Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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.