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Mutations in the APC gene are rate-limiting for sporadic and familialcolorectal cancers1,2. APC germline

mutations result in familial adenomatouspolyposis (FAP), one of the major hereditarypredispositions to colorectal cancer, andsomatic APC mutations are found in themajority of the sporadic colorectal tumours.Functional analysis of the APC protein hasrevealed a broad spectrum of interactions

with several proteins and cellular compo-nents (Fig. 1). First, APC binds to and down-regulates β-catenin, a key effector of Wntsignal transduction and an important com-ponent of adherens junctions in epithelialcells. It also encompasses several nuclearlocalization and nuclear export functionsthought to be critical for the coordinatedshuttling and downregulation of β-cateninbetween the cytoplasm and the nucleus1,2.

APC also interacts with the microtubulecytoskeleton3,4 and concentrates in granulesat the growing (plus) ends of microtubulesat membrane protrusions5,6. The interac-tion between APC and microtubules alsoseems to be related to its capacity to bindmicrotubule-associated proteins of theEB/RP family, such as EB1 (ref. 7). Bindingof APC to EB1 is critical for its localizationat the kinetochore (the attachment site of

The multiple functions of tumour suppressors: it’s all in APC

Riccardo Fodde

The multi-functionality of the adenomatous polyposis coli (APC) tumour suppressor gene keeps surprisingcancer molecular biologists. Signal transduction, cytoskeletal organization, chromosomal segregation andcell adhesion are just some of the putative cellular functions previously assigned to this gene and thoughtto be related to its tumour-suppressing activity. New data on yet another tumour-related function of APC,namely the coordinated regulation of cell adhesion and motility, adds to its host of cellular activities.

protein in promoting Cdc14 release, and thefact that the presence of protease-inactiveseparase did not stimulate the protease-requiring step of sister chromatid separation,are highly suggestive of a proteolysis-inde-pendent involvement of separase in promot-ing mitotic exit.

How separase promotes mitotic exit isstill an open question. Separase cleaves theFEAR component Slk19 (Fig. 1c), but this isdispensable for mitotic exit7,12. Uhlmannand colleagues noted that overexpression ofthe catalytically inactive separase inducedphosphorylation of Net1/Cfi1, which wasshown previously to be Cdc5-dependentand to reduce the affinity between Net1/Cfi1and Cdc14 (refs 13, 14). It is possible thatthe FEAR pathway activates mitotic exit, atleast in part by stimulating Cdc5 activitytowards Net1/Cfi1 (Fig. 1b). This would beconsistent with the observation that Cdc5only promotes phosphorylation ofNet1/Cfi1 after anaphase initiation, coinci-dent with the activation of separase1,13,14.

It is perhaps not unusual for an enzymeto have more than one type of activity, butso far separase is unique in this regardamong the CD clan of cysteine proteases.Moreover, it seems that the ability of sepa-rase to promote mitotic exit is limited tobudding yeast. The inactivation ofSchizosaccharomyces pombe, Caenorhabditiselegans, Drosophila melanogaster and mam-malian separases did not interfere withmitotic exit, but this does not imply that thesole function of separase in other organ-isms is to promote anaphase initiation.

Clearly, additional separase substrates exist.At least one other conserved separase sub-strate in many organisms (but not in bud-ding yeast) is separase itself5,15,16 (Fig. 1c). Inthis case, the cleavage products remainassociated and the complex formed by thetwo peptides is catalytically active5,15. As thecleavage sites are conserved, presumablyauto-cleavage of separase has an importantfunction, perhaps in downregulating itsown activity. Attempts to demonstrate adifference in activity in vitro using the Scc1cohesin subunit as a substrate have notbeen successful5,15. However, mutating theseparase cleavage site in Drosophila ThreeRows (THR) protein, which is analogous tothe amino terminus of separase from otherorganisms, resulted in maternal-effectlethality in developing embryos16. The mostpronounced defect was a massive loss ofnuclei at stage 14 of cellularization, whenScc1 cleavage is unlikely to have a role, butthe cause for the abnormal phenotype wasconsistent with an elevated protease activi-ty of the Drosophila separase16. Separase isalso required for spindle integrity in bothfission yeast17 and budding yeast12. In thelatter case, this function may involve theproteolytic cleavage of Slk19 (Fig. 1c). InC. elegans, the depletion of separase activ-ity by inhibitory RNA (RNAi) approachesresults in the loss of anterior–posteriorpolarity in early embryonic divisions18, butthe target of separase and whether sepa-rase’s proteolytic activity is involved, arestill unknown. Although a ubiquitousnon-proteolytic function for separase is

yet to be demonstrated, there is com-pelling evidence that separase is involvedin more than just separating sister chro-matids. What these processes are and howseparase is involved in their execution isstill a mystery.Karen E. Ross and Orna Cohen-Fix are in the

Laboratory of Molecular and Cellular Biology,

National Institute of Diabetes and Digestive Kidney

Disease, The National Institutes of Health, 8 Center

Drive, Bethesda MD, 20892, USA

e-mail: kross@helix.nih.gov or ornacf@helix.nih.gov

1. Sullivan, M. & Uhlmann, F. Nature Cell Biol 5, 249–254 (2003).

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© 2003 Nature Publishing Group

the microtubules to the chromosomes dur-ing metaphase) and may have a role in thefidelity of chromosomal segregation1.

Moreover, APC has been found to asso-ciate with the plasma membrane, where it isapparently transported in an actin-depend-ent fashion8. These observations have led tothe hypothesis of two distinct peripheralpools of intracellular APC: the first consti-tuted by the microtubule-bound granulesfound at membrane protrusions, the sec-ond bound to the plasma membrane in anactin-dependent manner6,8. Motile cellspreferentially show microtubule-boundAPC clusters6, whereas in less motile epithe-lial layers, APC reaches the membranethrough its interaction with the actincytoskeleton8. Notably, β-catenin is alsofound in two distinct pools in the epithelialcell: the membrane-bound pool where β-catenin participates in adherens junc-tions together with E-cadherin and α-catenin (thus enhancing cell adhesion),and a more soluble pool that shuttlesbetween cytoplasm and nucleus (thustransducing the canonical Wnt signal)1,2.Accordingly, E-cadherin and APC competedirectly for binding to β-catenin; the lattermediates the interaction of the alternativeE-cadherin and APC complexes to the actincytoskeleton through binding to β-catenin9.

The APC tumour suppressor was alsofound to bind a Rac-specific member of theDbl family of guanine nucleotide-exchangefactors (GEFs), termed Asef10. GEFs interactwith small GTP-binding proteins of theRho family, such as Rac and are involved inreorganization of the actin cytoskeletonduring the formation of lamellipodia andmembrane ruffling. APC releases the nega-tive auto-regulation of Asef through directbinding and stimulates Asef, promotingreorganization of the actin cytoskeletal net-work and altered cell morphology. In thisissue of Nature Cell Biology, Akiyama andcolleagues, who originally reported theinteraction between Asef and APC, provideadditional data on the role of the APC–Asefcomplex in cell migration and in E-cad-herin-mediated cell adhesion11. By usingadenoviruses expressing wild-type andmutant Asef, they show that Asef activitynegatively regulates cell adhesion bydecreasing the pool of E-cadherin and β-catenin localized at cell–cell contacts andincreasing their cytoplasmic pool, and thatGEF activity is necessary for this function.In addition, cell motility is positively affect-ed by the GEF activity of Asef and this effectis further enhanced by cotransfection withthe Asef-binding Armadillo (Arm) repeatdomain of APC11.

This multi-functionality of the APCtumour suppressor is interesting in view ofits rate-limiting role in colorectal tumourformation and possibly tumour progres-sion. The majority of APC mutations resultin truncated proteins that lack the binding

sites for microtubules, EB1 and some of therepeats that interact with β-catenin, axinand conductin1,2. These truncated APC pro-teins retain the Arm domain and have beenshown to be stable in vivo. Akiyama andcolleagues show that inhibition of APC andAsef through overexpression of specificdominant-negative mutants or throughinhibitory RNA approaches (RNAi) inhibitsthe migration of colorectal cancer cells(SW480) containing endogenous truncatedAPC, but not of colorectal cancer cells con-taining intact APC and mutant β-catenin(HCT116). Exogenous expression of trun-cated APC constructs increase motility ofnormal (MDCK) cells, whereas full-lengthAPC does not. So, truncated APC proteinsseem to preferentially trigger the Asef-mediated cell migration, whereas wild-typeAPC is a less efficient stimulator11.

As discussed above, the APC geneencodes for a multifunctional protein thatparticipates in several cellular processes,including cell adhesion and migration, sig-nal transduction, cytoskeletal organizationand chromosome segregation. However,despite the fact that each of these roles ispotentially linked with cancer, it seems thatthe main tumour-suppressing function ofAPC resides in its capacity to properly reg-ulate intracellular β-catenin levels1,2. In thenormal intestinal epithelium, β-catenin iselevated and accumulates in the nucleus inthe proliferative compartment, whereas it isdecreased and localized to the basolateralmembrane of epithelial cells in the uppertwo-thirds of the crypt12. Vice versa, APCcytoplasmic staining is markedly increasedin post-replicative cells within the upperportions of the crypt, suggesting anincreased level of expression with matura-tion, whereas it is virtually absent in thecrypt region where cells are actively divid-ing13. This pattern of expression is in agree-ment with the role of β-catenin signallingin maintaining stem cell properties andcontrolling differentiation in the intes-tine12,14,15. Activation of β-catenin down-stream targets, including c-Myc, Tcf-1,cyclin D1, CD44 and many others(http://www.stanford.edu/~rnusse/path

ways/targets.html), is required to main-tain proliferative capacity in the stem cellcompartment at the bottom of the crypt.Moving upwards along the crypt–villusaxis, an increase of APC expression coun-teracts β-catenin signalling and allows cel-lular differentiation. Constitutive activationof Wnt signalling through loss of APCfunction disturbs this finely tuned geneticprogramme that coordinates the balancebetween cell proliferation, differentiationand apoptosis along the intestinalcrypt–villus axis. This results in anenlarged stem cell compartment anddecreased cell differentiation. However, ifcontrol of Wnt signalling indeed representsthe only tumour-suppressing function ofAPC, one would expect APC and β-cateninmutations to be functionally equivalent.Notably, it has been reported that smalladenomas with β-catenin mutations donot seem to be as likely to progress to larg-er adenomas and invasive carcinomas asadenomas with APC mutations16. This sug-gests that although tumour initiationthrough loss of APC or oncogenic β-catenin mutations is functionally equiv-alent, inactivation of the additional APCfunctions in cell adhesion, motility andchromosomal stability might underliemalignant progression in the colorectum.In this scenario, APC mutations triggertumour formation by constitutively acti-vating β-catenin signalling and thus confera stem cell phenotype to the intestinalepithelial cell. However, this enlargementof the proliferative compartment in thecrypt does not suffice for adenoma progres-sion. The authors show that truncated APCproteins are also likely to cause decreasedcell–cell adhesion and aberrant cell migra-tion11, thereby supporting tumour progres-sion towards malignancy.

The multi-functionality of APC is a com-mon feature of several tumour suppressorgenes. Mismatch repair (MMR) genes thatare frequently inactivated in colorectal can-cer, such as MLH1 and MSH2 (ref. 17),BRCA1 (ref. 18), the Von Hippel-Lindau(VHL) gene19 and many other tumour sup-pressor genes, have similarly been shown to

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NATURE CELL BIOLOGY VOL 5 MARCH 2003 www.nature.com/naturecellbiology 191

Asef binding

• Cell migration& adhesion

β-catenin binding& regulation

• Wnt signalling

Axin andconductin binding

• Wnt signalling

Microtubule binding

• Cytoskeletonregulation

• Chromosomeregulation

EB1 binding

• Cytoskeletonregulation

• Chromosomesegregation

Figure 1 The many functions of APC. Schematic representation of the APC protein, show-ing regions that interact with other proteins, and the proposed functional significance ofthese interactions.

© 2003 Nature Publishing Group

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encode for multifunctional proteins thatpossibly coordinate signal transduction withother cellular activities. In the gut, the APCgene (rather than the β-catenin–TCF com-plex) is the master switch that controls cellu-lar proliferation, adhesion, migration anddifferentiation in the self-renewing cryptsand villi. This master regulatory role isreflected by the consequences of APC muta-tion in intestinal tumour initiation and pro-gression. As cancer is often the result ofabnormalities in multiple and distinct cellu-lar functions, inactivation of multi-func-tional genes may efficiently trigger tumour

formation and promote progression towards malignancy.Riccardo Fodde is in the Center for Human &

Clinical Genetics, Leiden University Medical

Center, Leiden, 2300 RA, The Netherlands

e-mail: r.fodde@lumc.nl

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