Colorectal cancers show distinct mutation spectra in members of the canonical WNT signaling pathway...

GENES, CHROMOSOMES & CANCER 49:746–759 (2010)

Colorectal Cancers Show Distinct Mutation Spectrain Members of the Canonical WNT SignalingPathway According to Their Anatomical Locationand Type of Genetic Instability

Cristina Albuquerque,1* Celia Baltazar,1 Bruno Filipe,1 Filipa Penha,1 Teresa Pereira,2 Ron Smits,3 Marılia Cravo,4

Pedro Lage,4,5 Paulo Fidalgo,4 Isabel Claro,4,5 Paula Rodrigues,5 Isabel Veiga,6 Jose Silva Ramos,7

Isabel Fonseca,2 Carlos Nobre Leitao,4 and Riccardo Fodde8

1Centro de Investigac~ao de Patobiologia Molecular (CIPM),Instituto Portugues de Oncologia de Lisboa Francisco Gentil,EPE,Lisboa,Portugal2Servico de Anatomia Patolo¤ gica,Instituto Portugues de Oncologia de Lisboa Francisco Gentil,EPE,Lisboa,Portugal3Gastroenterologyand Hepatology,Erasmus MCUniversity Medical Center,Rotterdam,The Netherlands4Servico de Gastrenterologia,Instituto Portugues de Oncologia de Lisboa Francisco Gentil,EPE,Lisboa,Portugal5Cl|�nica de Risco Familiar,Instituto Portugues de Oncologia de Lisboa Francisco Gentil,EPE,Lisboa,Portugal6Servico de Gene¤ tica,Instituto Portugues de Oncologia do Porto Francisco Gentil,EPE- Porto,Portugal7Servico de Gastrenterologia,Hospital dos Capuchos,Lisboa,Portugal8Departmentof Pathology,Josephine Nefkens Institute,Erasmus MC,Rotterdam,The Netherlands

It is unclear whether the mutation spectra in WNT genes vary among distinct types of colorectal tumors. We have ana-

lyzed mutations in specific WNT genes in a cohort of 52 colorectal tumors and performed a meta-analysis of previous

studies. Notably, significant differences were found among the mutation spectra. We have previously shown that in familial

adenomatous polyposis, APC somatic mutations are selected to provide the ‘‘just-right’’ level of WNT signaling for tumor

formation. Here, we found that APC mutations encompassing at least two b-catenin down-regulating motifs (20 a.a.

repeats) are significantly more frequent in microsatellite unstable (MSI-H) than in microsatellite stable (MSS) tumors where

truncations retaining less than two repeats are more frequent (P ¼ 0.0009). Moreover, in cases where both APC hits are

detected, selection for mutations retaining a cumulative number of two 20 a.a. repeats became apparent in MSI-H tumors

(P ¼ 0.001). This type of mutations were also more frequent in proximal versus distal colonic tumors, regardless of MSI

status (P ¼ 0.0008). Among MSI-H tumors, CTNNB1 mutations were significantly more frequent in HNPCC than in spo-

radic lesions (28% versus 6%, P < 10-6) and were preferentially detected in the proximal colon, independently of MSI sta-

tus (P ¼ 0.017). In conclusion, the observed spectra of WNT gene mutations in colorectal tumors are likely the result

from selection of specific levels of b-catenin signaling, optimal for tumor formation in the context of specific anatomical

locations and forms of genetic instability. We suggest that this may underlie the preferential location of MMR deficient

tumors in the proximal colon. VVC 2010 Wiley-Liss, Inc.

INTRODUCTION

The majority of colorectal tumors are thought

to be initiated by mutations in the APC (adeno-

matous polyposis coli) tumor suppressor gene

whose loss of function underlies the transition

from the normal polarized intestinal epithelium

to dysplastic adenomatous lesion (Fodde et al.,

2001). The APC protein encompasses a broad

spectrum of functions, ranging from control of

the WNT signal transduction pathway, to cell ad-

hesion, migration, cell cycle regulation, apoptosis,

chromosomal segregation and more recently in

cell proliferation and differentiation (Fodde,

2003). Despite its multi-functionality, APC’s

main tumor suppressing function resides in its

capacity to down-regulate intracellular b-cateninlevels along the WNT signal transduction path-

way (Fodde et al., 2001). APC, together with the

Ser/Thr kinases GSK3b (glycogen synthase ki-

nase 3 beta) and CK1 (casein kinase 1), and the

scaffold proteins Axin1 and Conductin (Axin2),

Supported by: Science and Technology Foundation (FCT),Grant number: POCTI/CBO/47823/2002; Calouste GulbenkianFoundation.

*Correspondence to: Cristina Albuquerque, Centro de Investigacaode Patobiologia Molecular, Instituto Portugues de Oncologia deLisboa Francisco Gentil, Rua Prof. Lima Basto 1099-023 Lisboa.E-mail: [email protected]

Received 29 November 2009; Accepted 9 April 2010

DOI 10.1002/gcc.20786

Published online 7 May 2010 inWiley InterScience (www.interscience.wiley.com).

VVC 2010 Wiley-Liss, Inc.

forms the so-called ‘‘destruction complex’’ that

binds to b-catenin and promotes its down-regula-

tion, thus preventing signaling activity to the

nucleus. In the absence of functional APC, b-cat-enin is not efficiently degraded and it translocates

to the nucleus where, together with distinct

members of T-cell family of transcription factors

(TCFs), modulates expression of several Wnt

downstream target genes (Polakis, 2000) (http://

www.stanford.edu/�rnusse/pathways/targets.html).

Virtually all APC mutations result in truncated

proteins that lack all Axin/Conductin binding

motifs and a variable number of 20 a.a. repeats

associated with the down-regulation of intracellu-

lar b-catenin levels (Miyoshi et al., 1992; Powell

et al., 1993; Miyaki et al., 1994; van der Luijt

et al., 1997; Laurent-Puig et al., 1998). Thus,

APC mutations affect the stability and functional-

ity of the destruction complex leading to intracel-

lular accumulation of b-catenin and constitutive

signaling to the nucleus (Korinek et al., 1997).

APC mutations have been found in the major-

ity of MSS (microsatellite stable) tumors and in

20–50% of MSI-High cancers, both from sporadic

and HNPCC (hereditary nonpolyposis colorectal

cancer) patients carrying germline mutations in

MMR genes (Huang et al., 1996, 2004; Homfray

et al., 1998; Rowan et al., 2000). Colorectal

tumors with an intact APC gene, mostly MSI-H,

often carry oncogenic b-catenin (CTNNB1) muta-

tions which make the resulting protein resistant

to proteolytic degradation (Kitaeva et al., 1997;

Mirabelli-Primdahl et al., 1999; Morin et al.,

1997; Muller et al., 1998; Sparks et al., 1998;

Miyaki et al., 1999; Lovig et al., 2002; Johnson

et al., 2005). Moreover, mutations in other WNT

genes like axin (AXIN1) and conductin (AXIN2)have also been reported in MSI tumors and

shown to result in the intracellular accumulation

of b-catenin (Salahshor et al., 1999; Liu et al., 2000;

Satoh et al., 2000; Shitoh et al., 2001; Shimizu

et al., 2002; Suraweera et al., 2006).

Notably, somatic APC mutations in polyps from

patients affected by familial adenomatous polypo-

sis (FAP) are selected based on their residual

b-catenin regulatory activity (Rowan et al., 2000;

Albuquerque et al., 2002a; Crabtree et al., 2003;

Cho et al., 2006). Likewise, the spectra of muta-

tions in WNT genes may follow specific patterns

depending on the sporadic and hereditary nature

of the colorectal tumors, their MSS and MSI sta-

tus, and their location along the GI tract. To

address these issues in a more systematic fashion,

we have performed somatic mutation analyses of

the APC, CTNNB1, and AXIN2 genes in a cohort

of 52 colorectal tumors stratified by their sporadic

or hereditary nature, MSI status, and anatomical

location. In parallel, we performed a meta analy-

sis by employing the mutation analysis data from

colorectal tumors, stratified by MSI status and an-

atomical location, reported to date in the scien-

tific literature (Aoki et al., 1994; Huang et al.,

1996, 2004; Kitaeva et al., 1997; Olschwang et al.,

1997; Yagi et al., 1997; Homfray et al., 1998; Mul-

ler et al., 1998; Otori et al., 1998; Miyaki et al.,

1999; Salahshor et al., 1999; Rowan et al., 2000;

Shitoh et al., 2001; Aust et al., 2002; Lovig et al.,

2002; Shimizu et al., 2002; Smith et al., 2002;

Miyaki et al., 2003; Giaretti et al., 2004; Johnson

et al., 2005; Suraweera et al., 2006).

MATERIALS AND METHODS

Patients and Tumor Samples

Thirty patients with sporadic colorectal cancer

(CRC) and seventeen patients with HNPCC

were included in this study. All sporadic CRC

patients had no known familial history suggestive

of HNPCC or FAP. All HNPCC patients either

belonged to families fulfilling the Amsterdam cri-

teria for HNPCC classification or carry previously

identified germline mutations in DNA mismatch

repair (MMR) genes (Table 2).

Fifty-two colorectal neoplastic lesions were

included: 20 sporadic MSS carcinomas, 10 spo-

radic MSI-H carcinomas, and 12 carcinomas and

10 adenomas from HNPCC patients.

Fresh colorectal adenomas and carcinomas

were obtained from surgical or colectomy speci-

mens from patients who underwent surgery or

colonoscopy in the Portuguese Institute of Oncol-

ogy Francisco Gentil in Lisbon. Sections from

corresponding areas from the specimens submit-

ted for diagnosis were cut in two parts: one was

snap-frozen in liquid nitrogen immediately after

resection until processing for DNA isolation,

while the other was formalin-fixed and paraffin-

embedded for IHC analysis. Dysplasia grading, tu-

mor differentiation, according to the WHO guide-

lines, and the immunohistochemical analysis were

performed by an experienced pathologist (IF).

Moreover, a systematic review of the literature

using Pubmed and the Catalogue of Somatic

Mutations in Cancer of the Sanger Institute was

conducted and a meta-analysis of previous studies

reporting APC, CTNNB1, or AXIN2 mutations was

performed by evaluating the frequency and type

MUTATION SPECTRA IN WNT GENES 747

Genes, Chromosomes & Cancer DOI 10.1002/gcc

of the mutations according to the MSI status,

sporadic or hereditary nature, and anatomical

location along the colon and rectum.

DNA Isolation

Genomic DNA was extracted from each tumor

sample and matched normal colonic mucosa as

previously described (Albuquerque et al., 2002a).

In some cases, total genomic DNA was isolated

from blood by using a commercial kit (Puregene,

Gentra Systems).

Microsatellite Instability Analysis

Microsatellite instability (MSI) was analyzed

using the Bethesda microsatellite markers (Rodri-

guez-Bigas et al., 1997): D2S123, D5S346, D17S250,BAT-25, and BAT-26. Each tumor and paired nor-

mal DNA was amplified by PCR and analyzed by

denaturing polyacrylamide gel electrophoresis as

previously described (Albuquerque et al., 2002b).

Analysis of APC Allelic Loss

Allelic loss at the APC locus was analyzed using

microsatellite markers on chromosome arm 5q

selected from the Human Genome Database

based on their proximity to APC, their degree of

heterozygosity, and size range of the alleles. The

microsatellite markers D5S346, D5S656, D5S421,and D5S1965, were analyzed with the Applied

Biosystems ABI PRISM 310 genetic analyzer

using the GENESCAN software. For each

marker an allelic ratio was calculated by dividing

the intensity of the two alleles in normal control

DNA. This allelic ratio was compared with the

allelic ratio obtained for the two alleles of each

tumor sample. A comparative ratio greater than

1.5 was interpreted as significant, i.e., indicative

of allelic loss.

APC Mutation Analysis

APC mutation analysis was performed using

the protein truncation test (PTT) for exon 15,

and denaturing gradient gel electrophoresis

(DGGE) for exons 1-14. PTT was performed as

previously reported (Albuquerque et al., 2002a).

DGGE was performed as previously described

(Fodde et al., 1992; Olschwang et al., 1993)

except that electrophoresis was performed at

160 V in TAE buffer (40 mM Tris-acetate pH 7.5,

20 mM sodium acetate, 1 mM Na2EDTA) at 60�Cduring 4 hr.

CTNNB1 Mutation Analysis

Genomic DNA from each tumor sample was

amplified by PCR for SSCP (single strand confor-

mational polymorphism) analysis of exon 3 of the

CTNNB1 gene using previously described primers

(Koch et al., 1999). The amplified products were

analyzed in a MDE (mutation detection enhance-

ment) gel (FMC) and visualized by silver stain-

ing. The analysis of large deletions encompassing

exon 3 of the CTNNB1 gene was performed as

previously described (Koch et al., 1999). PCR

products were analyzed in a 1.2% agarose gel,

stained with ethidium bromide.

AXIN2 Mutation Analysis

Specific primers were designed to amplify the

region containing the (G)7, (C)6, and (C)5 tracts

in the AXIN2 gene, where a high incidence of

mutations was previously reported (Liu et al.,

2000). The primers used were as follows: AXIN2

F- 50-AGTCTGCCCGCTCGTCTCCA-30 and

AXIN2 R- 50-GTCAGGGGAGGCATCGCAGG-30.The amplified fragments were electrophoresed in a

7% polyacrilamide gel containing 32.5% formamide

and 6.9 M urea at 48W and visualized by silver

staining.

Sequencing Analysis

All mutations detected in the WNT genes,

were sequenced using the BigDye terminator

cycle sequencing kit (Applied Biosystems) and

analyzed on the ABI PRISM 310 genetic analyzer

(Applied Biosystems) using the SEQUENCING

ANALYSIS software.

Immunohistochemical Analysis

b-catenin, MSH2, MLH1 and MSH6 expres-

sion was evaluated using immunohistochemistry

(IHC) as previously described (Albuquerque

et al., 2002b). Negative and positive controls

were used to score for the presence/absence of

b-catenin expression, and to evaluate its relative

intensity (scored as either negative, weak/focal

(1þ), moderate (2þ) or strong/diffuse (3þ)) and

subcellular location (classified as membrane-

bound, cytoplasmic and nuclear). Controls were

analyzed simultaneously with the tumor samples.

Statistical Analysis

Differences in mutation frequencies among

distinct groups of colorectal tumors, stratified by

748 ALBUQUERQUE ETAL.


MSI status and anatomical location, were eval-

uated by the Fisher’s exact test.

RESULTS

Mutation analysis of the APC, CTNNB1,

and AXIN2 Genes

Tables 1 and 2 summarize the results of the

mutation, MSI, and immunohistochemistry analyses

of our CRC cohort in relation to tumor location

along the GI tract, histological type, and, in the

case of HNPCC tumors, MMR germline mutation.

We have detected mutations in APC, CTNNB1(b-catenin) or AXIN2 in most colorectal tumors

irrespective of their MSI status and sporadic or

hereditary nature. Overall, mutations in these

genes were found in 85% (17/20) of sporadic

MSS carcinomas, 60% (6/10) of sporadic MSI-H

carcinomas, and in 73% (16/22) of the HNPCC

tumors (both adenomas and carcinomas). Immuno-

histochemistry (IHC) analysis of intracellular

b-catenin accumulation underscored the central

role played by constitutive activation of the

WNT/b-catenin signaling pathway in CRC,

regardless of their MSI status and sporadic or he-

reditary nature. In contrast with its membrane-

bound distribution in normal colonic epithelium,

b-catenin was detected in the cytoplasm and/or in

the nucleus of the vast majority (32/36; 89%) of

the tumors carrying APC, CTNNB1, or AXIN2

TABLE 1. Results of the Mutation Analysis of APC, CTNNB1 and AXIN2 in Sporadic Colorectal Carcinomas Stratifiedfor MSI Status, Anatomical Location, and Histo-Pathological Classification

Carcinomas Histology Location

APC

CTNNB1 AXIN2Point mutation (codon

affected and type of change) Allelic loss

MSS39 Md Ascending N N þ (partial del of exon 3) N15 Md Splenic flexure G1303X C!T N N N61 Md Descending 1300 insA (TAAATAC) N N N47 Md Descending R1114X C!T þ N N51 Muc Descending E1309X G!T NI N N7 Md Sigmoid 903 del2bp N N N27 Md Sigmoid 1309 delAAAGA þ N N29 Wd Sigmoid N N N N37 Md Sigmoid 785 insTC (TCTCTCAT);

Q1367X C!TN N N

55 Wd Sigmoid N N N N57 Md Sigmoid N þ N N59 Md Sigmoid N N N N63 Md Sigmoid R1450X C!T N N N65 Wd Sigmoid E1451X G!T N N N69 Wd Sigmoid E1397X G!T N N N25 Md Rectum E868X G!T N N N31 Md Rectum R232X, C!T N N N33 Wd Rectum R1114X C!T þ N N35 Wd Rectum R232X C!T; 1317

delG (GCTGAA)N N N

53 Wd Rectum IVS 833-6 A!G (splice site) NI N NMSI-H

E-425T Md Caecum N NI S45F N45 Wd Ascending G1477X C!T NI N N97 Pd Ascending N NI N ins G139 Wd, muc Ascending 1495 a) NI N NE-443T Wd Ascending R876X C!T; R1450X C!T NI N N49 Wd, muc Ascending N NI N N5 Wd Descending N NI N N83 UN UN N NI N N3 Wd Rectum N N N NE-429T Wd Rectum R1450X C!T NI N N

Wd, well differentiated; md, moderately differentiated; pd, poor differentiated; muc, mucinous; N, normal; NI, not informative (due to homozigozity

and/or to MSI); þ, positive; ins, insertion; del, deletion; UN, unknown.aNot sequenced due to technical reasons.



mutations with IHC patterns ranging from weak/

focal (1þ) to strong/diffuse (3þ) (Table 3). Overall,

intracellular b-catenin accumulation was detected

in the majority of colorectal tumors analyzed,

namely in 100% (20/20) of sporadic MSS tumors,

89% (8/9) of the sporadic MSI-H tumors, and in

60% (6/10) and 78% (7/9) of the HNPCC adenomas

and carcinomas, respectively. Accordingly, a meta-

analysis of previously published data shows that

mutations in WNT genes are detected in the vast

majority of CRC (�90%), even in MSI-H CRC

(Table 4).

However, differences were observed in the fre-

quency and in the type of mutations in the WNT

genes according to the MSI status, sporadic or here-

ditary nature and to the anatomical location.

Association of Somatic WNT Gene Mutations

With MSI and Sporadic/Hereditary Status

Mutation frequencies

Differences in the WNT gene mutation fre-

quencies were found between MSS and MSI-H

tumors and, among the latter, between sporadic

TABLE 2. Results of the Mutation Analysis of APC, CTNNB1 and AXIN2 in HNPCC Tumors in Relation to MMR GermlineMutation and/or Loss of Tumor-Specific Expression, MSI Status, Anatomical Location, and Histological Classification

Tumors Location Histology

MMR germlinemutation or

expression deficiency

APC

CTNNB1 AXIN2

Point mutation(codon and

type of change)Allloss

CarcinomasMSI-H

13 Caecum md MLH1 E721X, T!A N NI N NT7 Caecum md MLH1 IVS1-11 T!A splic. N N S45P N287T Ascending md in course N NI S45F N639 Ascending md MSH2 R236X, C->T N N N N280T1 Hepatic flexure pd, muc MSH2 IVS1277-2 A!C splic. N NI N ins G101 Transverse pd, muc MLH1 R26X, C!T N N S45F N287T1 Transverse wd in course N N S45F N325 Rectum md, muc MSH2 849 insA (AAAAAA);

G1450X C!TNI N N

T16 Rectum UN in course R1114X C!T;1593 insA(AAAAAAA)

NP N N

HO1 UN UN NP R876X C!T;1455 delA(AAAAA)

NP N N

MSSL183T3 Splenic flexure muc MLH1 1976 G!T splic. N N N NL184T4 Splenic flexure muc MLH1 1976 G!T splic. N N N N

AdenomasMSI-H

287A2 Ascending TA, LGD MLH1 1þ <50% N N N N429A1 Transverse TA, LGD MLH1 1þ 90% N NI T41A N429A2 Transverse TA, LGD NP N N T41A NL397A Sigmoid TA, HGD MSH2 R236X, C->T N NI N del GL440A Rectum TA, LGD/HGD MSH2 2þ �20% N NI N N

MSS323A1 Ascending TA, LGD Normal expression G1429X C!T þ N NL447A Ascending TA, LGD/HGD MSH6 3þ �40% 997 delT (AGTTAT);

1494 del16 bpN N N

375A Sigmoid TA, LGD MSH2 2þ �80% R216X C!T þ N NL459A Sigmoid TA, LGD MSH2 2þ �20% R876X C!T;

1310 delAA(AGAAAAG)

N N N

L280A Sigmoid TA, LGD MSH2 - 1182 delA (AAAA);1420 delC(CCCC)

N N N

All loss, allelic loss; wd, well differentiated; md, moderately differentiated; pd, poor differentiated; muc, mucinous; TA, tubular adenoma; LGD, low

grade dysplasia; HGD, high grade dysplasia; N, normal; NI, not informative (due to homozygozity or to MSI); UN, unknown; NP, not performed; þ,

positive; ins, insertion; del, deletion; splic., splice site mutation. L183T3, L183T4 and L180A tumors were considered MMR mutant.



TABLE 3. Results of b-Catenin IHC Analysis in Sporadic and HNPCC Tumors

Tumor

Sporadic tumors HNPCC tumors

b-catenin expression b-catenin expression

Nucleus Cytoplasm Membrane Nucleus Cytoplasm Membrane

Carcinomas CarcinomasMSS MSI-H

39 3þ 3þ 1þ 13 – 2þ (50%) 3þ15 2þ 2þ – T7 – 1þ 3þ61 3þ (25%) 3þ 2þ 287T 2þ (50%) 3þ 3þ47 2þ 3þ – 639 – – 3þ51 2þ (rare) 2þ – 280T1 – 1þ 1þ7 2þ 3þ 1þ 101 3þ 3þ –27 – 2þ 2þ 287T1 NP NP NP29 3þ (10%) 3þ 1þ 325 – 2þ 3þ37 3þ (10%) 3þ 1þ T16 – – 3þ55 3þ (<50%) 3þ – HO1 NP NP NP57 3þ (20%) 3þ –59 – 2þ 3þ MSS63 2þ 3þ 1þ L183T3 2þ 3þ –65 3þ (10%) 3þ 2þ L184T4 NP NP NP69 3þ (20%) 2þ –25 3þ (20%) 3þ 1þ Adenomas31 – 2þ – MSI–H33 – 2þ 2þ 287A2 3þ 2þ –35 – 2þ 2þ 429A1 – 1þ 3þ53 3þ 3þ – 429A2 – 2þ 3þ

L397A – 2þ 2þMSI-H L440A – – 3þ

E-425T 3þ (50%) 3þ 2þ45 2þ (20%) 3þ 2þ MSS97 – 2þ 1þ 323A1 – – 3þ139 3þ (30%) 3þ 2þ L447A 1þ (focal) 1þ (focal) 2þ

E-443T – 3þ 1þ L459A – – 3þ49 – – 2þ 375A – 1þ 3þ5 – 1þ 2þ L280A – – 3þ3 3þ 3þ 1þE-429T 3þ 3þ 1þ83 NP NP NP

NP, not performed. b-catenin expression was scored as negative (-); weak (1þ); moderate (2þ) and intense (3þ). The percentages reported next

to a subset of scores indicate the proportion of tumor cells presenting with that particular score.

TABLE 4. Results of the Mutation Analysis of MSI-H Carcinomas, Comprising the Present and Previously Published Studies

Tumors

Mutations

APC CTNNB1 AXIN2 AXIN1

Sporadic MSI-HPresent study 4/10 (40%) 1/10 (10%) 1/10 (10%) –Previous studies (a) 64/144 (44%) 9/161 (6%) 4/14 (28%) 6/26 (23%)

Total 68/154 (44%) 10/171 (6%) 5/24 (20%) 6/26 (23%) � 93% WNT mutationsHNPCC MMR-mutant

Present study 3/12 (25%) 4/12 (33%) 1/12 (8%) –Previous studies (a) 30/72 (42%) 23/85 (27%) – 1/7 (14%)

Total 33/84 (39%) 27/97 (28%) 1/12 (8%) 1/7 (14%) � 89% WNT mutations

(a) Mutation data reported by Huang et al. (1996, 2004), Homfray et al. (1996), Kitaeva et al. (1997), Muller et al. (1998), Miyaki et al. (1999), Sal-

ahshor et al. (1999), Shitoh et al. (2001), Shimizu et al. (2002), Lovig et al. (2002), Johnson et al. (2005) and Suraweera et al. (2006). Despite the

extensive mutational data available in the literature for MSI-H tumors, these studies were selected based on their separate analysis of sporadic

MSI-H and HNPCC MMR-mutant tumors.



and HNPCC MMR deficient tumors. As

expected, APC loss of function defects, either

point mutations and/or allelic imbalance, were

significantly more frequent among sporadic MSS

carcinomas (16/20; 80%) than in sporadic MSI-H

carcinomas (4/10; 40%, P ¼ 0.034) and HNPCC

MMR mutant carcinomas and adenomas (4/18;

22%, P ¼ 0.00044). Likewise, CTNNB1 and

AXIN2 mutations were only detected in sporadic

MSI-H and HNPCC tumors, except for one

CTNNB1 deletion detected in 1/20 sporadic MSS

carcinomas. Similarly, whereas APC mutations

were detected in all HNPCC MSS adenomas (4/

4), only one was found among the six MSI-H/

MMR-deficient adenomas where, instead, two

CTNNB1 and one AXIN2 mutations were identi-

fied (Tables 1 and 2).

Among the MSI-H tumors, CTNNB1 mutations

appeared to be more frequent in HNPCC MMR

deficient than in sporadic MSI-H carcinomas (4/

12 versus 1/10). A subsequent CTNNB1 mutation

analysis of a series of paraffin embedded carcino-

mas (7 sporadic MSI-H and 13 HNPCC cases)

confirmed this trend: mutations were found in

14% (1/7) and 31% (4/13) of sporadic MSI-H and

HNPCC carcinomas, respectively (data not

shown). However, APC and AXIN2 mutation fre-

quencies did not appear to differ between spo-

radic and HNPCC MMR deficient carcinomas [4/

10 (40%) and 1/10 (10%) versus 3/12 (25%) and 1/

12 (8%), respectively] (Tables 1 and 2). A meta-

analysis of previous studies merged with the

results presented here confirmed that APC muta-

tion frequency does not differ between HNPCC

MMR deficient and sporadic MSI-H carcinomas

[33/84 (39%) versus 68/154 (44%)]. In contrast,

CTNNB1 mutations are significantly more fre-

quent in the HNPCC MMR-mutant cancers

when compared with sporadic MSI-H carcinomas

[27/97 (28%) versus 10/171 (6%), P < 10�6]

(Table 4). Notably, the frequencies of CTNNB1mutations appear to vary with the disease-causing

MMR gene (Tables 1 and 2): CTNNB1 mutations

were more frequently detected in tumors from

patients carrying germline MLH1 mutations than

in tumors associated to MSH2 germline defects

(4/4 versus 0/4, P ¼ 0.014, Fisher’s exact test).

The latter presented somatic mutations either in

APC (2/4) or in AXIN2 (2/4).

Type of mutations

In polyps from FAP patients, somatic APCmutations are known to be selected based on the

retention of different numbers of b-catenindown-regulating motifs (20 a.a. repeats) in the

corresponding truncated proteins (Rowan et al.,

2000; Albuquerque et al., 2002a; Crabtree et al.,

2003; Cho et al., 2006). A similar selection bias

seems to operate in our cohort of sporadic and

HNPCC colorectal tumors (Table 5 and Fig. 1).

APC mutations located 30 to codon 1397 (resulting

in truncated proteins retaining at least two 20 a.a.

repeats) were significantly more frequent among

sporadic MSI-H and HNPCC MMR-mutant

tumors (8/13; 62%) than in sporadic MSS tumors

(3/17; 18%) (P ¼ 0.016). In the latter group, APCmutations encompassing one or no 20 a.a. repeats

were prevalent (14/17; 82%). A meta-analysis of

previous studies was performed by evaluating

previously reported mutation data with respect to

the resulting number of 20 a.a. repeats. The out-

come of the meta-analysis were then merged

with the results obtained in this study which con-

firmed that APC mutations retaining two or more

20 a.a. repeats were significantly more frequent

among MSI-H tumors, either HNPCC or sporadic

(50/98; 51%), than among MSS tumors (105/311;

34%) (P ¼ 0.0009) (Table 5). Likewise, APCmutations retaining one or none 20 a.a. repeat are

prevalent among MSS tumors (206/311; 66%).

Moreover, the relative incidence of APC muta-

tions retaining one 20 a.a. repeat is clearly lower

than those encompassing 2 of the same motifs

among HNPCC and sporadic MSI-H tumors [18/

98 (18%) versus 50/98 (51%)]. In contrast, among

sporadic MSS tumors these two mutation types

are equally frequent [114/311 (37%) versus 105/

311 (34%), P ¼ 0.0001] (Table 5).

A more detailed analysis of the tumors from

our series where two independent APC mutations

were detected indicates that HNPCC MMR-mu-

tant and sporadic MSI-H tumors may differ from

sporadic MSS tumors in the type of interdepend-

ence between the two somatic hits. In the

HNPCC/sporadic MSI-H group, allelic losses or

mutations leading to truncated proteins lacking

all 20 a.a. motifs were frequently associated with

mutations encompassing 2 or more 20 a.a. repeats

(5/5 tumors). The latter differs from the results

obtained in sporadic MSS tumors where APCallelic losses or mutations leading to truncated

proteins lacking all 20 a.a. repeats are invariably

found in association with mutations that result in

truncated proteins with one or none 20 a.a. repeat

(4/5 tumors, P ¼ 0.023). By combining our results

with the meta-analysis data, selection for two in-

dependent mutations retaining in total at least



Figure 1. Schematic representation of the distribution of somaticmutations detected in sporadic MSS, sporadic MSI-H and HNPCC MMR-mutant tumors along the APC protein. APC mutations are depictedaccording to the number of 20 a.a. repeats remaining in the resultingtruncated protein. Mutations retaining two 20 a.a. repeats [orange (orgrey, in black and white version) bars] are significantly more frequent in

sporadic MSI-H and HNPCC MMR-mutant tumors (represented abovethe APC protein) than in sporadic MSS tumors (represented below theAPC protein) (62% versus 18%). In the latter, mutations retaining onlyone 20 a.a. repeat [blue (or black, in black and white version) bars] aremuch more frequent. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

TABLE 5. Distribution of APC Mutations in Sporadic MSS and Sporadic/HNPCC MSI-H Tumors Accordingto the Number of 20 a.a. Repeats Retained by APC Truncating Mutations

Number of 20 a.a. repeats retainedby each APC truncating mutation

Cumulative number of 20 a.a.repeats retained by APCmutations in tumors withtwo independent hits

Sporadic MSSSporadic/HNPCCMMR-MUTANT

SporadicMSS

Sporadic/HNPCCMSI-H

0 1 �2 0 1 �2 �1 �2 �1 �2

This study Sporadic MSI-H/HNPCCMMR-mutant

8 6 3 5 0 8 4 1 0 5

Olschwanget al. (1997)

Sporadic MSSCRC

36 17 20 – – – 19 20 – –

Huang et al.(1996)

Sporadic MSSand MSI-H

18 18 28 13 4 20 0 6 2 6

Homfrayet al. (1998)

Sporadic MSSand MSI-H andHNPCC MSI-H

4 14 11 4 13 3 2 4 1 8

Rowan et al.(2000)

MSS and MSI-HCRC cell lines

10 7 8 2 0 7 5 14 0 6

Lovig et al.(2002)

Sporadic MSSand MSI-H

16 52 35 1 0 6 3 4 – –

Huang et al.(2004)

HNPCCMSI-H

– – – 5 1 6 – – 0 2

All studies 92 114* 105* 30 18* 50* 33** 49** 3** 27**(30%) (37%) (34%) (31%) (18%) (51%) (40%) (60%) (10%) (90%)

206***(66%)

105***(34%)

48***(49%)

50***(51%)

MSS, microsatellite stable; MSI-H, microsatellite instability; CRC, colorectal carcinomas.

*P ¼ 0.0001; **P ¼ 0.001; ***P ¼ 0.0009.



two 20 a.a. repeats was observed in 27/30 (90%)

of the HNPCC and sporadic MSI-H tumors. This

APC mutation spectrum differs from what is

observed among sporadic MSS tumors where the

genotypes resulting in two or more 20 a.a. repeats

or in one or none 20 a.a. repeat are represented

in a 60:40 ratio [49/82 (60%) versus 33/82 (40%),

respectively, (P ¼ 0.001)] (Table 5). This sug-

gests a specific selection bias for truncated APC

proteins encoding for some residual b-catenindown-regulating activity among sporadic MSI-H

and HNPCC MMR deficient tumors (selection

for two or more 20 a.a. repeats), in contrast with

MSS cases.

With respect to the specific type of APC nucle-

otide substitutions, C-T transitions were exclu-

sively observed among HNPCC MMR deficient

and sporadic MSI-H tumors. The latter is likely

to represent a feature of loss of MMR function as

also indicated by the lower incidence of C-T

transitions (10/10 versus 7/11; P ¼ 0.055, Fisher’s

exact test) and the presence of G-T transversions

among MSS tumors (4/11; 36%). As expected,

among all APC point mutations here detected,

1-2 bp insertions/deletions within mono or dinu-

cleotide repeats were more frequently observed

among HNPCC MMR-mutant (5/8; 62%) tumors

when compared with sporadic MSI-H (0/4; P ¼0.07, Fisher’s exact test) and MSS (2/17, 12%; P¼ 0.016, Fisher’s exact test) cases.

Association of Somatic WNT Gene Mutations

With Anatomical Location

Besides the observed differences in the WNT

gene mutation spectra according to MSI status,

we also observed differences according to tumor

location along the colon and rectum, independ-

ently of MSI status. Accordingly, we included in

this analysis only the sporadic and HNPCC MSS

tumors from our cohort and merged them with

the results from the meta-analysis of sporadic

MSS tumors and consecutive series of sporadic

adenomas or carcinomas (among sporadic carcino-

mas, MSI frequency is usually lower than 15%,

thus lowering significantly the bias introduced by

MSI-H carcinomas). We observed that APC muta-

tions encompassing at least two 20 a.a. repeats

were significantly more frequent in tumors with a

proximal location (caecum, ascending and trans-

verse colon) than in those from the descending/

sigmoid colon [26/37 (70%) versus 25/79 (32%),

P ¼ 0.00008] or in the rectum [26/37 (70%) ver-

sus 9/31 (29%), P ¼ 0.00062] (Table 6). In the

TABLE 6. Distribution of APC Mutations Along the Colon and Rectum According to the Number of 20 a.a. Repeats Retained byEach Truncating Mutation in Sporadic Colorectal Carcinomas (CRC) and Adenomas, Excluding MSI-H Tumor Series

Number of 20 a.a. repeats retained by each APC truncating mutation

Proximal Descending/Sigmoid Rectum

0 1 �2 0 1 �2 0 1 �2

This study Sporadic MSS CRC/HNPCC MSS

1 0 2 5 6 3 5 1 0

Rowan et al. (2000) MSS CRC cell lines 0 1 2 3 1 1 1 2 1Giaretti et al. (2004) Sporadic CRC 2 0 1 6 4 2 1 1 2Aust et al. (2002) Sporadic colorectal

adenomas0 1 1 1 6 1 – – –

Yagi et al. (1997) Sporadic MSS CRC 0 1 4 0 5 4 – – –Otori et al. (1998) Sporadic colorectal

adenomas1 0 2 0 0 2 – – –

Aoki et al. (1994) Sporadic CRC – – 1 – – – – 1 –Miyaki et al. (2003) Sporadic carcinoma

and adenomas0 2 4 – – – – – –

Smith et al. (2002) Sporadic CRC 0 2 9 4 13 12 0 10 6All studies 4 7*,** 26*,** 19 35* 25* 7 15** 9**

(11%) (19%) (70%) (24%) (44%) (32%) (23%) (48%) (29%)11***,**** 26***,**** 54*** 25*** 22**** 9****(30%) (70%) (68%) (32%) (71%) (29%)

MSS, microsatellite stable. Only MSS sporadic tumors or random series of sporadic colorectal tumors with unknown microsatellite instability status

were included in this meta-analysis.

*P ¼ 0.0004, **P ¼ 0.0016, ***P ¼ 0.00008, ****P ¼ 0.00062.



descending/sigmoid colon and in the rectum,

truncating proteins encompassing either one or

none of the 20 a.a. repeats were more frequent

[54/79 (68%) and 22/31 (71%), respectively], with a

preference for one 20 a.a. repeat with respect to

none [35/79 (44%) versus 19/79 (24%) in the de-

scending/sigmoid colon and 15/31 (48%) versus 7/

31 (23%) in the rectum]. The analysis of the com-

bination of both APC hits with respect to anatomi-

cal location was not possible because of the lack of

information concerning tumor location in the ma-

jority of studies where both hits were identified.

The incidence of CTNNB1 mutations among spo-

radic MSI-H and HNPCC MMR deficient groups

also varied with the location of the tumor along

the colorectal tract (Tables 1 and 2). We detected

CTNNB1 mutations in 7/16 (44%) of the proximal

tumors but in none of the 10 distal or rectal tumors

(P ¼ 0.017).

DISCUSSION

It is generally accepted that constitutional acti-

vation of the WNT signaling pathway is a rate-

limiting step for colorectal tumor formation (Pola-

kis, 2000; Fodde et al., 2001; Fodde, 2003).

Although several studies have previously reported

mutational analysis of WNT genes in sporadic

MSS, MSI-H, and HNPCC tumors, most did not

address HNPCC and sporadic MSI-H tumors as

distinct entities and the relative role of the ana-

tomical location of the tumors. Here, we have

carried out a thorough mutation analysis of the

APC, CTNNB1, and AXIN2 genes in a cohort of

52 colorectal tumors comprehensive of sporadic

MSS and MSI-H carcinomas, and of HNPCC

adenomas and carcinomas, together with the IHC

analysis of b-catenin and the main MMR genes.

Overall, mutations in APC, CTNNB1, or AXIN2genes were found in the vast majority of tumors

of the present cohort, thus underscoring the cen-

tral role of the WNT signaling pathway not only

in MSS tumors but also in both hereditary and

sporadic MMR-deficient colorectal tumors, as also

validated by the presence of b-catenin intracellu-

lar accumulation in most HNPCC (13/19; 68%)

and sporadic MSI-H (8/9; 89%) tumors (Table 3).

In tumors characterized by intracellular b-cateninaccumulation, but without APC, CTNNB1 or

AXIN2 mutations, alternative (epi)genetic defects

in additional WNT genes not analyzed in our

study are likely to underlie tumor formation and

progression.

In parallel with the above mentioned mutation

data, we used the considerable body of mutation

analysis in colorectal tumors, stratified by MSI

status, hereditary nature and by anatomical loca-

tion, reported to date in the scientific literature.

As shown in Table 4, the overall incidence of

mutations in WNT genes in either sporadic or

HNPCC MSI-H groups adds up to approximately

90% of the total cases studied. This high fre-

quency among sporadic MSI-H tumors is similar

to that of MLH1 hypermethylation and BRAF on-

cogenic mutations (Higuchi and Jass, 2004).

Hence, in sporadic MSI-H tumors, genetic and

epigenetic mutation events at these genes are

likely to coexist in the same tumors. Accordingly,

in our cohort we detected the V600E BRAFmutation in 4/10 sporadic MSI-H tumors (data

not shown), two of which carry APC and AXIN2mutations, respectively. The same is true for the

G13D and G12D K-RAS oncogenic mutations

found in 3/10 sporadic MSI-H tumors (data not

shown), two of which also carry APC mutations.

MLH1 hypermethylation was also detected in

8/10 tumors, five of which carry APC (three

cases), CTNNB1 and AXIN2 mutations.

Notably, in addition to the high contribution of

WNT gene mutations in CRC irrespective of

MSI status, the WNT mutation spectra revealed

significant differences with respect to the fre-

quency and type of mutations according to the

type of genetic instability, hereditary nature and

tumor location. The analysis of the WNT genes

mutation spectra, combining the meta-analysis

with our results, confirmed that, whereas sporadic

MSS tumors mainly carry APC defects (80%),

HNPCC and sporadic MSI-H lesions revealed a

more heterogeneous spectrum with CTNNB1 and

AXIN2 mutations and with a lower frequency of

APC mutations (40%). However, the latter two

groups differ in the relative frequency of

CTNNB1 mutations with a significantly increased

incidence in HNPCC MMR deficient than in

sporadic MSI-H tumors (28% versus 6%, respec-

tively) (Table 4).

A more detailed analysis of the WNT genes

mutation spectra shows that the distribution of

APC mutations also differs between MSS and

MSI-H tumors. This observation is only partly

explained by the non-random distribution of short

repeats along the APC coding region, known to

represent preferential mutation targets in MMR-

deficient cells. Of note, we found a significant

increase in the incidence of mutations leading to

truncating proteins retaining two 20 a.a. repeats



(51%) among the MSI-H sporadic/HNPCC cases

when compared with mutations encompassing

one or none repeat (18% and 31%, respectively).

This was in sharp contrast with MSS tumors

where truncating proteins encompassing none,

one or two 20 a.a. repeats are more equally dis-

tributed (30%, 37% and 34%, respectively) (P ¼0.0009) (Table 5). The difference between trun-

cating proteins retaining two and one 20 a.a.

repeat (respectively, 51% versus 18% in the spo-

radic/HNPCC MSI-H group and 34% versus 37%

in the MSS group, P ¼ 0.0001) further highlights

selection for two, instead of one, 20 a.a. repeats

in the MSI-H group. Moreover, this difference in

APC mutation spectra between sporadic MSS and

sporadic/HNPCC MSI-H tumors is confirmed

when looking at the interdependence between

two somatic APC hits in the same tumor. Accord-

ingly, the preference for two 20 a.a. repeats as a

result of the combination of both APC hits is evi-

dent in the sporadic/HNPCC MSI-H group (90%

versus 10%), when compared with the MSS cases

(60% versus 40%, P ¼ 0.001) (Table 5).

Notably, truncating mutations retaining two 20

a.a. repeats are also more frequent in proximal

than in descending/sigmoid tumors (70% versus

32%, P ¼ 0.00008) (Table 6). The higher fre-

quency of this type of APC mutations among

proximal colonic tumors could result from the

preferential proximal location of sporadic MSI-H

and HNPCC tumors and from the non-random

distribution of mono and dinucleotide repeats,

more prone to MMR errors, in the proximity of

codons 1,400–1,500. However, this should not

play any major confounding role in our data set

as we included only MSS tumors in addition to

sporadic adenomas where MSI is rarely detected,

and sporadic carcinomas where the frequency of

MSI-H cases is approximately 15%. Accordingly,

CTNNB1 mutations were significantly associated

with a proximal location among MSI-H tumors

from the present cohort (P ¼ 0.017) which further

suggests that distinct WNT gene mutation spec-

tra are selected along the different segments of

the colon. A schematic representation of the col-

orectum showing the most frequent types of

WNT gene mutations in the proximal versus de-

scending/sigmoid colon, according to the nature

of the tumors (MSS, sporadic MSI-H and

HNPCC), is depicted in Figure 2.

The observed nonrandom distribution of APCtruncated alleles retaining different numbers of

20 a.a. repeats, and thus encoding different levels

of b-catenin signaling along the colon and rec-

tum, is reminiscent of the APC mutation spec-

trum in FAP adenomas where APC mutation are

selected to retain a total of one or two 20 a.a.

repeats (Albuquerque et al., 2002a). This correla-

tion between anatomical location and the spectra

of APC somatic mutations was also observed in

FAP adenomas where APC germline mutations

deleting all 20 a.a. repeats were associated with

somatic mutations retaining one or two 20 a.a.

repeats in the descending/sigmoid and in the

proximal colon, respectively. Germline defects

encompassing one repeat were associated with

second hits with no or one repeat, respectively, in

the descending/sigmoid and in the proximal colon

(Albuquerque et al., manuscript in preparation).

It has been described that truncated APC is

required for optimal cell proliferation and to modu-

late the transcriptional activity of b-catenin in a

cell cycle dependent manner (Schneikert and Beh-

rens 2006; Schneikert et al., 2007). Therefore, the

selection for truncated proteins encompassing a dif-

ferent number of 20 a.a. repeats between the prox-

imal and distal colon may reflect the requirement

for distinct b-catenin down-regulation activities.

Recently, Buchert et al. (2010) identified dif-

ferences in the b-catenin signaling threshold lev-

els that determine physiological and pathological

outcomes during embryogenesis, intestinal and

hepatic homeostasis. Similarly, in the human gas-

trointestinal tract an optimal signaling level will

ensure cell homeostasis. Therefore, APC trun-

cated proteins appear to be selected based on the

capacity to overcome the threshold level above

which this homeostasis is disturbed, thus allowing

clonal expansion and neoplastic transformation.

The absence of 20 a.a. repeats as a result of the

two APC mutational events, i.e., too high signal-

ing levels, is selected against, presumably due to

the induction of apoptosis. In agreement with

this, it has been reported that b-catenin overex-

pression and high levels of WNT transcriptional

activity are associated to high apoptotic rates

(Kim et al., 2000; Bordonaro et al., 2008)

As the level of b-catenin signaling appears to

be selected in cancer in a tissue-specific fashion,

as a function of the APC mutations, providing the

‘‘just right’’ level of WNT signaling for tumor for-

mation (Albuquerque et al., 2002a; Groves et al.,

2002; Cho et al., 2006; Gaspar et al., 2009;

Buchert et al., 2010), we suggest that the optimal

level of b-catenin signaling for tumor formation

appears to differ not only between MSS and

MSI-H tumors, sporadic and hereditary, but also

between tumors located in the proximal and



Figure 2. Schematic representation of WNT mutation spectrathroughout the colon and rectum in MSS, sporadic MSI-H andHNPCC MMR-mutant tumors. For each of these three groups oftumors, the frequency of somatic mutations in the WNT genes (APC,CTNNB1, and AXIN2) and the frequency of APC truncating mutationsretaining 1/0 or 2/3 20 a.a. repeats are represented by vertical andhorizontal graphic bars, respectively. In the colorectum image, each

mutation/type of mutation is depicted in the proximal or in the distalside of colon according to its prevalence in each of these two seg-ments. Mutations/types of mutations limited by a square and writtenin bold-type letter are the most prevalent whereas the remainingones are detected less frequently (�10% of the tumors). [Color fig-ure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]



distal colon. At the MSI level, this selection for

distinct levels of b-catenin signaling may be asso-

ciated with differences in apoptosis resistance,

known to be increased in MMR deficient com-

pared with proficient tumors. As for tumor loca-

tion, the different embryological origin and

histological features between proximal and distal

colon may reflect specific and distinct WNT sig-

naling threshold levels to trigger tumor initiation.

Accordingly, the decrease of absorptive cells and

the increase of goblet cells from proximal to dis-

tal colon reflect differences in cell differentiation

previously proposed to explain tumor regionality

in the mouse intestine (Haigis et al., 2004). Like-

wise, the specific differentiation pattern of the

proximal colon should lead to a specific threshold

level for which the just-right signaling for tumor

formation is most likely provided by two or three

20 a.a. repeats (mutations in codons 1400-1500).

This and the increased probability of MMR

errors in mono and dinucleotide repeats in the

proximity of codons 1,400–1,500, may compre-

hensively result in the observed preferential loca-

tion of MMR deficient tumors in the proximal

colon. More recently, it has been also reported

that the proximal and distal colon have distinct

and specific gene expression profiles (Glebov

et al., 2003) and follow different tumorigenic

pathways (Sugai et al., 2006), which further sup-

ports the selection for specific b-catenin signaling

levels in these two regions.

In conclusion, alterations in the WNT pathway

genes APC, CTNNB1, and AXIN2 are found in

the vast majority of colorectal tumors. However,

the frequency and the type of mutations, namely

the number of 20 a.a. repeats retained by the

truncated APC gene mutations, significantly differ

among MSS and MSI-H, sporadic and hereditary,

and proximal and distal colonic tumors. We sug-

gest that, as it was shown for adenomas in FAP

patients carrying APC germline mutations, the

observed distinct patterns of WNT gene muta-

tions in sporadic and HNPCC colorectal tumors

may result from selection for the ‘‘just right’’

level of b-catenin signaling optimal for tumor for-

mation depending on their anatomical location

(proximal or distal colon) and the type of genetic

instability (MSI-H or MSS).

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Colorectal cancers show distinct mutation spectra in members of the canonical WNT signaling pathway...

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