Wnt signalling in the mouse intestine - Nature

Oncogene (2006) 25, 7512–7521

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REVIEW

Wnt signalling in the mouse intestine AR Clarke Cardiff School of Biosciences, Museum Avenue, Cardiff University, Cardiff, UK

The ApcMin/ þ mouse has emerged as a powerful model of human intestinal tumour predisposition. As such, it has provided a platform for studying genetic and epigenetic modifiers of adenoma predisposition, and for assessing the chemotherapeutic potential of a plethora of different agents. The development of new conditional and hypomorphic Apc alleles, together with models carrying mutations in other Wnt pathway components, has greatly extended the scope of experimentation. Together these approaches are being used to identify and validate key critical targets of the Wnt pathway, such as Mash2, Tiam1 and the Eph/Ephrins. They have also established a fundamental role for Wnt in the development and maintenance of normal intestinal physiology, and in particular control of the stem cell niche. These activities are now being dissected at the level of individual Wnt components, with some surprising dependencies revealed. In terms of adenoma development, these models also support a ‘just right’ notion for tightly controlled b-catenin activity both in normal physiology and neoplastic development. They also indicate a two-stage dependency for some Wnt pathway targets, with an initial requirement that is subsequently overcome to permit progression. Finally, these models establish that the Wnt pathway does not operate in isolation, and that both normal and diseased physiology develops in a dynamic interplay with other pathways such as the Notch, Hedgehog and BMP pathways. The comprehensive understanding arising from these studies should lead the identification of novel prognostic markers and therapeutic targets, and also open the possibility of tissue engineering in the intestine. Oncogene (2006) 25, 7512–7521. doi:10.1038/sj.onc.1210065 Keywords: Wnt; Apc; intestine

Introduction Deregulation of the Wnt pathway is well recognized to underpin the early stages of colorectal neoplasia in both the human and the mouse. This is perhaps most clearly demonstrated by the recognition that mutations in the adenomatous polyposis coli gene (APC) underlie familial Correspondence: Professor AR Clarke, Cardiff School of Biosciences, Museum Avenue, Cardiff University, Cardiff, CF10 3US, UK. E-mail: [email protected]

adenomatous polyposis (FAP), a human syndrome characterized by the development of multiple polyps in the colon. In the mouse, genetic modelling of the involvement of the Wnt pathway was first made possible by the creation of the ApcMin/ þ mouse, which emerged out of an ethylnitrosourea mutagenesis programme and was subsequently characterized to carry a point mutation in the murine Apc gene (Su et al., 1992). The ApcMin/ þ mouse has proven to be a relatively good model of the human FAP syndrome, and has been used extensively to identify genetic and epigenetic modifiers of disease, as well as to assess therapeutic regimens such as exposure to a range of different chemopreventatives. In recent years this model has been joined by other Apc mutant mice, both constitutive and conditional, and also by a cohort of models targeting different elements of the Wnt pathway. These models have increasingly rationalized our understanding of the role played by the Wnt pathway in neoplasia, and are beginning to reveal the complex interplay that underlies normal intestinal physiology mechanisms as well as identifying putative novel disease markers and therapeutic targets. The ApcMin/ þ mouse Initially identified through the development of overt clinical symptoms of intestinal disease, the ApcMin/ þ mouse carries a mutation at codon 850 and develops multiple small intestinal adenomas, in addition to a smaller number of colonic polyps. The prevalence of small intestinal lesions represents one of the major differences between the human syndrome and the mouse model, the explanation for which remains unclear. The predisposition to intestinal polyposis also changes on different genetic backgrounds, a phenomenon which led to the identification of the first modifier of neoplasia, Mom-1 (Dietrich et al., 1993), subsequently identified as secretory phospholipase A2 (Pla2g2a, MacPhee et al., 1995). More recently other modifiers have been identified, including Mom-2 (Silverman et al., 2002) and a region on the distal arm of chromosome 18 (Haines et al., 2005). As a relatively accurate genetic model of human disease, the ApcMin/ þ mouse has been used extensively to characterize the response to potential therapeutic strategies, and a comprehensive list of the effects of 269 chemopreventative agents has been assembled at http://www.inra.fr/reseau-nacre/sci-memb/corpet/ indexan.html (Corpet and Pierre, 2003). The pertinent question arising from these studies is the extent to which

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these results relate to human disease. By virtue of the sheer number of studies performed in the ApcMin/ þ mouse, it is becoming possible to address this question, although there remain immense technical difficulties in direct mouse/man comparisons. It is therefore encouraging that a meta-analysis of chemopreventative agents used in mouse and human has shown relatively good correspondence for a number of agents, such as sulindac (Corpet and Pierre, 2005). Further encouragement that the ApcMin/ þ mouse is truly relevant to human disease derives from the observation that expression of the modifier Pla2g2a significantly correlates with survival for patients with gastric adenocarcinoma (Leung et al., 2002). Microarray studies also argue in favour of direct human relevance of the ApcMin/ þ mouse, with transcriptional profiles derived from the mouse successfully identifying novel gene targets in human colorectal tumours (Reichling et al., 2005). Somewhat fewer studies have been reported assessing genetic modification of the ApcMin phenotype, however this still represents a substantial and growing database of genetic backgrounds known to accelerate or suppress tumorigenesis. For example, deficiency of many DNA repair proteins accelerates polyp formation including the mismatch repair proteins (e.g. Reitmair et al., 1996), the thymidine glycosylase Mbd4 (Millar et al., 2002), and the base excision repair gene Myh (Sieber et al., 2004). In terms of the Wnt pathway, both heterozygosity and complete deficiency of the proposed Wnt target gene cyclin D1 have been shown to suppress adenoma formation, although adenomas do still develop in the absence of cyclin D1 (Wilding et al., 2002; Hulit et al., 2004). Indeed, the status of cyclin D1 as a Wnt target is somewhat debatable, as it appears not to be an immediate target of the Wnt pathway in vivo, becoming deregulated in a delayed manner in a subset of Wnt-activated cells (Sansom et al., 2005). For another proposed target, peroxisome proliferator-activated receptor (PPAR)-delta, the story is also rather unclear. In this case, both activation and inactivation of this pathway have been shown to accelerate intestinal tumour formation in the ApcMin/ þ mice, albeit to differing extents (Gupta et al., 2004, Harman et al., 2004, Reed et al., 2004). The interpretation here must be that activation of PPAR-delta accelerates neoplasia, but that this is not a relevant mechanism to normal adenoma formation in the ApcMin/ þ mouse. These rather complex results for both cyclin D1 and PPAR-delta underline the necessity of for validating proposed Wnt targets and Wnt-dependent mechanisms in an appropriate in vivo setting. Different Apc mutations: different phenotypes Analysis of the pattern of Apc mutations within human colorectal cancers has suggested a ‘just right’ model of activation of the b-catenin signalling cascade (Albuquerque et al., 2002). This interpretation is based on a comparison of the germline and somatic mutations in FAP patients, which reveals selection for a second hit consistent with the retention of some activity to

downregulation of b-catenin. The ‘just right’ model implies that different Apc mutations result in different levels of pathway activation and thereby differing degrees of tumour susceptibility. This strongly argues that not all Apc mutations are equivalent, a conclusion which also follows from the multifunctional nature of Apc, and which is supported by the phenotypes of mice bearing different mutations in Apc. Compared to the ApcMin/ þ mutation, mice bearing an earlier truncation (at codon 716) develop higher numbers of polyps (Oshima et al., 1995), whereas mice heterozygous for the later 1638N chain terminating mutation have an attenuated phenotype relative to the ApcMin/ þ mouse (Smits et al., 1997). The 1638N mice develop five to six adenomas of the small intestine during the first 6 months of life, and have also been reported to develop aberrant crypt foci in the colon that retain Apc protein, suggesting heterozygosity for this mutation is sufficient to initiate lesion development (Pretlow et al., 2003). Finally, mice have been constructed bearing hypomorphic alleles wherein expression of Apc is reduced to 20 and 10% of wild-type levels and polyp formation is reduced compared to the 716 mutation (Li et al., 2005). These hypomorphic alleles are characterized by levels of b-catenin expression that inversely correlate with Apc levels, and have been used to argue for a threshold level of Apc (15% of wild type) at which a single polyp will develop per mouse (Li et al., 2005). The primary consequences of inactivation of Apc: conditional models The development of the different constitutive Apc mutants has greatly advanced our understanding of the subtleties of altered Wnt signalling upon adenoma formation. However, these models have shed relatively little light on the precise mechanisms through which Wnt activation leads to adenoma initiation. This difficulty has been at least partially circumvented by the development of strategies to conditionally inactivate Apc within the adult intestinal epithelium. Two approaches have been used to achieve this, based on the use of a loxP-flanked Apc allele in conjunction with either an inducible Cyp1A-Cre transgene (Sansom et al., 2003) or a tamoxifen regulable, intestinal-specific Villin-CreER transgene (Andreu et al., 2005). The phenotypes reported by both systems are remarkably similar. Inactivation of Apc leads to the rapid relocalization of b-catenin to the nucleus and rapid changes in both the appearance of enterocytes and in the gross histology of the crypt. Cell movement within the crypt is completely blocked and there is also a failure of the differentiation programmes to mature goblet and enteroendocrine cells, together with mislocalization of Paneth cells. Coincident with the nuclear relocalization of b-catenin, many of the cells enter S-phase. Within a normal crypt, the location of the proliferating compartment is tightly controlled. However, following Apc loss, this position dependency is lost, with cells entering Sphase along the length of the perturbed crypt-villus axis. These studies establish that a single genetic event, mutation of Apc, is sufficient to confer a ‘crypt Oncogene


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progenitor’ phenotype, furthermore they indicate simple mechanisms, such as failed differentiation and failed cell movement, that can account for the conversion away from normal epithelium. It does however remain unclear if loss of Apc alone is sufficient for adenoma formation, or if there is subsequent selection for genetic or epigentic events to permit full adenoma development. Models carrying mutations in other Wnt pathway components The models of Apc mutation described above demonstrate the fundamental role of dysregulated Wnt signalling in adenoma initiation and begin to identify mechanisms through which this may occur. The corollary of this is that normal functioning of the Wnt pathway is predicted to be required for the normal physiological maintenance of the intestine. In support of this, overexpression of the Wnt inhibitor Dickkopf1 (Dkk-1) results in loss of proliferative cells in both foetal and adult intestine, coinciding with the loss of the crypt compartment (Pinto et al., 2003; Kuhnert et al., 2004). Expression profile analysis between the crypt and villus suggest that there are over a thousand differentially expressed genes, with Wnt signalling maximal in the proliferative compartment of the crypt (Mariadason et al., 2005). In situ hybridization studies have also shown a degree of compartmentalization of Wnt pathway components within the intestine (Gregorieff et al., 2005). Crypt epithelial cells show high expression of Wnt-3, Wnt-6, Wnt 9b, Frizzled 4, Frizzled 6, Frizzled 7 and Lrp5 sFrp5, whereas differentiated and mesenchymal cells showed stronger expression of Wnt2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4 and Frizzled 6. These observations imply contrasting roles for the different pathway components. Unfortunately, the majority of knockouts of these proteins have resulted in embryonic lethality and have therefore not been particularly informative. For example, inactivation of Lrp6 leads to death at birth with multiple developmental defects that are very similar to those seen in Wnt3a, Wnt7a and Wnt-1 mutants (Pinson et al., 2000). Mice null for Lrp5 are viable and exhibit only subtle defects in bone ossification with no reported intestinal phenotype (Gong et al., 2001; Kato et al., 2002). Combined mutation of both Lrp5 and Lrp6 has revealed critical compensatory roles for these genes in gastrulation, but their requirement in adult intestine remains unclear (Kelly et al., 2004). In a final example, Frizzled 4 null mice are viable but infertile (Hsieh et al., 2005), and again no intestinal phenotype has been reported. Turning to the internal components of the signalling pathway itself, inactivation of Axin has been show to result in early embryonic lethality (Chia and Costantini, 2005), and as yet there are no reports of conditional deletion of Axin in the mouse intestine. The Frat proteins had been proposed as potent activators of the Wnt pathway through inhibition of glycogen synthase kinase (GSK)3 activity. However, mice null for all three Frat homologues have been generated and surprisingly they show no gross abnormalities and no defects in b-catenin/T-cell factor (TCF) Oncogene

signalling (van Amerongen et al., 2005). Therefore, in terms of many of the components of the Wnt pathway, much remains to be established in relation to intestinal development and disease. However, the roles of several key mediators of the Wnt pathway have begun to be elucidated. These include b-catenin, Tcf-4 and c-Myc, although as can be seen from the discussion below, the role of the latter protein remains somewhat contentious. b-Catenin acts both as a downstream component of the Wnt signal transduction pathway and as a mediator of cell adhesion through its interaction with the cadherins. b-Catenin activity is regulated along the length of the crypt, as reflected by the pattern of nuclear accumulation of b-catenin, which decreases in a gradient from the base of the crypt to the interface with the villus (Batlle et al., 2002; van de Wetering et al., 2002). Inactivation of Apc results in the rapid relocalization of b-catenin to the nucleus and the subsequent transcriptional activation of downstream targets. Wong et al. (1998) used a chimaeric strategy to express a human NH2 terminal truncation mutant (DeltaN89) which phenocopies activation of the Wnt pathway in Xenopus and Drosophila. Expression of this construct mimics some of the phenotypes observed following conditional loss of Apc (Sansom et al., 2004, Andreu et al., 2005), but it does not replicate them entirely and critically did not lead to neoplastic transformation in the 10-month period of study. A more potent phenotype was observed by Harada et al. (1999), using mice bearing a mutant b-catenin allele whose exon 3 was flanked by loxP sequences. Intestinal-specific cre-mediated recombination of this allele leads to stabilization of b-catenin and subsequent polyp development. These experiments establish similarity in phenotype between the effects of Apc deletion and b-catenin stabilization; however the precise nature of the overlap remains to be established. If these two mutations can be shown to be phenotypically equivalent, this argues that the neoplastic predisposition following Apc loss is almost entirely mediated through b-catenin and is not strongly influenced by other proposed Apc functions (Kaplan et al., 2001). The converse experiments have also been performed, namely to establish the requirement for b-catenin in normal intestinal physiology. b-Catenin complexes with Tcf-4 in response to Wnt signalling and activates transcription of Wnt target genes. Tcf-4 complexes with b-catenin in response to Wnt signalling and activates transcription of Wnt target genes. Korinek et al. (1998) showed in 1998 that mice deficient in the gene encoding Tcf4 (Tcf7/2) show a histopathological abnormality – namely failure of the crypt to proliferate even although apparently normal transition of intestinal endoderm into epithelium occurs at E14.5. Mice lacking Tcf-4 have a depleted epithelial stem-cell compartment, implying b-catenin/Tcf-4 signalling is required for crypt maintenance. Conditional inactivation of b-catenin supports this, with deficient cells not tolerated in the intestine, becoming rapidly replaced by wild-type cells (Ireland et al., 2004). c-Myc has been proposed as a critical mediator of Wnt signalling downstream of b-catenin/Tcf-4, and one


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might therefore also predict perturbation of the crypt following loss of c-Myc. However, Bettess et al. (2005) have used a tamoxifen-dependent conditional strategy to show that there is remarkably little requirement for c-Myc within the normal epithelium. Deletion of c-Myc during development does lead to the loss of a proportion of crypts, but c-Myc deficiency has apparently no effect upon normal adult intestinal physiology. These results are in contrast to a parallel study (Muncan et al., 2006) that again uses a conditional strategy to delete c-Myc from the adult intestine. In this latter study, deletion of c-Myc leads to the death of the crypt over 14 days, with enterocytes metabolically compromised before this point. These studies therefore differ markedly in the severity of c-Myc deficiency. The reason for this discrepancy is currently unclear, but must lie either in differences in the targeted c-Myc alleles or in the pattern of cre-mediated deletion. Whatever the basis of the difference, it is clear that the consequences of c-Myc deficiency are not as severe as those following b-catenin loss. Wnt pathway targets Comprehensive lists of putative Wnt target genes have been developed by several different groups (e.g. Batlle et al., 2002; Sansom et al., 2004), and these lists have aided in the validation of a genome wide predictor of Wnt target genes (Hallikas et al., 2006). As well as revealing a plethora of genes implicated in the Wnt response, these lists are also helping to refine our understanding of the mechanisms underlying tumour initiation and suppression. Some these genes have now been validated in vivo including the homeobox (HMG)box transcription factor Sox9 (Blache et al., 2004) and the HMG gene Cdx-1 (Lickert et al., 2000). Expression of the imprinted gene Mash2/Ascl2 is immediately induced following loss of Apc (Sansom et al., 2004), and Jubb et al. (2006) have confirmed Mash2 as a Wnt target in tumour cell lines by showing downregulation following transfection with constructs carrying a dominant negative Lef, the APC homologues APC2, or short-interfering RNA against b-catenin. This study also confirmed upregulation of Mash2 in both human colorectal adenocarcinomas and in mouse adenomas. Tiam1 is a selective Rac GTPase activator, that is expressed in the base of intestinal crypts and has been shown to be upregulated in both human and mouse intestinal tumours. Tiam1 null mice have been used to show that deficiency of this target gene significantly reduces polyp formation in the ApcMin/ þ mouse, but enhances invasion (Malliri et al., 2006). These studies suggest an immediate requirement for Tiam1 in adenoma formation that presumably must subsequently be overcome to permit progression to adenocarcinoma. Activation of the Wnt pathway has also been reported to upregulate a negative feed back loop in both human colorectal and liver tumours mediated by, among others, Axin2/conductin (Lustig et al., 2002). The existence of such a loop argues that the Wnt pathway is maintained at a finely controlled level in physiologically normal

tissue, a scenario reminiscent of the ‘just right’ notion for tumorigenesis. In support of this, an ability to switch off Wnt signalling has been shown to be critical to normal gastrointestinal specification. Thus, expression of the HMG gene Barx1 is confined to the stomach mesenchyme, where it directs expression of the two secreted Wnt antagonists sFRP1 and sFRP2. In Barx1 null mice, Wnt signalling is not inhibited resulting in visceral homeosis, with disorganized gastric epithelium and expression of intestinal genes (Kim et al., 2005a). More Wnt pathway targets: the Eph/Ephrins Roles in patterning the crypt-villus axis have been demonstrated for other Wnt target genes, such as those belonging to the EphB/Ephrin-B family. The Eph proteins are receptor tyrosine kinases which mediate bi-directional signalling through their membrane bound ligands, the ephrins. In the intestine, the normal expression profile is a gradient of decreasing Eph expression from the base of the crypt upwards, with an inverse gradient of Ephrin expression from the villus downwards. The pattern has been shown to be Wnt/ b-catenin dependent, with EphB2 and EphB3 direct targets of the pathway (Batlle et al., 2002, Sansom et al., 2004). Combined disruption of EphB2 and EphB3 leads to the intermingling of the proliferative and differentiated cell populations. EphB3-deficient mice show perturbed Paneth cells positioning, with cells no longer migrating downwards, instead scattering along the cryptvillus axis (Batlle et al., 2002). More recently, Holmberg et al. (2006) have extended these findings to indicate that the Eph/Ephrins also influence proliferative capacity, with approximately half the mitogenic activity of both the small and large intestine regulated by EphB signalling. Perturbation of Eph/Ephrin signalling has also been reported in Foxl-1 null mice. Foxl-1 is a winged helix transcription factor normally expressed in the mesenchyme. Constitutive inactivation of Foxl-1 leads to the mesenchymal induction of a subset of Wnt family genes and the generation of aberrant small intestinal crypts, characterized by widely distributed Paneth cells and ectopic, increased expression of both EphB2 and EphB3 (Takano-Maruyama et al., 2006). Foxl-1 has therefore been suggested to mediate a mesenchymal–epithelial interaction that is essential for normal Wnt regulation and subsequent Eph/Ephrin activity. Accordingly, deficiency of Foxl-1 leads to a marked increase in tumour multiplicity in the colon of ApcMin/ þ mice and the development of gastric tumours (Perreault et al., 2005). Somewhat similar epithelial–mesenchymal interdependency has been demonstrated for the mesenchymal forkhead transcription factors Foxf1 and Foxf2 (Ormestad et al., 2006). Inactivation of both Foxf2 alleles, or a single allele each of Foxf1 and Foxf2, results in multiple intestinal defects. These lesions are characterized by elevated mesenchymal Wnt5a expression and ultimately lead to epithelial cell depolarization and tissue disintegration. These observations have clear implications for tumorigenesis in the intestine, and it was therefore perhaps Oncogene


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predictable that modifying EphB expression would influence adenoma formation in the ApcMin/ þ mouse (Batlle et al., 2005). However, given that the EphB proteins are Wnt targets and that they positively regulate proliferation, it is surprising that loss of EphB activity should accelerate tumorigenesis, resulting in the formation of aggressive adenocarcinomas. This apparent contradiction may arise out of a need to overcome some of the phenotypes associated with immediate activation of the Wnt pathway. For example, loss of Apc completely blocks cell movement in the crypt. Such a block must clearly be overcome for an adenoma to progress to invasion. In a scenario similar to that observed for Tiam-1, this implies a two-stage process, with deregulation of the Wnt pathway providing a necessary platform from which adenomas/adenocarcinomas can only develop by refinement of the immediate Wnt phenotype. Further support for this notion derives from measurements of cell death following Apc loss, with an immediate and potent induction of cell death contrasting with the lower levels observed in aberrant crypt foci and adenomas (Sansom et al., submitted). This again argues for a strong selection process, refining the Wnt phenotype to permit adenoma formation. Wnt signalling and intestinal stem cell fate Several different pathways have been implicated in the control of pluripotency in mouse embryonic stem (ES) cells. Leukaemia inhibitory factor (LIF) was the first of these factors, mediating self-renewal through the janus kinase-signal transducer and activator of transcription (Jak-Stat) pathway. A role has also been established for the Wnt pathway, with activation of the pathway following exposure to the GSK3 inhibitor 6-bromoindirubin-30 -oxime sufficient to maintain self-renewal of both human and mouse ES cells (Sato et al., 2004). Similarly Wnt3a, notably in conjunction with LIF, has been shown to support self-renewal (Ogawa et al., 2006; Singla et al., 2006). There also appears to be a dosedependency in Wnt mediated maintenance of selfrenewal, as Kielman et al. (2002) have used various Apc and b-catenin mutations to demonstrate increasing inhibition of differentiation with increasing doses of b-catenin. Hao et al. (2006) have also shown that the STO (S, SIM; T, 6-thioguanine resistant; O, ovabain resistant) feeder cells, used to maintain ES cells in an undifferentiated state, produce both Wnt5a and Wnt6, and that activation of the Wnt pathway in ES cells upregulates the messenger RNA (mRNA) for Stat3, data that again supports convergence of the Wnt and Jak/Stat pathways on ES cell self-renewal. The above experiments establish a Wnt dependency to the most fundamental stem cell, the ES cell, and raise the possibility that the Wnt programme also directly regulates the stem cell compartments within somatic tissues, such as the crypts of lieberkuhn in the intestine. Several studies have been undertaken using laser capture dissection in conjunction with microarray analysis to define the molecular profile of the stem cell niche. These studies have become increasingly refined, dissecting out Oncogene

populations enriched for the intestinal stem cells and epithelial lineage progenitors, both within normal crypts and crypts depleted for Paneth cells (Mills et al., 2002; Stappenbeck et al., 2003, Giannakis et al., 2006). These studies identified, respectively, 147, 163 and 3324 enriched transcripts in the niche. These profiles showed similarities to those that define mouse haematopoietic stem cells and were enriched for growth factor response pathways, such as insulin-like growth factor-1, and proteins required for mRNA processing and cytoplasmic localization. Critically for the discussion here, these studies also identified genes involved in the Wnt signalling cascade. One remarkable link has recently been reported between the mechanisms governing ES cell and intestinal stem cell maintenance. Ectopic, intestinal expression of the POU-domain transcription factor Oct-4, normally required for maintenance of ES cell pluripotency, results in dysplasia and an expansion of crypt progenitor cells (Hochedlinger et al., 2005). This phenotype is highly comparable with that obtained following conditional loss of Apc (Sansom et al., 2004), implying that the Wnt pathway plays a direct role in maintaining the intestinal stem cell population. The observed increase in b-catenin following ectopic Oct4 expression suggests that the Wnt pathway is activated downstream of Oct4, although this remains to be fully established. Although parallel intestinal overexpression studies have not yet been reported for Stat3, this regulator of ES self-renewal has also been implicated in colorectal cancer, with persistent STAT3 activation associated with cell proliferation and tumour growth (Corvinus et al., 2005). Somewhat controversial data has been generated from experiments using B6o>129/Sv chimaeric mice that bear a Lef-1/b-catenin fusion construct in the 129/ Sv component. These have suggested that enhanced b-catenin signalling is lethal to the intestinal stem cell population (Wong et al., 2002). Surprisingly, no changes in cell division were observed, but the 129/Sv cells were characterized by apoptosis and were lost by the time crypt development was completed. These experiments contradict our own observations following conditional loss of Apc, where we see coincident elevation of both apoptosis and proliferation and ultimately retention of Apc-deficient cells in both the adult and the embryonic intestine (Sansom et al., 2004 and unpublished). These differences presumably reflect differences in the level of activation of the Wnt pathway, although they may also reflect a more wide-ranging role for Apc. As discussed above, Tcf-4 maintains the epithelial stem-cell compartment (Korinek et al., 1998). More recently van Es et al. (2005a) have shown that expression of a Paneth cell gene programme is also dependent on Tcf-4 in the embryonic intestine and upon Frizzled-5 in the adult intestine. Tcf-1-deficient mice do not display an overt intestinal phenotype, instead showing an incomplete arrest in T lymphocyte differentiation. However there is clear redundancy operating among Wnt pathway members, as Tcf4/Tcf1/ mice show severe hindgut phenotypes (Gregorieff et al., 2004), and Lef1/Tcf1/ mice develop a severe


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developmental phenotype that resembles the phenotype of Wnt3a/ mice (Galceran et al., 1999). Based on the above, it is clear that Wnt signalling is involved in regulating intestinal homeostasis and that although deregulation of the Wnt programme can have diverse effects upon stem cell fate, the predominant effect of activation of the Wnt pathway is to impose a ‘crypt – progenitor’ phenotype. The challenge emerging from these observations is to understand the subtleties of Wnt dependency within the stem cell compartment, which if successful should open the way to tissue engineering through the manipulation of this compartment. Epigenetics and the Wnt pathway It is increasingly clear that tumorigenesis mediated through the Wnt pathway is modulated at an epigenetic level. For example, loss of imprinting at the Igf2 locus results in a doubling of the adenoma burden in ApcMin/ þ mice (Sakatani et al., 2005). In terms of understanding such epigenetic control, most attention has been placed on analysing the genes responsible for imposing and maintaining epigenetic control. Deficiency of Dnmt1, the predominant DNA methyltransferase, results in early embryonic lethality. However, viable, hypomorphic alleles have been generated which dramatically suppress polyp formation in both the ApcMin/ þ mouse and in mice lacking DNA mismatch repair (Eads et al., 2002; Trinh et al., 2002). Surprisingly, this effect appears tissue-specific, as Dnmt1 hypomorphism has also been reported to accelerate liver tumour development in ApcMin/ þ mice, possibly by promoting loss of heterozygosity (Yamada et al., 2005). The methyl binding domain proteins also impact upon the transcriptome, as they interpret methylation signals and subsequently recruit chromatin-modifying complexes. Deficiency of one member of this family, Mbd2, has been shown to strongly suppress polyp formation in the ApcMin/ þ mouse (Sansom et al., 2003). Similar data has also been obtained for Kaiso, which binds both a sequence-specific Kaiso site, and also methylated cytosine-phosphateguanosine dinucleotides (Prokhortchouk et al., 2006). Furthermore, Kaiso mediates repression of the Wnt target gene matrilysin (Spring et al., 2005). In a somewhat similar vein, the transcriptional repressor HDAC2 has been shown to be upregulated following loss of Apc and interference with HDAC2 activity by valproic acid has been shown to reduce adenoma burden in the ApcMin/ þ mouse (Zhu et al., 2004). The precise mechanism by which these genes modulate tumorigenesis remains largely unclear. The most attractive hypothesis is that the transcription repression mediated by these proteins is ‘hijacked’ by the developing lesion to inactivate critical defensive genes. In support of this general concept, attenuated Wnt signalling has been reported following the epigenetic inactivation of the secreted frizzled-related proteins (Suzuki et al., 2004). However, it remains unclear if these are key targets of Dnmt1, Mbd2, Kaiso or HDAC2. Regardless of mechanism, these genes present themselves as

potential new therapeutic targets, especially where constitutive knockouts have demonstrated either no phenotype or a weak deleterious phenotype. Progression: the adenoma–adenocarcinoma transition FAP patients are characterized by the development of many thousands of colonic polyps, and the likelihood of progression to adenocarcinoma and carcinoma for each individual polyp is relatively low. A somewhat similar scenario is true for the ApcMin/ þ mouse, although the total adenoma burden is much reduced. Given that adenoma burden curtails the lifespan of the ApcMin/ þ mouse, this has made it difficult to study progression using this model. Genetically this problem has begun to be addressed by analysis of candidate gene loci, including the tumour suppressors Smad 2 and Smad4/ Dpc4, the beta-catenin interactor E-cadherin and oncogenic mutations of Ki-Ras. Heterozygosity for Smad2 has been reported to drive progression in Apc heterozygotes, with the development of multiple invasive cancers in the compound heterozygotes (Hamamoto et al., 2002). Smad4 þ / mice develop polyposis in the fundus and antrum at 6–12 months of age, with subsequent polyp development in both the duodenum and caecum. These polyps progress to both carcinoma in situ and ultimately invasion (Xu et al., 2000). This process appears to be driven by haploinsufficiency, although loss of remaining wild-type allele has been reported to occur variously in polyps or to be confined to more progressed lesions. (Takaku et al., 1999; Xu et al., 2000; Alberici et al., 2006). In the context of an Apc mutation, haploinsufficiency of Smad4 drives both adenoma formation and progression (Alberici et al., 2006). E-cadherin competes with Apc for the binding of b-catenin and so may directly influence the Wnt pathway. It may also affect tumorigenesis by altering cell adhesion and associated functions. Downregulation of E-cadherin has been implicated in the progression of a range of human tumours, including those of the intestine (Dorudi et al., 1993). Despite this association, results obtained from an Apc þ / 1638NE-cad þ / intercross have shown reduced E-cadherin levels to drive tumour initiation rather than progression (Smits et al., 2000). Mice transgenic for the most frequent oncogenic mutation of K-Ras (V12G) under control of the Villin promoter have been shown to spontaneously develop intestinal lesions ranging form aberrant crypt foci to invasive carcinomas. Surprisingly, these lesions show no mutation in Apc, but a proportion of lesions did show mutation of p53 (Janssen et al., 2002). Contrasting results have been reported for an endogenously targeted K-Ras (V12) allele (Guerra et al., 2003), expression of which does not perturb normal crypt villus architecture, and only leads to malignant transformation in a proportion of lung bronchiolo-alveolar cells. This difference is presumed to derive from differences in the nature of the mutations, in particular in differences in expression levels of the mutant K-ras alleles. The allele Oncogene


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used by Guerra et al. (2003) has now been crossed into a conditional Apc mutant background, where it has been shown not to perturb the immediate phenotype of Apc loss, but to accelerate both incidence and invasion in a heterozygous Apc context. (Sansom et al., 2006) It has also been argued that elements of the Wnt pathway directly drive genomic instability, which will also clearly impact upon progression. There are now several reports of increased genomic instability following aberrant Wnt signalling, suggested variously to occur through upregulation of conductin/Axin2 and subsequent compromise of the spindle checkpoint (Hadjihannas et al., 2006), or through deficiencies in microtubule/kinetochore interactions (Fodde et al., 2001; Kaplan et al., 2001; Rao et al., 2005). Pathway interactions Despite the paramount importance of the Wnt pathway in controlling intestinal physiology, it clearly interacts with many other pathways and cannot be considered in isolation. In humans, mutation of the BMP pathway component BMPR1A is associated with juvenile polyposis. He et al. (2004) have used Bmpr1a knockout mice to show a fundamental role for the BMP pathway in intestinal homeostasis that is mediated through suppression of Wnt signalling. Furthermore, Inhibition of BMP signalling by transgenic expression of noggin leads to the formation of numerous ectopic crypt units perpendicular to the crypt-villus axis. Haramis et al. (2004). The Hedgehog pathway has also been shown to be critical to intestinal development, with both Sonic and Indian Hedgehog expressed throughout the morphogenetic events that mark development of the small intestinal mucosa. Transgenic mice expressing the panHedgehog inhibitor Hhip show compromised epithelial remodelling and villus formation by driving signalling within subepithelial myofibroblasts and smooth muscle cells. This has a secondary effect upon the epithelium, enhancing Wnt activity and leading to increased proliferation and the development of ectopic precrypt structures on the villus tips. The interaction with the Wnt pathway is further underlined by data from van den Brink et al. (2004), who show Hedgehog signalling to restrict expression of Wnt targets to the base of the crypt and identify Indian Hedgehog as a negative regulator of the Wnt pathway (Madison et al., 2005). The Wnt pathway also cross-talks with the Notch pathway. Inactivation of the Notch pathway by either conditional deletion of the pathway transcription factor CSL/RBP-J or the use of the gamma-secretase inhibitors results in rapid conversion of proliferative cells

into post-mitotic goblet cells (Milano et al., 2004; van Es et al., 2005b). Interestingly, a rather similar perturbation has been reported for the conditional expression of an activated Notch mutant, which again results in an increase in goblet cell numbers (Zecchini et al., 2005). These data suggest the level of Notch signalling to be critical to epithelial cell fate, and that maintenance of undifferentiated, proliferative cells requires concerted action by both the Wnt and Notch pathways. Beyond these critical pathways, many other elements have been shown to modify Wnt signalling in the intestine and so modify either morphology or tumour predisposition. Examples of these molecules include Kruppel like factor 4, which interacts with and represses b-catenin mediated gene expression (Zhang et al., 2006); and the R-Spondin proteins, potent and specific mitogens for the intestinal epithelium that at least partially mediate their activity through stabilized b-catenin. (Kim et al., 2005b). A direct Wnt interaction has also been demonstrated for the proto-oncogene and AP-1 component c-Jun, which forms a ternary complex with Tcf4 and b-catenin to drive target gene transcription. Consistent with this, conditional inactivation of c-Jun reduces adenoma burden in the ApcMin/ þ mouse (Nateri et al., 2005).

Summary Since the earliest association with human FAP and the development of the ApcMin/ þ mouse, the Wnt pathway has been associated with intestinal neoplasia in both the human and mouse. Since its development, this model has proven particularly powerful in validating potential chemopreventative regimens and in determining genetic and epigenetic factors that enhance or decrease predisposition. The development of increasingly sophisticated Apc mutants, together with the use of mice bearing mutations in other component of the Wnt pathway has accelerated this process, and has begun to shed more light on the basic mechanisms of neoplasia. These models are now delivering a plethora of potential new therapeutic targets, each of which requires its own careful validation. These approaches have also begun to reveal a more fundamental role for the Wnt pathway in the normal physiology of the intestine, where it determines cell fate in interplay with the Notch, Hedgehog and BMP pathways. Nowhere is this more critical than its emerging role in controlling stem cell fate in the intestinal crypt, which is opening the possibility of manipulating the Wnt pathway to engineer the intestine.

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