstein, K.W. Kinzler, Trends Genet. 9 (1993) 138 – 141.
[269] C. Lengauer, K.W. Kinzler, B. Vogelstein, Nature 386 (1997)
623 – 627.
[270] K.W. Kinzler, B. Vogelstein, Science 280 (1998) 1036 – 1037.
[271] A. Lindblom, Curr. Opin. Oncol. 13 (2001) 63 – 69.
[272] K.B. Kaplan, A.A. Burds, J.R. Swedlow, S.S. Bekir, P.K. Sorger, I.S.
Nathke, Nat. Cell Biol. 3 (2001) 429 – 432.
[273] D.B. Levy, K.J. Smith, Y. Beazer-Barclay, S.R. Hamilton, B. Vogelstein, K.W. Kinzler, Cancer Res. 54 (1994) 5953 – 5958.
[274] B.M. McCartney, H.A. Dierick, C. Kirkpatrick, M.M. Moline, A.
Baas, M. Peifer, A. Bejsovec, J. Cell Biol. 146 (1999) 1303 – 1318.
[275] S. Hayashi, B. Rubinfeld, B. Souza, P. Polakis, E. Wieschaus, A.J.
Levine, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 242 – 247.
[276] Y. Kawasaki, T. Senda, T. Ishidate, R. Koyama, T. Morishita, Y.
Iwayama, O. Higuchi, T. Akiyama, Science 289 (2000) 1194 – 1197.
[277] I.S. Nathke, C.L. Adams, P. Polakis, J.H. Sellin, W.J. Nelson, J. Cell
Biol. 134 (1996) 165 – 179.
[278] S. Munemitsu, B. Souza, O. Muller, I. Albert, B. Rubinfeld, P.
Polakis, Cancer Res. 54 (1994) 3676 – 3681.
[279] K.J. Smith, D.B. Levy, P. Maupin, T.D. Pollard, B. Vogelstein, K.W.
Kinzler, Cancer Res. 54 (1994) 3672 – 3675.
[280] L.K. Su, M. Burrell, D.E. Hill, J. Gyuris, R. Brent, R. Wiltshire,
J. Trent, B. Vogelstein, K.W. Kinzler, Cancer Res. 55 (1995)
2972 – 2977.
[281] J. Deka, J. Kuhlmann, O. Muller, Eur. J. Biochem. 253 (1998)
591 – 597.
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[282] M.M. Mogensen, J.B. Tucker, J.B. Mackie, A.R. Prescott, I.S.
Nathke, J. Cell Biol. 157 (2002) 1041 – 1048.
[283] Y. Mimori-Kiyosue, N. Shiina, S. Tsukita, J. Cell Biol. 148 (2000)
505 – 518.
[284] J. Zumbrunn, K. Kinoshita, A.A. Hyman, I.S. Nathke, Curr. Biol. 11
(2001) 44 – 49.
[285] Y. Mimori-Kiyosue, S. Tsukita, J. Cell Biol. 154 (2001) 1105 – 1109.
[286] T. Jimbo, Y. Kawasaki, R. Koyama, R. Sato, S. Takada, K. Haraguchi, T. Akiyama, Nat. Cell Biol. 4 (2002) 323 – 327.
[287] M.H. Wong, M.L. Hermiston, A.J. Syder, J.I. Gordon, Proc. Natl.
Acad. Sci. U. S. A. 93 (1996) 9588 – 9593.
[288] J.R. Miller, R.T. Moon, J. Cell Biol. 139 (1997) 229 – 243.
[289] P. Herter, C. Kuhnen, K.M. Muller, A. Wittinghofer, O. Muller, J.
Cancer Res. Clin. Oncol. 125 (1999) 297 – 304.
[290] F. Hamada, M. Bienz, Nat. Cell Biol. 4 (2002) 208 – 213.
[291] T.A. Chan, Z. Wang, L.H. Dang, B. Vogelstein, K.W. Kinzler, Proc.
Natl. Acad. Sci. U. S. A. 99 (2002) 8265 – 8270.
[292] J. Kawanishi, J. Kato, K. Sasaki, S. Fujii, N. Watanabe, Y. Niitsu,
Mol. Cell. Biol. 15 (1995) 1175 – 1181.
[293] M. van Noort, J. Meeldijk, R. van der Zee, O. Destree, H. Clevers, J.
Biol. Chem. 277 (2002) 17901 – 17905.
[294] C.S. Potten, M. Loeffler, Development 110 (1990) 1001 – 1020.
[295] E.D. Hay, A. Zuk, Am. J. Kidney Dis. 26 (1995) 678 – 690.
[296] E. Batlle, J.T. Henderson, H. Beghtel, M.M.W. van den Born, E.
Sancho, G. Huls, J. Meeldijk, J. Roberston, M. van de Wetering,
T. Pawson, H. Clevers, Cell 111 (2002) 251 – 263.
[297] D.G. Wilkinson, Nat. Rev., Neurosci. 2 (2001) 155 – 164.
[298] Q. Xu, G. Mellitzer, V. Robinson, D.G. Wilkinson, Nature 399
(1999) 267 – 271.
[299] H. Oshima, M. Oshima, M. Kobayashi, M. Tsutsumi, M.M. Taketo,
Cancer Res. 57 (1997) 1644 – 1649.
[300] B. Hinz, K. Brune, J. Pharmacol. Exp. Ther. 300 (2002) 367 – 375.
[301] T.C. He, T.A. Chan, B. Vogelstein, K.W. Kinzler, Cell 99 (1999)
335 – 345.
[302] M. Oshima, J.E. Dinchuk, S.L. Kargman, H. Oshima, B. Hancock,
E. Kwong, J.M. Trzaskos, J.F. Evans, M.M. Taketo, Cell 87 (1996)
803 – 809.
[303] S.K. Boolbol, A.J. Dannenberg, A. Chadburn, C. Martucci, X.J.
Guo, J.T. Ramonetti, M. Abreu-Goris, H.L. Newmark, M.L. Lipkin,
J.J. DeCosse, M.M. Bertagnolli, Cancer Res. 56 (1996) 2556 – 2560.
[304] Y. Beazer-Barclay, D.B. Levy, A.R. Moser, W.F. Dove, S.R. Hamilton, B. Vogelstein, K.W. Kinzler, Carcinogenesis 17 (1996)
1757 – 1760.
[305] G. Lal, C. Ash, K. Hay, M. Redston, E. Kwong, B. Hancock, T.
Mak, S. Kargman, J.F. Evans, S. Gallinger, Cancer Res. 61 (2001)
6131 – 6136.
[306] S.M. Prescott, R.L. White, Cell 87 (1996) 783 – 786.
[307] R. van Stolk, G. Stoner, W.L. Hayton, K. Chan, B. DeYoung, L.
Kresty, B.H. Kemmenoe, P. Elson, L. Rybicki, J. Church, K. Provencher, D. McLain, E. Hawk, B. Fryer, G. Kelloff, R. Ganapathi,
G.T. Budd, Clin. Cancer Res. 6 (2000) 78 – 89.
[308] G.D. Stoner, G.T. Budd, R. Ganapathi, B. DeYoung, L.A. Kresty,
M. Nitert, B. Fryer, J.M. Church, K. Provencher, R. Pamukcu,
G. Piazza, E. Hawk, G. Kelloff, P. Elson, R.U. van Stolk, Adv.
Exp. Med. Biol. 470 (1999) 45 – 53.
[309] F.M. Giardiello, V.W. Yang, L.M. Hylind, A.J. Krush, G.M. Petersen, J.D. Trimbath, S. Piantadosi, E. Garrett, D.E. Geiman, W. Hubbard, G.J. Offerhaus, S.R. Hamilton, N. Engl. J. Med. 346 (2002)
1054 – 1059.
[310] I. Chau, D. Cunningham, N. Engl. J. Med. 346 (2002) 1085 – 1087.
[311] L.R. Howe, A.J. Dannenberg, Semin. Oncol. 29 (2002) 111 – 119.
[312] G. Steinbach, P.M. Lynch, R.K. Phillips, M.H. Wallace, E. Hawk,
G.B. Gordon, N. Wakabayashi, B. Saunders, Y. Shen, T. Fujimura,
L.K. Su, B. Levin, N. Engl. J. Med. 342 (2000) 1946 – 1952.
[313] R.W. Wilkinson, E.L. Ross, R. Poulsom, M. Ilyas, J. Straub, D.
Snary, W.F. Bodmer, S.J. Mather, Proc. Natl. Acad. Sci. U. S. A.
98 (2001) 10256 – 10260.
23
[314] K.M. Hajra, E.R. Fearon, Genes Chromosomes Cancer 34 (2002)
255 – 268.
[315] K.W. Kinzler, M.C. Nilbert, B. Vogelstein, T.M. Bryan, D.B. Levy,
K.J. Smith, A.C. Preisinger, S.R. Hamilton, P. Hedge, A. Markham,
et al., Science 251 (1991) 1366 – 1370.
[316] M. Ilyas, I.P. Tomlinson, A. Rowan, M. Pignatelli, W.F. Bodmer,
Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 10330 – 10334.
[317] J.M. Wheeler, B.F. Warren, N.J. Mortensen, H.C. Kim, S.C. Biddolph, G. Elia, N.E. Beck, G.T. Williams, N.A. Shepherd, A.C.
Bateman, W.F. Bodmer, Gut 50 (2002) 218 – 223.
[318] S. Sekine, T. Shibata, Y. Yamauchi, Y. Nakanishi, T. Shimoda, M.
Sakamoto, S. Hirohashi, Virchows Arch. 440 (2002) 381 – 386.
[319] M. Fujimori, S. Ikeda, Y. Shimizu, M. Okajima, T. Asahara, Cancer
Res. 61 (2001) 6656 – 6659.
[320] N. Sunaga, T. Kohno, F.T. Kolligs, E.R. Fearon, R. Saito, J. Yokota,
Genes Chromosomes Cancer 30 (2001) 316 – 321.
[321] A.L. Pereira-Suarez, M.A. Meraz, M. Lizano, C. Estrada-Chavez, F.
Hernandez, P. Olivera, E. Perez, P. Padilla, M. Yaniv, F. Thierry, A.
Garcia-Carranca, Tumour Biol. 23 (2002) 45 – 53.
[322] S.C. Abraham, C. Reynolds, J.H. Lee, E.A. Montgomery, B.L.
Baisden, A.M. Krasinskas, T.T. Wu, Human Pathol. 33 (2002)
39 – 46.
[323] G. Garcia-Rostan, R.L. Camp, A. Herrero, M.L. Carcangiu, D.L.
Rimm, G. Tallini, Am. J. Pathol. 158 (2001) 987 – 996.
[324] K. Ishigaki, H. Namba, M. Nakashima, T. Nakayama, N. Mitsutake,
T. Hayashi, S. Maeda, M. Ichinose, T. Kanematsu, S. Yamashita, J.
Clin. Endocrinol. Metab. 87 (2002) 3433 – 3440.
[325] Y. Udatsu, T. Kusafuka, S. Kuroda, J. Miao, A. Okada, Pediatr. Surg.
Int. 17 (2001) 508 – 512.
[326] K. Shitoh, F. Konishi, T. Iijima, T. Ohdaira, K. Sakai, K. Kanazawa,
M. Miyaki, J. Clin. Pathol. 52 (1999) 695 – 696.
[327] B.A. Alman, C. Li, M.E. Pajerski, S. Diaz-Cano, H.J. Wolfe, Am. J.
Pathol. 151 (1997) 329 – 334.
[328] R. Koesters, R. Ridder, A. Kopp-Schneider, D. Betts, V. Adams, F.
Niggli, J. Briner, M. von Knebel Doeberitz, Cancer Res. 59 (1999)
3880 – 3882.
[329] S. Maiti, R. Alam, C.I. Amos, V. Huff, Cancer Res. 60 (2000)
6288 – 6292.
[330] Y. Tanaka, K. Kato, K. Notohara, H. Hojo, R. Ijiri, T. Miyake, N.
Nagahara, F. Sasaki, N. Kitagawa, Y. Nakatani, Y. Kobayashi, Cancer Res. 61 (2001) 8401 – 8404.
[331] S.C. Abraham, D.S. Klimstra, R.E. Wilentz, C.J. Yeo, K. Conlon,
M. Brennan, J.L. Cameron, T.T. Wu, R.H. Hruban, Am. J. Pathol.
160 (2002) 1361 – 1369.
[332] S.C. Abraham, T.T. Wu, R.H. Hruban, J.H. Lee, C.J. Yeo, K. Conlon, M. Brennan, J.L. Cameron, D.S. Klimstra, Am. J. Pathol. 160
(2002) 953 – 962.
[333] T. Saito, Y. Oda, A. Sakamoto, S. Tamiya, N. Kinukawa, K.
Hayashi, Y. Iwamoto, M. Tsuneyoshi, J. Pathol. 192 (2000)
342 – 350.
[334] H. Sato, T. Hasegawa, Y. Kanai, Y. Tsutsumi, Y. Osamura, Y. Abe,
H. Sakai, S. Hirohashi, Virchows Arch. 438 (2001) 23 – 30.
[335] T. Saito, Y. Oda, A. Sakamoto, K. Kawaguchi, K. Tanaka, S. Matsuda, S. Tamiya, Y. Iwamoto, M. Tsuneyoshi, J. Pathol. 196 (2002)
445 – 449.
[336] V.J. Wielenga, R. Smits, V. Korinek, L. Smit, M. Kielman, R. Fodde,
H. Clevers, S.T. Pals, Am. J. Pathol. 154 (1999) 515 – 523.
[337] J.S. Kim, H. Crooks, T. Dracheva, T.G. Nishanian, B. Singh, J. Jen,
T. Waldman, Cancer Res. 62 (2002) 2744 – 2748.
[338] N. Miwa, M. Furuse, S. Tsukita, N. Niikawa, Y. Nakamura, Y.
Furukawa, Oncol. Res. 12 (2001) 469 – 476.
[339] M. Fujita, Y. Furukawa, T. Tsunoda, T. Tanaka, M. Ogawa, Y. Nakamura, Cancer Res. 61 (2001) 7722 – 7726.
[340] O. Tetsu, F. McCormick, Nature 398 (1999) 422 – 426.
[341] M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D’Amico, R.
Pestell, A. Ben-Ze’ev, Proc. Natl. Acad. Sci. U. S. A. 96 (1999)
5522 – 5527.
24
R.H. Giles et al. / Biochimica et Biophysica Acta 1653 (2003) 1–24
[342] B. Mann, M. Gelos, A. Siedow, M.L. Hanski, A. Gratchev, M. Ilyas,
W.F. Bodmer, M.P. Moyer, E.O. Riecken, H.J. Buhr, C. Hanski,
Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1603 – 1608.
[343] M.E. Conacci-Sorrell, T. Ben-Yedidia, M. Shtutman, E. Feinstein, P.
Einat, A. Ben-Ze’ev, Genes Dev. 16 (2002) 2058 – 2072.
[344] F.T. Kolligs, M.T. Nieman, I. Winer, G. Hu, D. Van Mater, Y. Feng,
I.M. Smith, R. Wu, Y. Zhai, K.R. Cho, E.R. Fearon, Cancer Cell 1
(2002) 145 – 155.
[345] L. Levy, C. Neuveut, C.A. Renard, P. Charneau, S. Branchereau, F.
Gauthier, J. Tran Van Nhieu, D. Cherqui, A.F. Petit-Bertron, D.
Mathieu, M.A. Buendia, J. Biol. Chem. 27 (2002) 27.
[346] M. Rodova, M.R. Islam, R.L. Maser, J.P. Calvet, J. Biol. Chem. 277
(2002) 29577 – 29583.
[347] T.J. Koh, C.J. Bulitta, J.V. Fleming, G.J. Dockray, A. Varro, T.C.
Wang, J. Clin. Invest. 106 (2000) 533 – 539.
[348] B. Lustig, B. Jerchow, M. Sachs, S. Weiler, T. Pietsch, U. Karsten,
M. van de Wetering, H. Clevers, P.M. Schlag, W. Birchmeier, J.
Behrens, Mol. Cell. Biol. 22 (2002) 1184 – 1193.
[349] T. Yamada, A.S. Takaoka, Y. Naishiro, R. Hayashi, K. Maruyama,
C. Maesawa, A. Ochiai, S. Hirohashi, Cancer Res. 60 (2000)
4761 – 4766.
[350] H.C. Crawford, B.M. Fingleton, L.A. Rudolph-Owen, K.J. Goss,
B. Rubinfeld, P. Polakis, L.M. Matrisian, Oncogene 18 (1999)
2883 – 2891.
[351] T. Brabletz, A. Jung, S. Dag, F. Hlubek, T. Kirchner, Am. J. Pathol.
155 (1999) 1033 – 1038.
[352] T. Muller, G. Bain, X. Wang, J. Papkoff, Exp. Cell Res. 280 (2002)
119.
[353] M. Oshima, H. Oshima, M. Kobayashi, M. Tsusumi, M.M. Taketo,
Cancer Res. 55 (1995) 2719 – 2722.