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BIOLOGY OF NEOPLASIA

B i o l o g y o f t h e A d e n o m a t o u s P o l y p o s i s C o l i Tu m o r S u p p r e s s o r

By Kathleen Heppner Goss and Joanna Groden

Abstract: The adenomatous polyposis coli ( APC )gene was rst identied as the gene mutated in aninherited syndrome of colon cancer predispositionknown as familial adenomatous polyposis coli (FAP).Mutation of APC is also found in 80% of all colorectaladenomas and carcinomas and is one of the earliestmutations in colon cancer progression. Similar to othertumor suppressor genes, both APC alleles are inactivated by mutation in colon tumors, resulting in the lossof full-length protein in tumor cells. The functional sig-nicance of altering APC is the dysregulation of severalphysiologic processes that govern colonic epithelial cell

homeostasis, which include cell cycle progression, mi-gration, differentiation, and apoptosis. Roles for APC insome of these processes are in large part attributable to

its ability to regulate cytosolic levels of the signalingmolecule beta-catenin and to affect the transcriptionalprole in cells. This article summarizes numerous ge-netic, biochemical, and cell biologic studies on themechanisms of APC-mediated tumor suppression.Mouse models of FAP, in which the APC gene has beengenetically inactivated, have been particularly useful intesting therapeutic and chemopreventive strategies.These data have signicant implications for colorectalcancer treatment approaches as well as for under-standing other disease genes and cancers of othertissue types.

J Clin Oncol 18:1967-1979. 2000 by AmericanSociety of Clinical Oncology.

T HE ADENOMATOUS polyposis coli ( APC ) tumorsuppressor gene was identied 10 years ago throughits association with an inherited syndrome of colorectalcancer known as familial adenomatous polyposis coli(FAP). The genetics of FAP and its similarity to othersyndromes of site-specic tumor predisposition, such asretinoblastoma, led to the designation of APC as a tumorsuppressor gene. This designation was conrmed by muta-

tional inactivation of APC in both familial and sporadictumors and by its ability to inhibit tumor cell growth.Functionally, the APC gene product modulates the onco-genic Wnt signal transduction cascade through its effects oncellular levels of beta-catenin. In addition, dependent orindependent of its ability to titrate -catenin, APC affectsdiverse physiologic processes from cell growth to apoptosisin a number of cell types and organisms.

This article summarizes many experiments that focus onthe role of APC mutation in tumor development and thebasic mechanisms of APC -mediated tumor suppression.Such research has far-reaching implications for our under-standing of how tumors form and how they are treated, aswell as for promoting our understanding of basic cellbiology and biochemistry.

THE ROLE OF APC IN TUMORIGENESIS: INHERITEDSUSCEPTIBILITY TO CANCER

Characterization of the etiology of FAP has providedinsight into the cause of this inherited cancer predispositionand of sporadic colon cancer. FAP is a rare, autosomal,

dominantly inherited syndrome affecting approximately onein 10,000 individuals and is characterized by the develop-ment of multiple (hundreds to thousands) colorectal adeno-mas at a young age (before 20 years). Because of the largenumber of adenomas and the well-established propensity of at least a subset of adenomas to become malignant, prophy-lactic resection of the colon is the recommended approachto managing the disease. Extracolonic manifestations of FAP are common and include adenoma and adenocarci-noma of the stomach, periampullary region, pancreas andthyroid, osteomas, desmoids, dental abnormalities, epider-mal cysts, congenital hypertrophy of the retinal pigmentedepithelium (CHRPE) and CNS tumors. Conventionally, theclinical association of FAP with desmoid tumors andosteomas is referred to as Gardner syndrome, whereasTurcot syndrome is characterized by the association of FAPwith CNS tumors, in particular medulloblastoma. The

From the Howard Hughes Medical Institute, Department of Molec-ular Genetics, Biochemistry, and Microbiology, University of Cincin-nati College of Medicine, Cincinnati, OH.

Submitted July 21, 1999; accepted November 3, 1999.Supported in part by National Institutes of Health grant no.

CA-63507 and the Howard Hughes Medical Institute. Address reprint requests to Joanna Groden, PhD, Department of

Molecular Genetics, College of Medicine, University of Cincinnati, 231 Bethesda Ave, Cincinnati, OH 45267; email [email protected].

2000 by American Society of Clinical Oncology.0732-183X/00/1809-1967

1967 Journal of Clinical Oncology,Vol 18, No 9 (May), 2000: pp 1967-1979Downloaded from jco.ascopubs.org on December 6, 2010. For personal use only. No other uses without permission.

Copyright 2000 American Society of Clinical Oncology. All rights reserved.


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calculated risk of medulloblastoma for an FAP patient isgreater than 90-fold of that of a normal individual. 1

Identication of the gene mutated in FAP was accom-plished by linkage analysis of families with the syndrome 2,3and conventional positional cloning strategies. 4-7 The APC gene was rst localized to human chromosome 5q by theidentication of a chromosome 5q deletion in a Gardnersyndrome patient several years earlier. 8 The APC geneincludes 21 exons contained within a 98-kilobase locus. 9

The largest exon, 15, comprises more than 75% of the 8,535base pairs (bp) of coding sequence (Fig 1) and is the targetof most germline mutations in FAP patients and somaticmutations in tumors. In addition to the conventional form of APC encoded by exons 1 to 15, alternatively expressedexons of the gene (0.3, BS, 0.1, 0.2, 1, 9, and 10A) 10-12

encode alternate protein isoforms.13

Although the tissue-specic expression patterns of these isoforms differ fromconventional APC, 14 their function is not yet understood.They are found generally in postmitotic tissues and termi-nally differentiated cell lines 14 and differ most signicantlyfrom conventional APC in their amino-termini and pre-dicted ability to homo- or heterodimerize.

Most APC mutations are chain-terminating, the majorityof mutations created by frameshifts, which result in trun-cated gene products without the carboxy-terminus. APC

mutation follows the classical two-hit model of tumorsuppressor inactivation, in that patients with FAP inheritone germline mutation and develop tumors from those cellsin which a second hit, or loss of the other allele of APC , issomatically acquired. More than 1,400 mutations in APC ,both germline and somatic, have been described. 15 Two of the most common germline mutations are 5-bp deletions atnucleotides 3927 and 3183 (codons 1309 and 1061), whichcomprise 18% and 12% of all germline APC mutations,respectively. 15 These deletions occur in short, direct repeatswithin APC and have been identied in patients with andwithout a family history of the disease, suggesting that theycan occur as new mutations. 16 Laken et al 17 described apolymorphism of APC at nucleotide 3920 that is found inapproximately 6% (47 of 766) of Ashkenazi Jews and 28%

(seven of 25) of Ashkenazi Jewish colon cancer patientswith a family history of colon cancer. This T 3 Atransversion does not result directly in a truncated geneproduct but seems to generate a hypermutable poly-adeninetract theoretically prone to slippage during DNA replica-tion, thereby accounting for a higher than expected fre-quency of somatic mutation within this general region(approximately 500 bp) of APC . The clinical signicance of this polymorphism remains uncertain; however, these datapoint to a novel mechanism by which nucleotide polymor-

Fig 1. TheAPC gene, mutational spectrum, clinical correlates, and APC protein structure. (A) The conventional form of theAPC gene contains 15 exons, with

the most 3 exon containing over three-quarters of the 8,535 bp of coding sequence. The alternatively spliced exons 1, 9, and 10a are also shown. (B) Germlinemutations associated with AAPC are clustered at the 5and 3 ends of the gene. Most AP C mutations occur within the central third of the gene, designated themutation cluster region. This region contains two of the most commonly found mutations, 5-bp deletions creating stop codons at positions 1061 and 1309, anda nucleotide polymorphism (ASH) in the Ashkenazi Jewish population that generates a poly-adenine tract.17 Regions of the gene where mutations areassociated with congenital hypertrophy of the retinal pigmented epithelium (CHRPE) and desmoid tumors are shown. The location of a 3mutation associated with hereditary desmoid disease is also indicated. (C) The APC protein contains 2,843 residues with several structural motifs, including the Armadillo repeats,15 and 20amino acid repeats, and carboxy-terminal basic region. Approximate binding regions of known protein partners of APC are shown below theprotein structure.

1968 GOSS AND GRODEN

Downloaded from jco.ascopubs.org on December 6, 2010. For personal use only. No other uses without permission.Copyright 2000 American Society of Clinical Oncology. All rights reserved.


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phisms can affect somatic mutations in APC and otherdisease-causing genes.

The precise location of germline mutation within APC can predict disease phenotype (Fig 1). Mutations betweenbases 3747 and 3990 (codons 1249 to 1330) are associatedwith a profuse phenotype of colonic tumors (ie, greater than5,000 adenomas), whereas mutations 5 or 3 to this regionare correlated with a sparse phenotype (ie, fewer than 1,000adenomas). Furthermore, mutations at the very 5 end of thegene (codons 78 to 163) result in an attenuated adenomatouspolyposis coli syndrome (AAPC) characterized by thedevelopment of fewer adenomas (often less than 100) at alater age. Although it is not understood how more severeAPC truncations result in a less severe phenotype, recentstudies of AAPC kindreds suggest that AAPC alleles mayencode protein with residual function because some AAPC alleles acquire somatic mutations or are lost entirely duringtumor development. 18 Some germline mutations in the 3region of exon 15 are associated with a similarly attenuatedphenotype. 19

Extracolonic manifestations are also associated with mu-tation position. For example, CHRPE usually occurs in FAPpatients with mutations 3 of exon 9A, 20 whereas desmoidsare more often associated with APC mutation between bases4335 and 4734 (codons 1445 to 1578). 21 Additionally,patients with high numbers of desmoids but without polyp-osis have been identied and carry germline APC mutationsat the 3 end of the gene. 22

THE ROLE OF APC IN TUMORIGENESIS: SPORADICTUMOR FORMATION

Study of FAP has provided insight into the cause of thisrare inherited disease but, perhaps even more importantly,has unveiled a common mechanism for sporadic colorectalcancer. Colorectal cancer is one of the most commoncancers in the developed world and this year will accountfor over 130,000 new cases and 57,000 deaths in the UnitedStates alone. APC mutation is extremely common in colo-rectal tumors; somatic inactivation of APC occurs in 50%and 80% of sporadic colon adenomas and adenocarcinomas,

respectively.23

A mutation cluster region exists within the 5end of exon 15, between nucleotides 3000 and 4800 (codons1000 to 1600) and represents approximately 60% of re-ported somatic mutations. 24 The majority of sporadic colontumors carry mutations of both APC alleles, the frequencyof which remains constant between benign and malignanttumors. 25 Furthermore, APC mutations are the earliestknown genetic alterations in colorectal cancer progression,as they have been identied in the smallest detectableadenomas as well as aberrant crypt foci. 26 These data

strongly support a role for APC mutation in colorectal tumorinitiation rather than in progression of a benign tumor tomalignancy.

THE ROLE OF APC IN TUMORIGENESIS: STRUCTURA ANALYSIS OF THE APC PROTEIN AND PROTEIN

PARTNERS

The APC gene product, known as APC, is large andcomprised of 2,843 amino acids with a molecular mass of approximately 310 kd (Fig 1). Our current understanding of APC function comes from a dissection of its proteinstructure and putative functional motifs and from analysis of its protein partners.

The predicted tertiary structure of the rst third of APCcontains a coiled-coil motif, characterized by a series of heptad repeats of hydrophobic residues that most likelymediate oligomerization of the protein. The rst 170 aminoacids are sufcient for APC homodimerization in vitro; thisassociation requires the rst 45 amino acids, which corre-spond to the rst heptad repeat. 27,28 Homodimerization of APC at the amino-terminus implies a possible dominant-negative mode of action for mutant APC in heterozygouscells, in which shorter proteins can functionally inactivatethe full-length, normal protein. Although this hypothesis isnot supported by mutational data (ie, two APC mutations orloss of heterozygosity are usually observed in adenomas), itis supported by other ndings. Mice carrying one mutant Apc allele display a signicant decrease in enterocytemigration in the intestinal villus. 29 In vitro studies demon-strate that normal APC activity is severely abrogated onintroduction of mutant APC and, to a lesser extent, an AAPC mutant gene. 30 Conversely, a dominant-negative mode of action for APC is not supported by other experiments. FAPpatients carrying cytogenetic deletion, not just mutation, of the APC gene have been identied. 2,8,31-33 In addition,transgenic mice overexpressing truncated APC in the intes-tinal epithelium fail to develop intestinal tumors. 34 Thesmallest pathologic lesions in mice with mutant APCdemonstrate loss of the wild-type allele, supporting thenotion that loss of full-length APC, not the acquisition of

mutant APC, is rate-limiting for tumor formation.35-37

The amino-terminus of APC includes at least one func-tional nuclear export signal (NES) sequence. 38,39 Thispeptide (67-DLLGRLKGLNLD-78) is similar to other well-characterized NES sequences (such as in HIV Rev, FMRP,and MAPKK) and, by itself, can direct exclusion of greenuorescent protein (GFP) from the nuclei of transfectedcells. The function of this NES is sequence-specic andtemperature-sensitive, consistent with GTP-dependent, re-ceptor-mediated export. 38 Although nuclear localization of

1969BIOLOGY OF THE APC TUMOR SUPPRESSOR



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APC has been reported in some cell types, 40 no functionalnuclear localization signals have been identied. Because

APC is far too large to diffuse passively into the nucleus, itis possible that APC is shuttled into the nucleus by anunconventional mechanism or that the import and export of APC are tightly regulated by protein conformation orbinding partners.

The rst third of APC contains seven Arm repeats namedfor an amino acid motif repeated 13 times in the Drosophilahomolog of -catenin, Armadillo. Implicated in mediatingprotein-protein interactions, Arm repeats are present in anumber of other proteins, including the desmosomal pro-teins plakoglobin, plakophilin, and band 6 protein, p120cas,the importin family of nuclear import receptors, and the

PF16 microtubule-associated protein. In the APC proteinpartner -catenin, similar repeats are required for bindingAPC, E-cadherin, and the architectural transcription factorsbelonging to the Tcf family. 41-43 Structurally, the Armrepeats in -catenin form a superhelix resulting in a posi-tively charged groove that associates with a stretch of acidicamino acids in its partner. 44 The function of these armrepeats in APC is unknown because protein partners thatspecically bind APC in this region have yet to be de-scribed.

Three 15amino acid and seven 20amino acid repeatsare present within the central third of APC. The 15amino

acid repeats associate with the multifunctional -cateninprotein and the related protein, plakoglobin. 45 The residuesof the 20 amino acid repeats are highly conserved bothbetween repeats and animal species and demonstrate se-quence similarity to the 15amino acid repeats. Smallfragments of APC containing the 20amino acid repeats caninteract with -catenin and mediate its downregulation 41

(Fig 2), which will be described further in the next section.To bind -catenin, these fragments of APC require phos-phorylation by the serine-threonine kinase GSK3 ,46 sug-gesting that APC and GSK3 together modulate levels of cytoplasmic -catenin. The axin family of proteins, includ-

ing axin, axil and conductin, also are found in the APC/ GSK/ -catenin complex and regulate the degradation of

-catenin. 47-49 The F-box protein beta-TrCP is a componentof an E3 ubiquitin ligase that facilitates -catenin degrada-tion at the proteosome, 50,51 although the specic role of APC in the degradation process is not known.

The importance of APC-mediated -catenin degradationis highlighted by the location of the mutation cluster regionwithin the region of the APC gene encoding the 20aminoacid repeats (Fig 1). Activating mutations of -catenin in

Fig 2. APC modulates-catenin/Tcf transcriptional activation and Wnt signal transduction. (A) In the presence of APC or in the absence of Wnt ligand,-catenin is localized to the adherens junction where it is associated with E-cadherin,-catenin, p120cas, and indirectly with the cytoskeleton. GSK3

phosphorylates -catenin in a complex that contains-catenin, APC, and axin family members, and-catenin is rapidly degraded by ubiquination at theproteosome. (B) WhenAPC is mutated, -catenin accumulates in the cytoplasm and the nucleus. Similarly, binding of Wnt ligand to its receptor, known asfrizzled, inactivates the GSK3kinase through dishevelled, generating a cytosolic pool of-catenin. -catenin associates with members of the Tcf family oftranscription factors and modulates the transcription of target genes with Tcf recognition sequences. In some instances,-catenin increases transcription oftarget genes by competing for Tcf binding with corepressors, such as Groucho and CBP, to relieve transcriptional repression.




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the GSK phosphorylation site required for its degradationresult in accumulation of -catenin 52 and have been docu-mented in approximately 50% of colorectal tumors that donot contain APC mutations. 53 There are limitations, how-ever, to a model in which the sole function of APC is toregulate cytoplasmic levels of -catenin. A narrower muta-tion spectrum of APC would be expected if this associationwere the only critical function for APC-mediated tumorsuppression. Moreover, studies in manipulatable develop-mental systems indicate additional functions of APC. The Drosophila APC homolog is not required for Armadillo (the Drosophila homolog of -catenin) signaling, 54 whereas in Xenopus , APC regulates -catenin signaling cooperativelyrather than antagonistically. 55 Whether or not the predom-inant function of APC is to regulate -catenin levels, oneconsequence of -catenin accumulation in the cytosol is thedysregulation of target gene transcription.

The carboxy-terminal end of APC, particularly residues2200 to 2400, is enriched in basic amino acids. These basicresidues may confer binding to microtubules because thisregion is sufcient for microtubule colocalization whentransiently it is overexpressed in colon cancer cells 56,57 andpromotes microtubule polymerization in vitro. 57 Speci-cally, residues 2219 to 2580 of APC bind nonassembledtubulin and bundle microtubules in vitro. 58 Although adirect interaction between APC and tubulin has not beenshown, APC is likely to associate indirectly with microtu-bules through EB1. EB1, a member of the EB/RP family of tubulin-binding proteins, was identied as a protein partnerof APC through a yeast two-hybrid library screen usingresidues 2186 to 2843 of APC as bait. 59 Given that aS. cerevisiae homolog of EB1 is required for a microtubule-dependent cytokinesis checkpoint, 60 it is exciting to postu-late that APC and EB1 function to maintain genomicstability by governing mitotic spindle integrity or properchromosome segregation. Recent experiments indicate thatAPC/EB1 complexes are indeed cell cycle-regulated. 61

APC localization to the leading edge of migrating epithelialcells in culture where microtubules are concentrated 62 alsosupports an additional role for APC/microtubule complexesin cell migration and/or adhesion. RP1, another member of

the EB/RP protein family binds the carboxy-terminal regionof APC, 63 again suggesting that APC may exist in amultiprotein complex with microtubules.

The carboxy-terminal 15 residues of APC bind the humanhomolog of the Drosophila tumor suppressor, discs large(DLG). Association of APC with DLG was identiedthrough a yeast two-hybrid library screen and conrmedbiochemically and by colocalization in vivo. 64 DLG con-tains a PDZ domain that specically associates with acarboxy-terminal XS/TXV peptide on a partnering protein.

The carboxy-terminus of APC contains this motif, speci-cally VTSV. In mammalian epithelial cells, DLG localizesto regions of cell-cell contact, 65 as do other PDZ-containingproteins such as ZO-1 and ZO-2. This association providesanother means by which APC may affect epithelial cellmigration and/or motility. DLG and APC colocalize atsynaptic junctions along neuronal processes. 64 DLG/APCcomplexes may have an important role in the CNS, whereboth proteins are highly expressed and APC mutation iscorrelated with predisposition to brain tumors. As theNMDA receptor and voltage-gated K channel associatewith DLG and the closely related SAP90 protein, 66,67 it isexciting to postulate that APC modulates neuron functionby associating or competing with DLG/NMDA/K channelcomplexes.

APC is a phosphoprotein with consensus phosphorylationsites for GSK3 , MAPK, cyclin-dependent kinases(CDKs), protein kinase A, casein kinase I and II, andcalmodulin kinase. APC is phosphorylated on serine andthreonine residues and is hyperphosphorylated during theM-phase of the cell cycle. 68,69 This observation is consistentwith an increase of histone H1-kinase activity associatedwith full-length APC during M-phase. 70 The CDK p34 cdc2

can be immunoprecipitated with APC and can phosphory-late APC in vitro. 70 Mutation of consensus CDK siteswithin the carboxy-terminal 700 residues of APC suggeststhat phosphorylation of APC by p34 cdc2 is a mechanism todisassociate APC/EB1 complexes, specically during mito-sis (J.G., unpublished data). 61 These phosphorylation sites,as well as the EB1 binding site, are absent when APC ismutated, suggesting that regulation of the APC/EB1/micro-tubule complex may be critical to the tumor-suppressingactivity of APC. Furthermore, APC can be phosphorylatedby GSK3 in vitro, 69 modications that are required for theinteraction of APC with -catenin. 46

THE ROLE OF APC IN TUMORIGENESIS: MECHANISMOF TUMOR SUPPRESSION

Our understanding of APC-mediated tumor suppressioncomes predominantly from the identication of its proteinpartners. Using the colonic crypt as a paradigm, one canenvision numerous processes in which a regulator of epi-thelial homeostasis, such as APC, may be important. Theseprocesses include proliferation or cell cycle control, migra-tion, differentiation, and apoptosis. Additionally, the regu-lation of -cateninmediated transcription is another wayfor APC to affect each of these processes indirectly.

Transcription

-catenin was rst identied as an essential component of the adherens junction complex, although its role in modu-




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lating gene expression has recently generated much moreattention. As mentioned, APC is a key regulator of -cate-nin because it titrates cytoplasmic -catenin by regulatingits degradation (Fig 2). In the absence of functional APC,free -catenin accumulates in the cytoplasm. This free poolof -catenin allows it to associate with members of the Tcf family of architectural transcription factors. Nuclear importof the complex occurs by recognition of the nuclear local-ization signal in Tcf by the import receptor importin-alpha. 42,71-74 This model becomes more complicated, how-ever, because free -catenin can be imported into thenucleus independent of Tcf binding and because nuclearlocalization of Tcf and -catenin can be insufcient fortranscriptional activation. 75 -catenin/Tcf complexes spe-cically bind Tcf consensus binding sites (5 -A/T A/TCAAAG-3 ), bend DNA to alter the local promoter envi-ronment, and change the transcriptional activity of specictarget genes.

There are strong indications that the -catenin/Tcf path-way is critical to tumor development. As discussed, APC and -catenin are frequently mutated in colorectal cancer,and -catenin mutations have been found in other tumortypes including skin, stomach, and pancreas. 76,77 APC and

-catenin are components of the Wnt signaling pathway,shown in Fig 2. Importantly, binding of the oncogenic Wntligand to its transmembrane receptor, Frizzled, activates asignal transduction pathway that results in GSK3 inhibi-tion and a similar accumulation of cytosolic -catenin aswhen APC is inactivated. 78 Activation of the Wnt family of ligands is sufcient for tumorigenesis in mice; and, al-though a direct role for Wnt in human cancer has not beenestablished, aberrant expression of Wnt has been detected inseveral tumor types. 79 Lastly, Tcf4 mutations have beenidentied in 40% to 50% of colorectal cancer cell lines andtumors demonstrating microsatellite instability. 80

The association of the Wnt signaling pathway and cancersuggests that transcriptional targets of this pathway governcell growth, migration, differentiation, or apoptotic pro-cesses. To date, a handful of -catenin/Tcf transcriptionaltargets have been described, each activated by -catenin/Tcf binding. These include the proto-oncogene and cell cycle

regulator c-myc ,81

the G1/S-regulating cyclin D1 gene,82

and the gene encoding the matrix-degrading metalloprotein-ase, matrilysin .83 The AP-1 transcription factors c-jun and fra-1 and the urokinase-type plasminogen activator receptorare also upregulated by -catenin/Tcf signaling. 84 The listof physiologically relevant targets will grow certainly, butthe challenge will be to demonstrate that transcriptionalchanges are causally involved in early-stage tumorigenesisas a consequence of APC mutation. The balance of Tcf and

-catenin available for DNA binding is most likely critical

to net transcriptional activity. In fact, promoter binding byTcf without -catenin represses transcription, 82,83,85 per-haps by Tcf association with transcriptional corepressorssuch as Groucho 86,87 and CBP. 88 Such data suggest that thispathway has several levels of regulation and that smallchanges in local protein concentrations can dramaticallychange the transcription prole of critical regulatory genes.An illustration of this point is that the Tcf family memberTcf1 is a target of -catenin/Tcf4 transactivation and mayact as a feedback repressor of transcription because it lacksthe domain required for -catenin association. 89

Cell Cycle Control

Similar to other tumor suppressors, such as Rb or p53,APC plays a role in controlling cell cycle progression. Earlyexperiments introduced full-length, wild-type APC intohuman colon adenocarcinoma cell lines with varied suc-cess. 90 The doubling time, ability to form colonies in softagar, tumorigenicity in mice, and morphologic characteris-tics were signicantly altered in some cell lines on additionof APC , whereas stable transfectants were difcult toestablish in other lines. 90 Overexpression of APC inNIH3T3 broblasts inhibits progression of the cells fromG0 /G1 to S phase of the cell cycle in response to serumstimulation. 91 Consistent with these data, more recent ex-periments demonstrate a G1/S arrest in the APC-decientcolon cancer cell line SW480 when transiently transfectedwith a GFP-APC fusion protein (Fig 3A and 3B) (Heinen etal, manuscript submitted for publication). This arrest can bepartially alleviated by overexpression of constitutively ac-tive -catenin or components of the Rb pathway (Heinen etal, manuscript submitted for publication). These experi-ments indicate that maintenance of the G 1 -S checkpoint byAPC is mediated through its effect on components of the Rbpathway and is attributable, at least in part, to regulation of

-catenin/Tcfmediated transcription of S-phase regulatorssuch as cyclin D1 and c-myc (Fig 3D). A role for APC at theG2 -M transition is also likely given the observations thatAPC is hyperphosphorylated during M-phase 69 and is atarget of the M-phase kinase p34 cdc2 .70 Localization of a Drosophila APC homolog to the mitotic spindle and cen-

trosome apparatus is also consistent with this hypothesis.92

Migration

The identication of -catenin and plakoglobin as proteinpartners of APC rst implicated APC in epithelial celladhesion and migration. -catenin localizes to the adherens junction, and plakoglobin localizes to the desmosome, andboth structures are involved in cell-cell contact. More recentcharacterization of the -catenin/Tcf signaling pathway hasleft the role of APC in adhesion and migration all but




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forgotten. Nevertheless, several lines of evidence supportsuch functions of APC, which may or may not be mediatedby -catenin regulation.

Localization of full-length APC is predominantly cyto-plasmic. Nathke et al, 62 as well as others, 40 immunolocal-ized APC to the leading edges of epithelial cells. This

staining of endogenous APC can be recapitulated with aGFP-fused exogenous APC (Fig 4). 38 Accumulation of APC at the leading edges of actively migrating cells isdependent on the integrity of the microtubules but not actinlaments, 62 alluding to a function for APC in cell motilityor adhesion through its association with microtubules.Additional support for this hypothesis comes from theanalysis of intestinal tissue from mice with geneticallyaltered levels of the Apc gene. Intestinal cell migrationalong the crypt-villus axis is altered in histologically normal

heterozygous Apc animals. 29 These epithelial cells show aconcomitant increase in -catenin levels, 29 suggesting thataltered migration is facilitated by -catenin dysregulation.Migration of intestinal epithelial cells is similarly disor-dered in mice with overexpression of full-length APC. 93 Itis certainly possible that APC mutation contributes to

tumorigenesis by altering the relative adhesiveness of co-lonic epithelial cells and misregulating the integrity of thecadherin-catenin complexes. An indirect role for APC inmigration may result from transcriptional activation of target genes that modulate cell motility and adhesion, suchas E-cadherin 71 and matrix-remodeling enzymes. 83 In manyways, embryonic development is analogous to tumor devel-opment in that it involves dramatic cell proliferation andmigration and extracellular matrix remodeling. Mice withtwo mutant Apc alleles die very early during embryonic

Fig 3. APC expression inhibits G1 -S cell cycle progression. (A) Expression of an APC/GFP fusion protein in the human colon cancer cell line SW480, whichcontains only mutant APC , results in the degradation of endogenous-catenin (Heinen et al, manuscript submitted for publication). Cells expressing GFP alonehave high levels of -catenin, whereas cells expressing APC/GFP do not (arrows). Magnication: 400. (B) Overexpression of the APC/GFP fusion protein inSW480 cells blocks the progression of S phase compared with GFP expression (Heinen et al, manuscript submitted for publication). This arrest is partially relieved by coexpression of a constitutively active-catenin (S37A). Rescue of the APC-mediated arrest by -catenin is dependent on -catenin/Tcftranscriptional activation as a dominant-negative Tcf mutant (N67) abrogates this rescue. (C) APC can regulate the G1/S transition by controlling

-catenin/Tcf-mediated transcription of target genes, such ascyclin D1and c-myc , that affect the Rb pathway. In addition, it is possible that APC controlsS-phase entry in a manner independent of -catenininduced effects on the Rb pathway.




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development from an inability to complete gastrulation, 94

supporting a critical requirement for APC in cell migration

and/or proliferation.

Differentiation

Although a role for APC in regulating intestinal celldifferentiation has not been proven, the pattern of APCexpression along the colonic crypt axis supports such afunction. Smith et al 95 rst demonstrated APC protein inepithelial cells of the lumenal half of the colonic crypt, aregion of the crypt containing terminally differentiated,nondividing cells. Alternatively expressed APC isoforms(ie, containing any of the 5 exons 0.3, BS, 0.2, or 0.1 but

not exon 1) are detectable only in postmitotic, differentiatedtissues. 13 Consistent with these results, APC expression isinduced early during the differentiation of mouse myoblastcultures into myotubes. 14 It is possible that genes regulatingdifferentiation may be targets of -catenin/Tcfmediatedtranscription. In fact, mice lacking Tcf4 have a defect inintestinal stem-cell proliferation, 96 suggesting that APCmay control differentiation and/or proliferation of the intes-tinal epithelium by preventing aberrant -catenin/Tcf sig-naling.

Apoptosis

In human and rodent intestine, APC expression is re-stricted to the lumenal half of the crypt, a nonproliferative,differentiated zone of enterocytes. 95 Cells are also shed into

the lumen from this region of the crypt after programmedcell death or apoptosis. Cell turnover is an essential mech-anism in maintaining intestinal homeostasis and one inwhich dysregulation could facilitate tumor formation even if normal cell cycle control is maintained. The role of APC inregulating apoptosis has been tested by a number of exper-iments. Inducible APC expression in a colorectal cancer cellline carrying only mutant APC increases apoptosis approx-imately 10-fold. 97 In Drosophila , however, germline inac-tivation of Apc results in retinal degeneration because of unscheduled apoptosis of retinal neurons. 98 This retinaldefect can be rescued by inactivation of Armadillo or Tcf

mutation,98

suggesting that -catenin/Tcfmediated pro-cesses are required for apoptosis in this system in whichAPC is absent. APC may have opposing effects on apopto-sis in these two systems because of differences in theorganisms and cells under study or the complexities of dissecting these processes in vivo or in tissue culture.

Our group has used cell-free Xenopus egg extract to studythe effect of APC on apoptosis in vitro (Steigerwald et al,manuscript in preparation). Recombinant human APC ac-celerates the rate of apoptosis using exogenous nuclei assubstrate. This effect can be mimicked by a region of APCsufcient for -catenin binding and downregulation and can

be ablated by caspase-8 inhibitors. This effect is transcrip-tion independent because the RNA II polymerase inhibitor-amanitin does not change the experimental outcome. In

this case, APC and -catenin, through an unknown signaltransduction mechanism involving at least one caspase-mediated pathway and not involving gene transcription,may regulate cell death.

THE ROLE OF APC IN TUMORIGENESIS: THERAPEUTIMPLICATIONS OF APC BIOLOGY

The goal of many investigations into the biologic func-tion of APC and the consequences of APC mutation in colontumorigenesis is to develop meaningful prognostic indica-tors and therapeutic strategies for managing colorectalcancer. APC remains an attractive target for therapeuticintervention because its mutation is a common and earlyevent in the continuum of colorectal tumor progression.

Generation and Use of Mouse Models

Both gene targeting by homologous recombination andchemical mutagenesis have established a number of mouse

Fig 4. APC localizes to the leading edge of migrating cells. An APC/GFPfusion protein was transiently expressed in COS-1 African green monkey kidney cells and photographed under UV illumination with a uoresceinisothiocyanate conjugated lter (magnication: 400).




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models of FAP. The Apcmin mouse is the best characterizedof these models and was generated by chemical mutagenesisthat introduced a chain-terminating mutation at nucleotide2549 in mApc .99,100 As in FAP patients, heterozygous Apcmin mice develop numerous intestinal adenomas inwhich the remaining wild-type allele is somatically inacti-vated during adenoma development. 35,37 Unlike the humandisease, however, Apcmin mice develop adenomas predom-inantly throughout the small intestine instead of the colonand rectum. Other mouse models of FAP have been createdby gene targeting of mApc and include Apc 716 and Apc1638N , both of which mimic the Apcmin phenotype inadenoma location but vary signicantly in tumor numberand life span. 36,101,102 Extracolonic manifestations areprominent in the Apc1638N animals specically; theseanimals develop desmoid tumors, cutaneous cysts, andretinal pigment epithelium abnormalities, all of which arecommon in FAP patients. 103,104 Phenotypic differences inthe three models may result from mutation location withinthe Apc gene or other environmental differences (ie, back-ground strain, diet, and microora). Interestingly, adenomaformation in the colon was achieved in mice by generating Apc mutations with a conditional targeting strategy and anadenovirus expressing cre -recombinase introduced rectal-ly. 105 Together, these mouse models of FAP will allow thedissection of the molecular mechanisms of Apc-mediatedtumorigenesis and will facilitate the analysis of putativetherapeutic targets in vivo.

A handful of drugs have been tested using these mousemodels that have shown signicant benets with respect totumor burden and life span. The nonsteroidal anti-inam-matory drugs (NSAIDs), piroxicam and sulindac, wereadministered to Apcmin mice and dramatically reducedadenoma formation. 106-109 Historically, epidemiologic evi-dence indicated a chemopreventive role for NSAIDs inpatients with sporadic colorectal tumors 110 and familialpolyposis. 111,112 These data are concordant with the obser-vation that tumor formation is suppressed in Apc 716 micein which a target of NSAIDs, cyclooxygenase-2 (COX-2), isgenetically ablated. 113 Furthermore, COX-2 is overex-pressed in mouse and human intestinal adenomas 114 and has

been implicated in intestinal cell apoptosis115

and tumorangiogenesis. 116 Such a protein provides an attractive targetfor abrogating APC-mediated tumorigenesis. The mecha-nism by which COX-2 is upregulated early in tumorigenesisis unknown, although, recent data suggest that it may be atarget of -catenin/Tcf transcriptional activation. 117

Other therapeutic agents used to treat mouse models of FAP include the matrix metalloproteinase inhibitor batimas-tat, 118 Bowman-Birk protease inhibitor, 119 and the DNAmethyltransferase inhibitor 5-azacytidine. 120 All of these

agents are effective at inhibiting adenoma formation in themouse and are in clinical trials or presently being used asstandard cancer therapy.

Gene Therapy

A limited number of experiments have addressed thepotential of replacing mutant APC using gene therapyapproaches. Introduction of human APC into the colon of

Apcmin

mice has been accomplished using cationic lipo-somes, where expression of the transgene was maintained inthe epithelium for at least 3 days. 121 Hargest et al 122 havealso used lipofection to establish prolonged APC expressionin the mouse colon. Further analysis is required to determinewhether expression of normal APC in this context canprevent tumor formation and to what extent. These resultspredict that restoring normal APC function to the colonicepithelium of FAP patients with a germline APC mutation isa feasible therapeutic strategy.

Fig 5. APC may regulate colonic epithelial cell homeostasis by affectingthe cell cycle, migration, differentiation, and apoptosis. The colonic cryptsare composed of an epithelial layer of cells that include stem cells undergo-ing mitosis ( ), columnar absorptive cells ( ), mucin-producing goblet cells( ), and enteroendocrine cells ( ). Cells differentiate and migrate to thelumenal surface of the crypt where they are extruded into the lumen of theintestine by programmed cell death ( ). It is likely that APC participates in allof these processes directly or indirectly by modulating transcription proles within the intestinal epithelial cells.




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In summary, the tumor suppressor APC participates inseveral cellular processes, from proliferation to apoptosis, inthe colonic epithelium (Fig 5). Some of the functions of APC are attributable to its ability to control -catenin levelsand the transcription of target genes. APC mutation is anearly and common event in sporadic colorectal tumorformation and is present in the germline of patients with aninherited predisposition to colon cancer known as familialadenomatous polyposis coli. Therefore, APC and its gene

product are attractive targets for the design of therapeuticand chemopreventive strategies for colorectal cancer pa-tients. Additional investigation into the biology, biochem-istry, and genetics of APC will no doubt result in therealization of these goals.

ACKNOWLEDGMENTWe thank Jennifer Kordich, Andrew Lowy, and Therese Tuohy for

critical review of the manuscript.

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