The many ways of Wnt in cancer

The many ways of Wnt in cancer Paul Polakis

More than20years ago,the oncogenicity of a Wnt ligand was revealed in a series of experiments originating with random proviral integration in mice.The signi?cance of Wnt signaling in human cancer has since been buttressed by the identi?cation of mutations in genes coding for the Wnt pathway components Axin,APC,and b-catenin.This review summarizes the reported genetic defects in the Wnt pathway,with an emphasis on their functional contribution to human tumor progression.

Addresses

Department of Research,Genentech Inc.,1DNA Way,South San Francisco,CA94080,USA

Corresponding author:Polakis,Paul(ppolakis@https://www.360docs.net/doc/a213016890.html,)

Current Opinion in Genetics&Development2007,17:45–51

This review comes from a themed issue on

Genetic and cellular mechanisms of oncogenesis

Edited by Sara A Courtneidge and Benjamin G Neel

0959-437X/$–see front matter

#2006Elsevier Ltd.All rights reserved.

DOI10.1016/j.gde.2006.12.007

Introduction

Mutations in the Adenomatous polyposis coli(APC)gene were identi?ed as the basis for familial adenomatous polyposis coli(FAP),a heritable predisposition to color-ectal cancer[1,2].Mutations in APC were subsequently identi?ed in the majority of sporadic colorectal tumors, where they appear early in the progression to cancer.The ?nding that wild type APC facilitated the destabilization of the b-catenin protein,whereas Wnt had the opposing effect,positioned APC as a negative regulator of Wnt signaling[3,4].The oncogenic relationship between b-catenin and APC was further forti?ed by the discovery of mutations in b-catenin that prevented its regulation by APC[5–7].Axin was later revealed to be an additional member of the b-catenin regulatory complex,and it too was found to be mutated in human cancers[8,9].Thus, the foundation of a new human cancer pathway was established,complete with extracellular ligands,cell sur-face receptors,intracellular transducers,and transcription factors that activate and repress genes that contribute to neoplasia(Figure1).

Pathway activation involves interaction of a Wnt ligand with a seven-transmembrane Frizzled molecule and an LRP5or LRP6co-receptor.The ensuing phosphorylation of the LRP co-receptor generates direct binding site for Axin,which is thereby recruited to the plasma membrane and ultimately degraded.The APC–Axin complex is disrupted along with its ability to mark b-catenin for destruction through its phosphorylation and targeting to the proteosome.The stabilized b-catenin interacts with the T-cell factor(TCF)and lymphoid enhancer factor(LEF)transcription factors in a manner that dis-places genetic repressors and recruits coactivators.Acti-vation of Frizzled and its co-receptors LRP5and LRP6by Wnt ligands leads to inhibition of the b-catenin regulatory complex by what remains an arcane mechanism.There are many ways for cancer to co-opt this program,wresting control from extracellular cues and recklessly driving the expression of genes governing growth,survival and cell fate determination.Here,I examine individual com-ponents of the Wnt pathway with respect to the genetic defects that affect them.

APC

Mutations in the APC gene typically truncate the protein in a manner consistent with selection against negative regulation of b-catenin[10].Also apparent from this mutational spectrum is a positive selection for the reten-tion of an APC fragment that extends about half way through the reading frame into the mutational cluster region(MCR)[11,12].The advantage conferred by these partial APC products remains unclear.Interference with wild type APC seems unlikely,because MCR mutations are frequently selected as a second hit in tumors that already harbor severely truncating germline APC mutations.Moreover,a recent functional test for domi-nant interfering activity of APC MCR fragments in Dro-sophila demonstrated that they had no effect on canonical Wnt signaling[13 ].A feature common to the APC MCR mutants is the retention of the armadillo repeat sequences that bind Asef(APC-stimulated guanine exchange fac-tor),an exchange factor for the Rac GTPase.Rac acti-vation,which has been linked to transformation,could drive positive selection for APC fragments if their activity were enhanced relative to that of wild type APC.Such evidence has been provided in a study showing that APC MCR was more potent than wild type APC in activating Asef,which in turn impacted cell migration and adhesion [14].This is probably not the whole story,though, because the truncated APC mutants selected for in cancer extend beyond the arm repeats into the b-catenin binding sites region.An alternative hypothesis relates to the retention of partial b-catenin regulatory activity by APC MCR fragments.This was in fact observed in the aforementioned?y study in which MCR-like mutants were expressed in an otherwise APC-null background

[13].Therefore,the curious forward selection for an APC MCR mutant in human cancer might result from a necessity to retain some level of b -catenin regulation.This was the premise for the ‘just-right’hypothesis,which posits that too much b -catenin activity might be detrimental to the developing cancer cell [15].

b -catenin

Although the APC gene is mutated in the majority of sporadic colorectal cancers,this rarely occurs in any other cancers originating outside of the gastrointestinal tract.The discovery of APC mutations in sporadic lung,ovarian and breast cancers,as well as extracolonic tumors associ-ated with FAP,attests to their oncogenic ability in these tissues [16–19],but their rarity suggests that they are not a preferred genetic route to cancer.Mutations in b -catenin that abrogate its regulation by APC represent an alternative route to Wnt activation and do occur more commonly in sporadic cancers outside of the gut.These mutations were ?rst identi?ed in sporadic colorectal cancers [5,6]and in melanoma [7],but subsequent sequencing efforts indi-cated that they occur quite infrequently in these cancers [20,21 ,22 ,23].A recent analysis found only two b -catenin mutations in 216unselected colorectal cancers,whereas another study reported only ?ve in 464colorectal cancers [22 ,23].Despite previous claims associating b -catenin mutations with microsatellite instability (MSI+),neither study corroborated this ?nding.A separate analysis failed to detect any b -catenin mutations in 112sporadic colorectal cancers,of which 34were MSI+[21 ].In this same study,

however,eight of 44colorectal cancers from hereditary nonpolyposis colorectal cancer (HNPCC)patients con-tained b -catenin mutations.HNPCC patients carry germ-line mutations in DNA replication repair genes,such as MSH2(MutS homolog 2)and MLH1(MutL homolog 1),and consequently present with MSI+tumors [24].These data indicate that mutations in b -catenin are more frequently associated with MSI+colorectal tumors,but perhaps only in the context of HNPCC.

Mutations in b -catenin are particularly common in endo-metrioid ovarian cancer,although their prevalence ranges widely (16–54%)across the various studies [25].It is now appreciated that the occurrence of these mutations is a function of endometrioid ovarian cancer staging,whereby they are more frequent in low-grade,highly differentiated endometrioid ovarian cancers and occur in nearly all early precursor lesions referred to as borderline endometrioid tumors [26].The endometrioid ovarian cancers with b -catenin mutations are associated with a favorable prog-nosis and might have reduced capacity to metastasize.Synchronous endometrioid carcinomas in the uterine cor-pus and ovary are thought to arise from either independent primary tumors or metastasis originating from a single cancer.In a genetic survey of these synchronous carci-nomas,b -catenin mutations were strongly associated with primary independent tumors and not found in any meta-static tumors [27 ].This suggests that the metastatic endometrioid cancers arise through a distinct genetic route,perhaps incompatible with mutations in b -catenin .

46Genetic and cellular mechanisms of oncogenesis

Figure

1

Representation of the Wnt signaling pathway.Negative regulators are depicted as red,positive regulators as green and those with uncertain impact yellow.

Two pediatric cancers,hepatoblastoma and Wilms’kid-ney tumor,frequently contain mutations in b-catenin,and gene expression markers were found to correlate with their presence.The Axin2gene is a direct target of Wnt signaling[28,29],and its expression in hepatoblastoma was associated with b-catenin mutations[28–30].Sim-ilarly,the GAD1(Glutamic acid decarboxylase1)transcript was mined out as a marker of Wilms’kidney tumors that contained defects in the WT-1(Wilm’s tumour1)gene[31]. The strong link between WT-1and b-catenin mutations led the authors to identify GAD1as a Wnt target that could be employed,along with other markers,to predict Wilms’tumors containing b-catenin mutations.Accordingly,some novel mutations in b-catenin were identi?ed in this study by analysis of GAD1-positive specimens.

Axin

The?delity of Axin2as a marker for Wnt signaling probably relates to its function as a dedicated negative regulator of the Wnt pathway.Accordingly,both Axin1 and Axin2are mutated in a variety of human cancers[32]. Clearly,the polypeptide chain-terminating mutations that ablate essential structure are inactivating,because elimination of just the DIX domain from the extreme C-terminus is suf?cient to cripple Axin1function[33].In the absence of experimental support,it is more dif?cult to assess the functional impact of the myriad reported single amino acid substitutions.Indeed,one of these substi-tutions,L396M,appears to be inactivating,because it was shown to prevent the binding of glycogen synthase kinase 3(GSK3)[34].Additional missense variants in the Axin1 gene have been detected in the germline of patients with multiple adenomatous polyps.Some of these variants were also found in unaffected individuals,but their increased frequency in cancer patients suggests that they contribute to a multifactorial inherited susceptibility to colorectal cancer[35].

An intriguing facet of the Axin2mutations is their pre-sence in tumors also containing either APC or b-catenin mutations.In colorectal cancer,Axin2mutations are mainly restricted to MSI+cancers,in which polynucleo-tide tracts in exon7are a common site for mutation[23]. Seven of the35MSI+cancers contained Axin2-truncating mutations,and four of these had concurrent mutations in APC.This argues against there being functionally equiv-alent mutations in APC and Axin2,and points to further selection for increased Wnt signaling even when APC is mutated.As noted above,some truncated APC mutants are only partially compromised in their activity[13]. Thus,the compensatory upregulation of Axin2,a tran-scriptional target of Wnt signaling,could present an additional locus for stepwise selection aimed at further upregulating Wnt activity.Accordingly,in three of the tumors with concurrent mutations,APC was truncated at codon1554,a mutant previously found to retain partial b-catenin regulatory activity[36].The association between Axin2and cancer was further strengthened by the identi?cation of truncating germline mutations that strongly predispose affected individuals to colorectal neoplasia[37].Interestingly,this was a serendipitous ?nding in a positional cloning effort intended to localize defective genes responsible for familial tooth agenesis. T-cell factor4

Although the Axins,APC and b-catenin are now widely recognized as the central culprits in the genetic activation of Wnt signaling,mutations in additional pathway com-ponents have also been reported.TCF4,one of the b-catenin binding transcription factors,is mutated in nearly half of the MSI+colorectal cancers[38–40].Con-sistent with the mutator mechanism characteristic of MSI+cancers,the TCF4mutations are all frameshift alterations targeting a polyA tract in the?nal exon. The functional consequences of these mutations remain controversial,but it was recently proposed that they eliminate the coding of TCF4isoforms capable of bind-ing the transcriptional repressor carboxy-terminal binding protein(CtBP)[41 ].

sFRPs

Additional but rare cases of genetic activation in the Wnt pathway include inactivating mutations in the secreted Frizzled-related protein sFRP1,which inhibits receptor activation by competitively binding to Wnt proteins.An analysis of10advanced colorectal cancers,selected on the basis of genomic interstitial deletions in the sFRP1 region,yielded three truncating mutations in exon1of the remaining allele[42].The nature of these mutations would be expected to ablate the inhibitory activity of sFRP1.The authors concluded that sFRP1mutations were rare,however,because no more were found upon examination of an additional51locally advanced color-ectal tumors.

Two conundrums arise when considering sFRP1as a tumor suppressor in colorectal cancer.First,most colorectal can-cers lack functional APC,and,in a linear view of the pathway,receptor activation would appear ineffective or redundant in this background.Second,secreted sFRP1 should act in both an autocrine and a paracrine manner, making it dif?cult to envision the growth advantage of a founder mutant cell.Resolution of the?rst conundrum might again relate to the residual activity of the mutant APC,hence allowing for further positive selection through receptor activation.Alternatively,it is conceivable that a mutation in sFRP1precedes that in APC,positive selection ensues,and then the sFRP1mutation remains vestigial thereafter.The second conundrum could be resolved if sFRP1had a signi?cantly stronger autocrine suppressive effect relative to its inhibition of neighboring cells.Finally, a?eld effect in which numerous neighboring cells in a tissue simultaneously lose sFRP1function could establish grounds for clonal expansion of mutants.This seems an

The many ways of Wnt in cancer Polakis47

unlikely scenario for genetic inactivation but could apply to epigenetic gene silencing of sFRP1.

Glycogen synthase kinase3

Glycogen synthase kinase3(GSK3)is a well-established negative regulator of Wnt signaling,yet mutations have never been identi?ed in cancer.Recent knockout studies from the Woodgett laboratory have demonstrated that constitutive activation of Wnt signaling,in the resulting mouse embryonic?broblasts,requires inactivation of both alleles of GSK3a and GSK3b(JR Woodgett,personal communication).The odds of this occurring spon-taneously in a single cell seem quite remote.Moreover, GSK3is essential to many cellular homeostatic functions apart from Wnt signaling,such as insulin signaling[43]. Finally,it was recently shown that GSK3b might also act in a positive manner in the Wnt pathway through phos-phorylation of the Wnt co-receptors LRP5and LRP6(low density lipoprotein receptor-related proteins5and6) [44 ].

ICAT

ICAT,a molecule that binds directly to b-catenin and thereby displaces the TCF and LEF transcription factors and the co-activator CBP,could also qualify as a potential tumor suppressor.A mutation that altered the ICAT initiation codon was identi?ed in1of37melanomas [45],a cancer in which ICAT is frequently downregu-lated.By contrast,no mutations where detected in a separate analysis of178melanomas[46].

PP2A

PP2A is family of heterotrimeric enzymes with phospha-tase activity.The PP2A phosphatase complex is another component of the Wnt pathway that is complicated by its participation in numerous other signaling processes.The PP2A regulatory subunit B56binds to APC,and the catalytic subunit binds to Axin[47,48],but whether PP2A is a positive or negative regulator of Wnt depends on the experiment and the interpretation thereof.That B56associated with APC,downregulated b-catenin and ventralized Xenopus embryos positioned it as a negative regulator[48,49].By contrast,another study reported that the PP2A catalytic subunit potentiated Xenopus Wnt signaling initiated by the expression of disheveled,when measured by dorsal axis duplication and siamois gene expression[50].However,the regulatory B-subunit acted as a negative regulator but did so independent of b-catenin stabilization[50].Furthermore,in mammalian cells,the dephosphorylation of Axin,which immediately follows Wnt stimulation,was attributed to PP2A,thereby positioning it as a positive regulator[51].Finally,the Drosophila Twins gene,which codes for a PP2A regulatory B-subunit,scored as a positive regulatory of Wnt[52].It is important to understand the signi?cance of PP2A in Wnt signaling,because it has a signi?cant role in cancer genetics over all[53].The potent transforming activity of the SV40virus is dependent upon its small t(ST) antigen,which targets and inhibits the PP2A complex. Transformation by ST was reversed by overexpression of B56g and phenocopied by its speci?c depletion[54]. Moreover,the regulatory A-subunits of PP2A are mutated in a functionally relevant fashion in some cancers[53].On balance,the PP2A complex appears to be tumor suppres-sive,but considering its plethora of substrate clients,it is dif?cult to conclude to what extent,if any,its involve-ment in Wnt signaling relates to this.

Wnt receptors

The Wnt co-receptors LRP5and LRP6seem like ripe candidates for genetic activation of the pathway,yet sporadic mutations have not been reported for human cancers.Hyperactivating mutations in LRP5have been identi?ed but are associated with a familial autosomal dominant syndrome characterized by high bone-density [55–57].These LRP5mutations attenuate Dickkopf1-mediated inhibition of Wnt signaling[58].Nevertheless, cancer susceptibility in affected members of these kin-dreds has not been noted.The development of infantile germ cell tumors is associated with a germline transloca-tion of MESD,which encodes an essential chaperone for LRP5and LRP6[59].The resulting MESD–SENP1 (Mesoderm development–Sentrin-speci?c protease1) fusion protein fails to localize to the endoplasmic reticu-lum,where MESD normally associates with the LRP5 and LRP6.It is intriguing that the hyperactive LRP5 G171V bone density mutant also fails to interact with MESD,as does the hyperactive mouse LRP6mutant associated with the crooked tail phenotype[60,61].Based on the oncogenic activation of the Frizzled-related hedgehog receptor smoothened,one might anticipate similar anomalies in the Frizzled receptors,but again they have not been found.Although a human germline mutation in frizzled4was discovered,it codes for a dominant-negative protein that appears to interfere with the wild type receptor,resulting in defective retinal vascularization[62].

Conclusion

In addition to the Wnt pathway genes discussed above, there exist additional components,such as Pygopus,Bcl-9 and others that might activate the pathway if mutated. Many of these remain to be investigated,and perhaps some new mutations will be discovered.However,recent experiences with high-throughput sequencing suggest that it is unlikely that highly prevalent mutations,such as those in APC in colorectal tumors,remain undiscovered [63 ].The constellation of mutations in human cancers is perhaps more vast and heterogeneous than once appreci-ated,and temporal order and combination also appear to be relevant to tumor progression.It is also apparent that epigenetic silencing of genes,such as sFRPs[64],plays a role in activating the Wnt pathway in cancer.There are many ways to activate Wnt signaling in cancer.In addition

48Genetic and cellular mechanisms of oncogenesis

to the so-called canonical pathway mediating b-catenin stability,Wnt activates alternate pathways involving acti-vation of Jun kinase,the Rho GTPase and calcium-dependent effectors[65].The Wnt ligands also bind to alternate receptors such as ROR2and Ryk.Although our appreciation of these alternate pathways in embryologic development is signi?cant,their contribution to cancer remains unclear.Despite the complexities of the genetic menu available to developing colorectal cancer cells, activation of the Wnt pathway remains the common denominator,and inhibiting its output remains a goal for therapeutic intervention.

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The many ways of Wnt in cancer Polakis51