J. Coloproctol. (Rio J.) vol.35 número3

w w w . j c o l . o r g . b r

Journal

of

Coloproctology

Original

Article

Immunohistochemical

study

of

the

canonical

and

non-canonical

Wnt

signaling

pathway

in

colorectal

carcinoma

and

non-neoplastic

mucosa

夽

Rodrigo

Felippe

Ramos

,

Celina

Tizuko

Fujyiama

Oshima

,

Thiago

Simão

Gomes

,

Ana

Maria

Amaral

Antonio

Mader

_,

_Caio

_Dal

_Moro

_Alves

_,

_Jaques

_Waisberg

b_{DepartmentofPathology,EscolaPaulistadeMedicina(EPM),UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

c_{DepartmentofPathology,FaculdadedeMedicinadoABC(FMABC),SantoAndré,SP,Brazil}

d_{DepartmentofSurgery,EscolaPaulistadeMedicina(EPM),UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3March2015 Accepted8June2015 Availableonline8July2015

Keywords:

Colorectalneoplasms Wntproteins Frizzledreceptors Axinsignalingcomplex Immunohistochemistry

a

b

s

t

r

a

c

t

ColorectalcancerislinkedtoseveralsignalingpathwayssuchasWntpathway.Our objec-tiveistodetectandverifytheintegrityofproteinmembersofWntsignalingpathwayin colorectalcarcinomaandnon-neoplasticcolorectaltissue.Sixty-fourpatientswith colorec-talcarcinomaprovidedsamplesofcolorectalneoplasiaandnon-neoplastictissues,which werepreparedintissuemicroarrayblocksandsubjectedtoimmunohistochemicalanalysis. TheprimaryantibodiesusedwereWnt-1,Wnt-2,Wnt-5aFrizzled-1,Frizzled-5andaxin. ImmunoexpressionofWnt-2proteinwassignificantlylowerincolorectaltumortissueand axinproteinimmunoexpressionwassignificantlyhigherintumortissue.Therewasno sig-nificantdifferenceintheexpressionofWnt-1,Wnt-5a,Frizzled-1andFrizzled-5proteins inbothtissues.ThehigherexpressionofWnt-2proteininnon-neoplasticcolorectal tis-suesuggeststheparticipationduringthehyperproliferativestageofcolorectalmucosa.The increasedaxinproteinimmunoexpressionincolorectaltumorsuggestsadecreaseinthe formationofthe␤-catenindestructorcomplex.

©2015SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.

Estudo

imunoistoquímico

da

via

de

sinalizac¸ão

canônica

e

não

canônica

da

proteína

Wnt

em

carcinoma

colorretal

e

em

mucosa

não

neoplásica

Palavras-chave: Neoplasiascolorretais

r

e

s

u

m

o

Ocâncercolorretalestáligadoaváriasviasdesinalizac¸ão,comoaviaWnt.Nossoobjetivo édetectareverificaraintegridadedasproteínasdaviadesinalizac¸ãoWntnocarcinoma

夽

StudycarriedoutattheInterdisciplinarySurgicalSciencePost-GraduationProgram,EscolaPaulistadeMedicina(EPM),Universidade FederaldeSãoPaulo(UNIFESP)intheLaboratoryofExperimentalMolecularPathology,DepartmentofPathology.

∗ _{Correspondingauthor.}

E-mail:rofelippe@terra.com.br(R.F.Ramos).

http://dx.doi.org/10.1016/j.jcol.2015.06.002

ProteínasWnt Receptoresfrizzled Complexodesinalizac¸ãoda axina

Imunoistoquímica

colorretalenotecidocolorretalnãoneoplásico.Sessentaequatropacientescom carci-nomacolorretalforneceramamostrasdeneoplasiaetecidosnãoneoplásicos,queforam colocadasemblocosdetissuemicroarrayesubmetidasàanáliseimuno-histoquímica.Os anticorposprimáriosutilizadosforamWnt-1,Wnt-2,Wnt-5aFrizzled-1,Frizzled-5eaxina. AimunoexpressãodaproteínaWnt-2foisignificativamentemenornotecidotumoralea imunoexpressãodaproteínaaxinafoisignificativamentesuperiornotecidodotumor.Não houvediferenc¸asignificativanaexpressãodeWnt-1,Wnt-5a,frizzled-1enasproteínas Friz-zled1e5emambosostecidos.AmaiorexpressãodeWnt-2daproteínanotecidocolorretal nãoneoplásicosugereaparticipac¸ãodestaproteínaduranteoestágiodehiperproliferac¸ão damucosacolorretal.Oaumentodaimunoexpressãodaproteínaaxinanotumorcolorretal sugereumadiminuic¸ãonaformac¸ãodocomplexodedestruic¸ãodaproteína␤-catenina.

©2015SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.

Introduction

Worldwide,colorectalcancer(CRC)isthethirdmostcommon

malignancyinmenandthesecondmostcommoninwomen.1

In the lower portion ofthe crypts of Lieberkühn, there

arestemcellsthatareresponsiblefortheproliferation, dif-ferentiationandself-renewingofthecolonepithelium.2_The

maintenanceofthestemcellcompartmentandthetransition

fromproliferationtodifferentiationareregulatedbyWntcell signalingreceptors(Winglessandwnt-relatedprotein).3

The Wnt pathway acts during embryogenesis in

verte-brates and invertebrates and in the regulation of cellular

proliferation,differentiationandapoptosis.4

Frizzled,LRP(low-densitylipoproteinreceptor-related pro-tein)5andLRP6areparticipatingreceptorsinthecanonical Wntsignalingpathway,inducingstabilizationofthe␤-catenin

protein in the cytoplasm. This regulation of stability is

mediatedbytheAPC(adenomatouspolyposiscoli)tumor

sup-pressorgenethroughacomplexofproteinsthatalsoincludes,

axin, GSK3 (glycogen synthase kinase 3) and CK1 (casein

kinase1),whichformtheso-calleddestructorcomplexof␤ -catenin. Axin/␤-catenininteraction isan importantcontrol pointfortheWntpathwaythatplaysaroleintumorgenesis.5 Theincreaseinthepoolof␤-catenininthecytoplasmbyWnt stimulationoccursduetoblockingofthedestructorcomplex bytheDisheveled(Dsh)protein,mediatingthetranscription

ofWnttargetgenesinthenucleus.6_{Deletionsormutations}

intheaxingenehavebeenobservedinvarioustumortypes,

includingCRCandhepatocellularcarcinoma.7

Thenon-canonicalpathwayisagenerictermforallWnt

signaling pathways that promote transcription that is not

mediatedby␤-catenin8_{andisactivatedbyFrizzledreceptors} independentlyoftheactivationofLRP5andLRP6.

Thecanonicalpathwayhasreceivedconsiderable

atten-tionovertheyearsduetoitsessentialroleinthehomeostasis

ofthecolonepitheliumandinthegenesisofCRC.However,

themechanismsofactionofthenon-canonicalWntsignaling

pathwayinCRCarenotyetcompletelyunderstood.9

In mammals, 19 types of Wnt proteins have been

described.9_{SomeWntproteinssuchasWnt-3andWnt-1can}

activateboththecanonicalpathwayandthenon-canonical

pathway,whileothers,suchasWnt-5aprotein,appeartobe

specifictothenon-canonicalpathway.8_{Frizzledreceptorscan} receivesignalsfromoneormoreproteinsoftheWntpathway, suchastheFrizzled-5receptor,whichreceivessignalsfrom

boththecanonicalpathway(Wnt-2) andthenon-canonical

pathway(Wnt-5a).10

InCRC, Wnt-1 andWnt-2proteinsare considered

stim-ulators of carcinogenesis,11,12 _{whereas the Wnt-5a protein}

appearstohavetumorsuppressorcharacteristics.10_However, therealroleofFrizzledreceptorsincolorectalcarcinogenesis remainsobscure.13

IdentificationofregulatoryproteinsinvolvedintheWnt

pathway offers opportunities to develop new therapies

directedatCRC14,15_{usingmonoclonalantibodiesagainst}

ther-apeutic targets suchastheWnt-1 andWnt-2 proteinsand

for the axin protein and Frizzled receptors.16 _{De Almeida}

etal.17_{showedthatasolublebiologicalreceptorwascapable} ofblockingtheautocrineWntsignalinvitro,andexperiments

inmiceshowednosignsoftoxicityafterseveralweeks.He

etal.18_{developedamonoclonalantibodycapableofblocking}

theWnt-1proteinandobservedincreasedapoptosisinCRC

celllines.AlthoughstillinphaseI,thesestudieshavealready

demonstratedthattheuseoftheWntsignalingpathwayasa

possibletherapeutictargetispromising.

Theaimofthisstudywastoinvestigatethecanonicaland

non-canonicalpathwaysofWntsignalingincolorectal

carci-nomaandadjacentnon-neoplasticcolorectalmucosathrough

theimmunoexpressionofWnt-1,Wnt-2,Wnt-5a,Frizzled-1,

andFrizzled-5andaxinproteins.

Method

Samples

ThisstudywasapprovedbytheResearchEthicsCommitteesof

theparticipatinginstitutions.Thestudywasconductedusing

samples from 64 patients with CRC who underwent

cura-tiveorpalliativesurgery.Thirty-four(53%)patientsweremen

and30(47%)werewomen.Themedianagewas69.2(range,

41–94years).Colorectaltissuesampleswereobtainedfrom64 patientswhounderwentsurgeryforCRCandweredividedinto

B– tissuesamplesobtainedfrom non-neoplasticcolorectal

mucosalocated10cmcranialtothetumor.

Histopathologicalanalysis

TissuesamplesfromCRCandnon-neoplasticcolorectal

tis-sueswere fixedin10% formalinand embeddedinparaffin

blocksforhistologicalanalysis.Conventionalhistological sec-tionswerepreparedfromeachblockat3-␮mthick.Allofthe

slideswerestainedwithhematoxylin–eosin(HE)andreviewed

byapathologistforconfirmationofthediagnosis.To charac-terizethehistology,thehistologicalgradeofthemalignancy accordingtotheInternational ClassificationofDiseasesfor

Oncology(ICD-O)wasused.

Inthe HE-stained slides,the surgicalmarginsand

best-preserved areas most representative of the tumor were

marked toobtain the cylinderused in TMA(tissue

micro-array). Areas of necrosis, hemorrhage and areas with low

cellularitywereexcluded.Next,theblockswereseparatedto verifythequalityandquantityofthetissuetobeusedand

tomarktheareasforbiopsy.TheTMAblockswereprepared

usingaBeecherdevice (BeecherInstruments,SilverSpring,

MD,USA).

Immunohistochemistry

For immunohistochemical studies, the streptavidin-biotin

technique(LSAB-DakoCytomation, CA,USA) wasused. The

polyclonalantibodiesusedwereasfollows:Wnt-1(G-19)(goat)

(R&DSystems, Minneapolis,MN,USA), Wnt-2 (H-20) (goat)

(R&DSystems, Minneapolis,MN,USA), Wnt-5a(goat) (R&D Systems,Minneapolis,MN,USA),Frizzled-1(F-13)(goat)(R&D Systems,Minneapolis,MN,USA),Frizzled-5(L-12)(goat)(R&D Systems,Minneapolis,MN,USA)andaxin(H-98)(rabbit)(1:100 SantaCruzBiotechnologyInc.,CA,USA).Alloftheantibodies wereusedata1:100dilution.

For immunohistochemicalstudies,4-␮mthick

histologi-calsectionswerepreparedaccordingtopreviouslydescribed

techniques.19_{Incubationwiththeantibodieswasperformed}

in amoist incubation chamberat 4◦_{C for atleast 16–18h}

(overnight).AfterthreewashesinPBSbufferpH7.2–7.6,

fur-therincubationwasperformedwithbiotinylatedsecondary

antibody(LSAB-DakoCytomation, CA, USA)in ahumidified

chamberatroomtemperaturefor30min.Subsequently,the

same process was performed using a

streptavidin-biotin-peroxidase kit (LSAB-DakoCytomation, CA, USA). Washes

were then madewith PBS buffer pH7.2–7.6,and the color

wasdevelopedwithliquidDAB(DakoCytomation,CA,USA)at

roomtemperaturefor5min.Afterwashinginrunningwater

for3min,counter-stainingwasperformedusingHarris hema-toxylinfor1min.Thesectionsweredehydratedin3absolute

ethanolbaths and 3xylenebaths and thenmounted with

cover slipsusingEntellan resin (SigmaChemical Co.,Saint

Louis,MO,USA)foranalysisinanopticalmicroscope.

The immunoreactivity of the Wnt-1, Wnt-2, Wnt-5a,

Frizzled-1,Frizzled-5proteinsandaxinwasanalyzedbased

on the proportion of the number of positive cells

(per-centageofpositivity)and the intensity of immunostaining

accordingtoastandardizedscale.Thepercentageofpositive cells orpositivitywas classified asfollows:zero=less than

5% immunostainedepithelial cells of the lesion;1=5–25%;

2=26–50%;3=51–75%;and4=morethan76%immunostained

epithelial cells in the lesion. The intensity of

immunoex-pression was evaluated as follows: 0=negative; 1+=weak;

2+=moderate;and3+=strong.Thefinalscorewascalculated bymultiplyingthescoreofthepercentageofpositivitybythe scoreforthereactionintensity.Thefinalscorewasclassified asreducedexpression(scorevaluebetween0and8)orstrong expression(scorevaluebetween9and12).20

Alloftheslideswereanalyzedbytwoindependent

exam-iners considering only the degree of immunostaining and

withoutaccesstoorknowledgeoftheanatomopathological

data.Incasesofdiscrepancyintheevaluation,theslideswere

re-evaluated,andaconsensusevaluationwasobtained.

Clinicalandanatomopathologicaldata

The following clinical and anatomopathological data were

collected from the medical records of the patients:

clin-ical characteristics (age, gender, ethnicity), macroscopic

tumorcharacteristics(location,appearance,size),microscopic characteristics (lymphnode invasion,degreeofcellular dif-ferentiation,andvenous,lymphaticandneuralinfiltration),

TNM21 _{classification, presenceof synchronousmetastases,}

andtissueimmunoexpressionoftheantibodiesused

(percent-ageofpositivityandintensityofimmunoexpression).

Statisticalstudy

Quantitative results were reported as the median and

standarddeviation.Qualitativedataweredescribedaccording tothefrequencyofdistribution.Associationsbetweena pro-tein’spositivityandtheclinicopathologicalcharacteristicsof interestwereevaluatedusingFisher’sexacttest.Thelevelof significanceadoptedwas5%(p≤0.05).Statisticalanalysiswas

performedusing SPSSsoftwareversion15.0(ThePredictive

AnalyticsCompany,Chicago,IL,USA).

Results

Fifty-nine patients (92.2%)underwent curativesurgery, and

5patients(7.8%)underwentpalliativesurgery.TheCRCwas

locatedinthecolonin40patients(62.5%)andintherectum

in24patients(37.5%).Neoadjuvanttherapywasadministered

in 10 patients (15.6%), all of whom had rectal carcinoma.

The average size of a colorectal neoplasm was 5.2 (range,

0.5–12cm).Thesizeofthelesioninitslargestdiameterwas >5cmin43patients(67.1%)and≤5cmin21patients(32.8%).

Lymphnodemetastasiswasdetectedin31cases(48.4%),while

33patients(51.6%)hadlymphnodesfreeofany

compromis-ingneoplasm.Venousvascularinvasionwasobservedin21

patients(32.8%),lymphatic vascularinvasionin24patients

(37.5%) and neural invasionin 12 patients(18.7%). In

rela-tiontothedegreeofcellulardifferentiation,11cases(17.2%)

were classified as well-differentiatedcarcinomas, 51 cases

(79.7%) as moderately differentiated and 2 cases (3.1%) as

poorly differentiated. The CRC had infiltrated superficially

Table1–Frequencyofimmunoreactivityscoresofnon-neoplasticcolorectaltissuesandcolorectalcarcinoma.

Proteinexpression NNCRT CRC pvalue

Strong Weak Strong Weak

Wnt-1 3/53(5.6%) 50/53(94.4%) 2/58(3.4%) 56/58(96.6%) 1.00

Wnt-2 19/60(33.3%) 41/60(66.7%) 10/62(16.1%) 52/62(83.9%) 0.05

Wnt-5a 0 49/49(100%) 0 58/58(100%) 0.28

Frizzled-1 4/52(7.7%) 48/52(92.3%) 3/42(7.2%) 39/42(92.8%) 0.70

Frizzled-5 0 50/50(100%) 0 61/61(100%) 0.35

Axin 13/49(26.5%) 26/49(73.5%) 34/61(55.7%) 27/61(44.3%) 0.04

NNCRT,non-neoplasticcolorectaltissue;CRC,colorectalcarcinoma;pvalueobtainedwithFisher’sexacttest.

metastasesintheliverand/orotherlocationswereobserved in14 cases(21.9%).Relapseoccurred in13patients(20.3%), and12patients(18.7%)died.Theaveragefollow-uptimewas 19.1months(range,3–36months).

ImmunoexpressionoftheWnt-1proteininnon-neoplastic

colorectal tissue and colorectal carcinoma showed no

sig-nificantdifference(p=1.0).ImmunoexpressionoftheWnt-2

protein in colorectal carcinoma was significantly lower

(p=0.05)thaninnon-neoplasticcolorectaltissue(Table1and

Fig. 1). Immunoexpression of the Wnt-5a protein in

non-neoplasticcolorectaltissueandcolorectalcarcinomashowed

no significant difference (p=0.28) between them.

Immuno-expressionoftheFrizzled-1andFrizzled-5proteinsshowed

no significant differences (p=0.7 and p=0.35,respectively).

Axin protein immunoexpression was significantly reduced

(p=0.039)innon-neoplasticcolorectaltissuerelativeto neo-plastictissue(Table1andFig.2).

Discussion

DespitetheevidenceonWntproteinsandtheirFrizzled recep-torsandtheirparticipationincolorectalcarcinogenesis,the interactionsandregulatorymechanismsoftheseproteinsare notcompletelyunderstood.22_{Severalmutationsinoncogenes,}

Fig.1–Photomicrographofcolorectalcarcinomawith positiveimmunoreactivityforWnt-2antibodyinthe cytoplasmoftumorcells(immunohistochemistry;200×).

Fig.2–Photomicrographofcolorectalcarcinomawith positiveimmunoreactivityantibodyaxininthecytoplasm oftumorcells(immunohistochemistry;100×).

tumor suppressor genes and other proteinsthat comprise

thesignaltransductionpathwayshavebeenassociatedwith

colorectalneoplasias.However,fewofthesemutations,when

known,occurinalltumorsandatallstagesofthedisease.

Although Wnt-1 isone ofthe moststudied proteinsof

thecanonicalWntsignalingpathway,thereare fewstudies

showingtheexpressionofthisproteinincolorectaltissues. Stanczaketal.23_{showedincreasedexpressionofWnt-1}

pro-teininnormalcolorectalmucosaanddecreasedexpressionof

thisproteinintumortissue.Holcombeetal.11_{foundabundant}

expressionofWnt-1proteininnormalmucosaandcolorectal

tumortissue.Inthepresentstudy,nosignificantdifference

wasfoundwhentheexpressionofWnt-1innon-neoplastic

colorectaltissuewascomparedwithcolorectaltumortissue.

Theseconflictingresultscouldsuggestthatothermechanisms maybeinvolvedincolorectalcarcinogenesisinadditiontothe activationofWnt-1protein.

Wnt-2proteinisanimportantinduceroftheWntcanonical signalingpathway.22 _Otherauthors11,12 _{haveshownlittleor}

eventheabsenceofexpressioninnormalcolorectalmucosa

butincreasedimmunoexpressionincolorectaltumortissue.

ThecurrentstudyshowedstrongexpressionofWnt-2protein

Of the proteins involved in the non-canonical Wnt

signaling pathway,Wnt-5a proteinappearstohave

tumor-suppressor behavior primarily for its ability to inhibit the

canonicalWntsignalingpathwayandreducethepoolof␤

-cateninproteininthe cytoplasm.10 _{Dejmeket al.}24_showed thatexpressionofWnt-5aisassociatedwithanincreased

five-yearsurvivalinpatientswithDukes’stageBCRC. Previous

studies11,22_{usingRT-PCRfoundWnt-5aproteinexpressionin} normalcell lines,particularlyatthe baseofthe crypts,but

notin CRC tissues. Thepresent sampling,however, found

markedlyreducedexpressionofWnt-5aproteininboth

neo-plasticandnon-neoplasticcolorectaltissues.Thisfactcanbe explainedbytheuseofnon-neoplastictissuesfromthesame

patientsascontrolsbecauseithasbeendemonstratedthat

lossofWnt-5agenestimulatesthecanonicalWntsignaling

pathwayinpatientswithCRC.25_Dejmeketal.24_{suggestedthat} expressionofWnt-5aproteininpatientswithCRCseemstobe associatedwiththeearlystagesofneoplasia,aresultdifferent fromthefindingsofthepresentstudyinwhichthemajority ofpatientswereinthemoreadvancedstagesofCRC.

TheinteractionofWntproteinswithFrizzledreceptorsis consideredcrucialtotheunderstandingofWntsignaling,both

inembryogenesisandindevelopmentoftumors.19 _Despite

intensiveresearchinto theWntsignaling pathwayandthe

participationofitsconstitutiveproteins,26_theroleofthe Friz-zledproteinsinthisconditionremainsunclear.Fewstudies

haveexaminedtheexpressionofFrizzledreceptorsin

colo-rectal tissue. You et al.27 _{observedthat the Frizzled-1and}

Frizzled-2receptorswereoverexpressedindysplasticmucosa

adjacenttoCRC,underexpressedinnormalmucosaandnot

expressedinneoplastictissue.InpatientswithsporadicCRC, Holcombeetal.11_{foundexpressionofFrizzled-1and} Frizzled-2receptorsonlyinpatientswithpoorlydifferentiatedlesions, suggestingthattheregulationoftheFrizzledproteinsisan importantstepintheprocessoftumorinvasion.Inthepresent

study,both the Frizzled-1and Frizzled-5receptorsshowed

decreasedexpressioninnon-neoplasticcolorectaltissueand CRC.

Theaxingeneactsasatumorsuppressorgenethrougha

negativefeedbacksystem,regulatingthesignalresponsesof

theWntsignalingpathway.28_{Normally,axinproteinreduces}

thelevelsof␤-catenininthecytoplasm.However,mutations

inthis geneleadto anincreaseinthe concentrationof␤

-catenin in colorectal tumors. Jin et al.29 _{analyzed 54 CRC}

specimensfrompatientsandfoundthat11%hadaxingene

mutations, suggesting that this change may be associated

withcolorectal carcinogenesis.Websteret al.30 _studiedthe axingenein4differentCRCcelllinesandshowedthat muta-tionofthisgenealtersitsbindingcapacitytotheGSK3␤gene

thereby preventingthe formation ofthe destructorprotein

complexof␤-catenin.

Thus,abnormallyhighlevelsofaxinproteinexpressioncan maskafunctionaldifferencebecausethelevelsofexpression ofthisgenetendtobelow.5_{Suchaneffectmayexplainthe} factthatstrongexpressionofthisproteinintumortissuewas

foundinthisstudy,whilereducedexpressionwasfoundin

non-neoplasticcolorectal tissue,afinding alsoobservedby

HughesandBrady.28

Someofthedifferencesfoundinthe resultsofthe

cur-rentstudy,whencomparedwiththosefoundintheliterature,

canbeexplainedbyfactorsrelatedtothemethodusedinthe research.Moststudies15,16,27_{usedanRT-PCRmethod,whereas}

the current study used an immunohistochemical method.

Another importantfactor wastheuse oftissueculturecell

linesforanalysisinthesestudies.Otherauthors12,26_showed

differences in the expression ofproteins inneoplastic

tis-suesofpatientscomparedwiththesametumorcelllinefrom

tissue culture.Moresophisticated methodssuchas insitu

hybridization or laser capture microdissection(LCM) could

betterelucidatetheseissues.

Thus, in the present study, differentdegrees of

expres-sion of Wnt, Frizzled receptors and axin proteins were

found inneoplastic colorectal tissuescompared with

non-neoplastictissues,findingsthatwerealsoobservedbyother authors.11,12,23,26_{BecausetheWntsignalingpathwayandAPC}

gene11 _{pathwayactinthetransformationofnormal}

epithe-liumintoproliferativeepithelium,itispossiblethatmostof

theproteinsofthesepathwaysinvolvedintumorigenesisare

expressedbothinthetumortissueandintheadjacentmucosa

ofthesesametumors.

Theresultsshowingtheabsenceofincreasedexpressionof

proteinsofthenon-canonicalWntpathway(Wnt-1,Wnt-5a,

Frizzled-1andFrizzled-5)suggestthatthisrouteisnotfully enabledinsporadicCRC,andthereforecontributeslesstothe

genesis ofCRC. However,the Wnt-2protein, typicalofthe

canonicalWntsignalingpathway,showedasignificant

differ-encebetweenthelevelsofimmunohistochemicalexpression

in non-neoplastic colorectal tissue and tumor tissue. The

higherexpressionofWnt-2proteininnon-neoplastic

colo-rectaltissuesuggeststheparticipationoftheproto-oncogene Wnt-2duringtheearlyand/orhyperproliferativestageof

pre-neoplasticchangesofthecolorectalmucosa.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

TheauthorsthankFundac¸ãodeAmparoàPesquisadoEstado

deSãoPaulo(FAPESP)forfinancialsupport.

r

e

f

e

r

e

n

c

e

s

1.FerlayJ,ShinHL,BrayF,FormanD,MathersC,ParkinDM. Estimatesofworldwideburdenofcancer2008:GLOBOCAM 2008.IntJCancer.2010;127:2893–917.

2.Ricci-VitianiL,FabriziE,PalioE,DeMariaR.Coloncancer stemcells.JMolMed.2009;87:1097–104.

3.FevrT,RobineS,LouvardD,HuelskenJ.Wnt/beta-cateninis essentialforintestinalhomeostasisandmaintenanceof intestinalstemcells.MolCellBiol.2007;27:7551–9.

4.MoonRT.Wnt/beta-cateninpathway.SciSTKE.2005;271:1–3.

5.ParveenN,HussainMU,PandithAA,MudassarS.Diversityof axininsignalingpathwaysanditsrelationtocolorectal cancer.MedOncol.2011;28:S259–67.

axinstabilityandWnt/␤-cateninsignaling.MolCellBiol. 2011;31:2053–65.

7. CadiganKM.Wnt/beta-cateninsignaling:turningtheswitch. DevCell.2008;14:322–3.

8. McDonaldSL,SilverA.TheopposingrolesofWnt-5ain cancer.BrJCancer.2009;101:209–14.

9. NusseR.Wntsignaling.ColdSpringHarbPerspectBiol. 2012;4:a011163.

10.TopolL,JiangX,ChoiH,Garrett-BealL,CarolanPJ,YangY. Wnt-5ainhibitsthecanonicalWntpathwaybypromoting GSK-3-independent␤-catenindegradation.JCellBiol. 2003;162:899–908.

11.HolcombeRF,MarshJL,WatermanML,LinF,MilovanovicT, TruongT.ExpressionofWntligandsandFrizzledreceptorsin colonicmucosaandincoloncarcinoma.MolPathol.

2002;55:220–6.

12.ViderBZ,ZimberA,ChastreE,PrevotS,GespachC,EstleinD, etal.EvidencefortheinvolvementoftheWnt2genein humancolorectalcancer.Oncogene.1996;12:153–8.

13.BurnsCJ,ZhangJ,BrownEC,VanBibberAM,VanEsJ,Clevers H,etal.InvestigationofFrizzled-5duringembryonicneural developmentinmouse.DevDyn.2008;237:1614–26.

14.UenoK,HiuraM,SuehiroY,HazamaS,HirataH,OkaM,etal. Frizzled-7asapotentialtherapeutictargetincolorectal cancer.Neoplasia.2008;10:697–705.

15.LuuHH,ZhangR,HaydonRC,RayburnE,KangQ,SiW,etal. Wnt/␤-cateninsignalingpathwayasnovelcancerdrug targets.CurrCancerDrugTargets.2004;4:653–71.

16.CurtinJC,LorenziMV.Drugdiscoveryapproachestotarget Wntsignalingincancerstemcells.Oncotarget.2010;1:563–77.

17.DeAlmeidaVI,MiaoL,ErnstJA,KoeppenH,PolakisP, RubinfeldB.ThesolublewntreceptorFrizzled8CRD-hFc inhibitsthegrowthofteratocarcinomasinvivo.CancerRes. 2007;67:5371–9.

18.HeB,ReguartN,YouL,MazieresJ,XuZ,LeeAY,etal. BlockadeofWnt-1signalinginducesapoptosisinhuman colorectalcancercellscontainingdownstreammutations. Oncogene.2005;24:3054–8.

19.BadiglianFilhoL,OshimaCT,deOliveiraLimaF,DeOliveira CostaH,DeSousaDamiãoR,GomesTS,etal.Canonicaland noncanonicalWntpathway:acomparisonamongnormal

ovary,benignovariantumorandovariancancer.OncolRep. 2009;21:313–20.

20.KrajewskaM,KrajewskiS,EpsteinJI,ShabaikA,SauvageotJ, SongK,etal.Immunohistochemicalanalysisofbcl-2,bax, bcl-X,andmcl-1expressioninprostatecancers.AmJPathol. 1996;148:1567–76.

21.TNM7thedition(2010).www.uicc.org/uiccold/resources/tnm

22.DimitriadisA,VincanE,MohammedIM,RoczoNN,Phillips WA,Baindur-HudsonS.ExpressionofWntgenesinhuman coloncancers.CancerLett.2001;166:185–91.

23.StanczakA,StecR,BodnarL,OlszewskiW,CichowiczM, KozlowskiW,etal.PrognosticsignificanceofWnt-1,

␤-cateninanE-caherinexpressioninadvancedcolorectal carcinoma.PatholOncolRes.2011;17:955–63.

24.DejmekJ,DejmekA,SäfholmA,SjölanderA,AndersonT. Wnt-5aproteinexpressioninprimaryDukesBcoloncancers identifiesassubgroupofpatientswithgoodprognosis. CancerRes.2005;65:9142–6.

25.SmithK,BuiTD,PoulsonR,KaklamanisWilliansG,HarrisA. Up-regulationofmacrophagewntgeneexpressionin adenoma-carcinomaprogressionofhumancolorectalcancer. BrJCancer.1999;81:496–502.

26.GilesRH,vanEsJH,CleversH.CaughtupinaWntstorm:Wnt signalingincancer.BiochimBiophysActa.2003;1653:1–24.

27.YouXJ,BryantPJ,JurnakF,HolocombeRF.ExpressionofWnt pathwaycomponentsfrizzledanddisheveledincoloncancer arisinginpatientswithinflammatoryboweldisease.Oncol Rep.2007;18:691–4.

28.HughesTA,BradyHJ.Regulationofaxin2expressionatthe levelsoftranscription,translationandproteinstabilityin lungandcoloncancer.CancerLett.2006;233:338–44.

29.JinLH,ShaoQJ,LuoW,YeZyLiQ,LinSC.Detectionofpoint mutationsoftheaxin1geneincolorectalcancers.IntJ Cancer.2003;107:696–9.