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ORIGINALARTICLESuppressionofTumorigenicity-14,encodingmatriptase,isacriticalsuppressorofcolitisandcolitis-associatedcoloncarcinogenesisPKosa,RSzabo,AAMolinoloandTHBuggeOralandPharyngealCancerBranch,NationalInstituteofDentalandCraniofacialResearch,NationalInstitutesofHealth,Bethesda,MD,USAColitis-associatedcolorectalcancersareanetiologicallydistinctsubgroupofcoloncancersthatoccurinindividualssufferingfrominammatoryboweldiseaseandariseasaconsequenceofpersistentexposureofhyperproliferativeepithelialstemcellstoaninammatorymicroenvironment.
Anintrinsicdefectintheintestinalepithelialbarrierhasbeenproposedtobeoneofseveralfactorsthatcontributetotheinappropriateimmuneresponsetothecommensalmicrobiotathatunderliesinammatoryboweldisease.
Matriptaseisamembrane-anchoredserineproteaseencodedbySuppressionofTumorigenicity-14(ST14)thatstrengthenstheintestinalepithelialbarrierbypromotingtightjunctionformation.
Here,weshowthatintestinalepithelial-specicablationofSt14inmicecausesforma-tionofcolonadenocarcinomawithveryearlyonsetandhighpenetrance.
Neoplasticprogressionisprecededbyachronicinammationofthecolonthatresembleshumaninammatoryboweldiseaseandispromotedbythecommensalmicrobiota.
Thisstudydemonstratesthatinammation-associatedcoloncarcinogenesiscanbeinitiatedandpromotedsolelybyanintrinsicintestinalpermeabilitybarrierperturbation,establishesSt14asacriticaltumor-suppressorgeneinthemousegastrointest-inaltractandaddsmatriptasetotheexpandinglistofpericellularproteaseswithtumor-suppressivefunctions.
Oncogene(2012)31,3679–3695;doi:10.
1038/onc.
2011.
545;publishedonline5December2011Keywords:inammatoryboweldisease;intestinalbarrier;coloncarcinogenesisIntroductionColitis-associatedcolorectalcancersareetiologicallyandmolecularlydistinctfromfamilialadenomatouspolyposiscoli-associatedcolorectalcancer,hereditarynon-polyposiscolicolorectalcancerandsporadiccolor-ectalcancer.
ThemalignancyoccursinindividualssufferingfromulcerativecolitisorCrohn'sdisease(collectively,inammatoryboweldisease)withanincidencethatisproportionaltothedurationofthedisease.
Theneoplasticprogressionofdisease-strickencolonicepitheliumisbelievedtobedrivenbythechronicinammatorymicroenvironment,whichpromotestheprogressivegenomicinstabilityofcolonicepithelialstemcellsbyinducingsustainedhyperproliferation(regen-erativeatypia)andbythecontinuouspresenceofhighlocalconcentrationsofDNA-damagingagents,suchasreactiveoxygenspecies(reviewedinthestudiesbyDaneseandMantovani(2010)andSalehandTrinchieri(2011)).
Althoughthereisconsiderabledebateabouttherelativeimportanceofthespecicfactorsthatcontributetothedevelopmentofinammatoryboweldisease,thereisaconsensusthatthediseaserepresentsaninappropri-ateimmuneresponsetothecommensalmicrobiotaingeneticallypredisposedindividuals(reviewedinthestudiesbySchreiberetal.
,2005;XavierandPodolsky,2007;VanLimbergenetal.
,2009;Kaseretal.
,2010;SalehandTrinchieri,2011).
Inthisregard,thecontributionofaberrantinammatorycircuitstothedevelopmentofinammatoryboweldiseasehasbeenclearlyestablishedbygeneticanalysis,includinggen-ome-wideassociationstudies,whichhavelinkedloss-of-functionmutationsorpolymorphismsingenesencodinginterleukins,interleukinreceptors,chemokinereceptors,nucleotide-bindingoligomerizationdomain-likerecep-tors,Toll-likereceptor4,intelectinsandprostaglandinreceptorstoulcerativecolitis,toCrohn'sdiseaseortoboth.
Furthersupportforaprincipalroleofderailedinammatorycircuitsisgainedfromthespontaneousinammatoryboweldiseaseobservedinmicedecientinvariousimmuneeffectors(reviewedinthestudiesbySchreiberetal.
,2005;XavierandPodolsk,2007;VanLimbergenetal.
,2009;Kaseretal.
,2010;SalehandTrinchieri,2011).
Muchlessexploredistheimportanceofindividualcomponentsoftheintestinalepithelialbarrierinpreventinginammatoryboweldisease,andthepotentialcontributionofintrinsicintestinalepithe-lialbarrierdefectstothedevelopmentofthesyndrome.
TheclearestindicationofthepotentialimportanceofprimarybarrierintegritycomesfromstudiesofmicewithgermlineablationofMuc2encodingthemajormucinthatshieldstheintestinalepitheliumfromdirectcontactwiththemicrobiota.
Thesemicedevelopcolitis,whichmayprogresstocolonadenocarcinomasinolderanimals(Velcichetal.
,Received6July2011;revised13October2011;accepted27October2011;publishedonline5December2011Correspondence:DrTHBugge,ProteasesandTissueRemodelingSection,OralandPharyngealCancerBranch,NationalInstituteofDentalandCraniofacialResearch,NationalInstitutesofHealth,30ConventDrive,Room211,Bethesda,MD20892,USA.
E-mail:thomas.
bugge@nih.
govOncogene(2012)31,3679–3695&2012MacmillanPublishersLimitedAllrightsreserved0950-9232/12www.
nature.
com/onc2002).
Additionalevidencehasbeenobtainedfromtransgenicmicewithintestinalepithelial-specicover-expressionofmyosinlight-chainkinase,whichdisplaydecreasedbarrierfunctionandincreasedimmuneactivation,althoughaninammatorybowel-likesyn-dromedidnotemergeintheabsenceofadditionalimmunologicalchallenges(Suetal.
,2009).
MatriptaseisamemberoftherecentlyestablishedfamilyoftypeIItransmembraneserineproteasesthatisencodedbytheSuppressionofTumorigenicity-14(ST14)gene(Kimetal.
,1999;Linetal.
,1999;Takeuchietal.
,1999;Tanimotoetal.
,2001;Buggeetal.
,2009).
ST14wasoriginallyproposedtobeacoloncancertumor-suppressorgene,duetoitsspecicdownregulationinadenocarcinomasofthecolon(Zhangetal.
,1998).
Matriptaseisexpressedinmultipleepitheliaoftheintegumental,gastrointestinalandurogenitalsystems,whereithaspleiotropicfunctionsinthedifferentiationorhomeostasisofbothsimpleandstratiedepithelia,atleastinpartthroughtheproteolyticactivationoftheepithelialsodiumchannelactivator,prostasin/PRSS8(SzaboandBugge,2011).
Inthesimpleepitheliumofthegastrointestinaltract,aprincipalfunctionofmatriptaseistopromotetheformationofaparacellularperme-abilitybarrier,possiblythroughtheposttranslationalregulationofthecompositionofclaudinsthatareincorporatedintotheepithelialtightjunctioncomplexofdifferentiatingintestinalepithelialcells(Listetal.
,2009;Buzzaetal.
,2010).
Throughlineage-specicloss-of-functionanalysisinmice,wenowhaveexaminedtheroleofSt14asatumor-suppressorgene.
Interestingly,wefoundthattheselectiveablationofSt14fromtheintestinalepitheliumresultsintheformationofadenocarcinomaofthecolonwithveryearlyonsetandhighpenetrance.
Neoplasticprogressionoccursintheabsenceofexposureofanimalstocarcinogensortumor-promotingagents,isprecededbychroniccolonicinammationthatresembleshumaninammatoryboweldiseaseandcanbesuppressedbyaggressiveantibioticstreatment.
Thisstudydemon-stratesthatinammation-associatedcoloncarcinogen-esiscanbeinitiatedsolelybyintrinsicparacellularpermeabilitybarrierperturbations,andestablishesthatSt14isacriticaltumor-suppressorgeneinthemousegastrointestinaltract.
ResultsMeta-analysisoftranscriptomesshowsdecreasedexpressionofST14inhumancolonadenomasandadenocarcinomasWerstperformedinsilicodataminingoftheOncominemicroarraydatabase(Rhodesetal.
,2004)tocorroboratetheinitialreportofreducedST14expressioninhumancoloncancer(Zhangetal.
,1998)(Figure1).
Interestingly,ST14wassignicantlydown-regulatedcomparedwiththenormalcolonin7ofthe14publishedstudieslistedinthedatabase(studiesAandC–H),whereas6studiesshowednochange(studiesBandJ–N)andasinglestudy(studyI)foundST14tobeupregulated(Figure1andSupplementaryTable1).
Ofthe14studies,studyA,whichcomparedgeneexpressionincolorectaldysplasticadenomatouspolypswiththenormalcolonicepithelium,wasconductedusinglasercapturemicrodissectedtissue(ST14downregulation,Po0.
0006)(Gasparetal.
,2008),andthereforeprovidedthemostreliableestimateofST14expressioninnormalanddysplasticcolonicepithelium.
TheSt14-ablatedcolonicepitheliumundergoesrapidandspontaneousmalignanttransformationTospecicallyexplorethefunctionalconsequencesofintestinallossofSt14oncoloncarcinogenesis,weinterbredmicecarryinganSt14LoxPallele(Listetal.
,2009)withmicecarryinganSt14nullallele(St14)andmicecarryingaCretransgeneunderthecontroloftheintestinal-specicvillinpromoter(villin-Cre)(Madisonetal.
,2002).
Thisresultedinthegenerationofvillin-Cre/0;St14LoxP/mice(hereaftertermed'St14'mice)andtheirassociatedlittermatesvillin-Cre/0;St14LoxP/,villin-Cre0;St14LoxP/andvillin-Cre0;St14LoxP/(hereaftertermed'St14'mice).
Asreportedrecently(Listetal.
,2009),thisstrategyresultedintheefcientdeletionofmatriptasefromtheentireintestinaltract,asshownbythelossofmatriptaseimmunoreactivityinthecolon(compareSupplementaryFigures1aandb)andthesmallintestine(compareSupplementaryFigures1candd),andbyhighlydiminishedSt14transcriptabundance(SupplementaryFigure1e).
St14micewereoutwardlyunremarkableatbirth,butdisplayedsignicantgrowthretardationafterweaning(SupplementaryFigure1f).
ExaminationofprospectivecohortsofSt14miceandtheirassociatedlittermatecontrolsrevealedthatintest-inalSt14ablationgreatlydiminishedlifespan(Supple-mentaryFigure1g).
Unexpectedly,histologicalanalysisofmoribundSt14micerevealedthepresenceofinvasiveadenocar-***2***************foldchange1AIB0NMLKJHGFEDCFigure1Matriptaseexpressionisdownregulatedinhumancolonadenomasandadenocarcinomas.
ExpressionofST14,encodingmatriptase,in14geneexpressionarraystudiesofhumancolonadenomasandadenocarcinomas.
Dataareexpressedasfoldchangerelativetothecorrespondingnormaltissue.
*Po0.
05,**Po0.
01,***Po0.
001.
SeeSupplementaryTable1fordetailsandreferences.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3680Oncogenecinomaofthecolonin8of24(33%)ofSt14miceexaminedat4–18weeksofage(Table1,Figures2a,bandd)anddysplasticcolonicepithelium(regenerativeatypia)intheremaining16mice(Table1andFigure2d).
Remarkably,inlightofthefactthatthesemicewerenotcarcinogentreatedorexposedtootherinsultstotheintestinaltract,adenocarcinomacouldbefoundinmiceo5weeksofage(Table1).
Alladenocarcinomashadprogressedtoinvadethemuscu-larismucosaeunderlyingthecolonicepitheliumandthesubadjacentmuscularisexterna(Figures2aandb).
Furthermore,insixofeight(75%)adenocarcinomasexamined,tumorcellshadinltratedthelymphaticvasculature,asrevealedbycombinedimmuno-histochemicalstainingwithpan-keratinantibodiesandantibodiesagainstthelymphaticendothelialmarker,LYVE-1(Table1andFigures2candd).
Tumorsdisplayedmanyhallmarksofhumancolitis-associatedcoloncancer,includingactivationofb-catenin(Figures3aanda0),dysorganizedbasementmembranedeposition(Figures3bandb0),brosis(Figures3candc0),severedysplasiawithabundantatypicalmitosis(Figure3d),epithelialhyperprolifera-tion(Figures4aanda0),lossofterminaldifferentiation(Figures4bandb0)andchronicinammatorycellinltrates(Figures4c,c0,dandd0).
Importantly,thesmallintestinewashistologicallyunremarkableinallmiceexamined(datanotshown),althoughSt14wasefcientlyablatedalsofromthistissue(Supplemen-taryFigures1dande).
Takentogether,thesedatashowthatSt14isacriticaltissue-specictumor-suppressorgeneinthemouseintestinethatsuppressestheformationofearly,invasiveadenocarcinomasofthecolon.
ImpairedbarrierfunctioninSt14-ablatedcolonWehavepreviouslyshownthateitherreducedmatriptaseexpressionorglobalpostnatalablationofmatriptasefromalltissuesresultsinimpairedintestinalbarrierfunction(Listetal.
,2009;Buzzaetal.
,2010),suggestingthatthiswouldalsobeafeatureofmicewithintestinalepithelial-specicembryonicdeletionofmatriptase.
Toexaminecolonicandsmallintestinalbarrierfunction,weinjectedareactivebiotintracerintotheintestinallumenof3-week-oldSt14andlittermateSt14miceandfollowedthefateofthemarkerusinguorescentstreptavidin.
Compatiblewithanintactintestinalbarrierfunction,thebiotinmarkerdecoratedthesurfaceofcoloniccryptsandvilliofthesmallintestine,butdidnotpenetrateintothetissueofSt14mice(Figures5aandd).
Incontrast,just3minafterintraluminalbiotininjection,thebiotintracercouldbefoundonthebasolateralmembranesandonconnectivetissuecellsofthecolonandsmallintestineofSt14micedemonstratingaprofoundfailuretoestablishafunctionalintestinalbarrier(Figures5bande).
NeoplasticprogressionoccurswithinachronicinammatorycolonicmicroenvironmentthatresemblesinammatoryboweldiseaseTheSt14-ablatedcolonictissuewashistologicallyunremarkablewhenexaminedatbirthandatpostnatalday5(compareFigures6a,a0,bandb0).
Therstobservablepathologicalmanifestation(day10)wasthedetachmentandapoptosis(anoikis)ofdistalcryptcells(compareFigures6candc0).
Thiswasfollowedbythefailureofcolonicepithelialcellstoundergoproperterminaldifferentiation,asevidencedbycessationofmucinformation(datanotshown).
Thereafter,St14colonsenteredaprogressivelyhyperplasticinammatoryTable1IntestinallesionsinSt14miceMouseGenderAge(days)DiagnosisofintestinallesionsLymphaticinvasionMCV41Male31RegenerativeatypiaNoMCV52Male33AdenocarcinomaYesMCV55Female55RegenerativeatypiaNoMCV56Female55RegenerativeatypiaNoMCV59Male25RegenerativeatypiaNoMCV66Male28RegenerativeatypiaNoMCV70Female24RegenerativeatypiaNoMCV76Male129RegenerativeatypiaNoMCV127Male111RegenerativeatypiaNoMCV153Male79AdenocarcinomaNoMCV159Female26RegenerativeatypiaNoMCV162Male105AdenocarcinomaYesMCV173Male44RegenerativeatypiaNoMCV180Female131AdenocarcinomaNoMCV192Male44RegenerativeatypiaNoMCV204Male48AdenocarcinomaYesMCV234Male50AdenocarcinomaYesMCV256Male56AdenocarcinomaYesMCV359Female51AdenocarcinomaYesMCV366Male36RegenerativeatypiaNoMCV397Male39RegenerativeatypiaNoMCV461Female30RegenerativeatypiaNoMCV468Male22RegenerativeatypiaNoMCV1250Male67RegenerativeatypiaNoEpithelialbarrierdefect-inducedcarcinogenesisPKosaetal3681Oncogeneandulcerativestatethateventuallyresultedinthegrossdistortionofcolonictissuearchitecture(compareFigures6d,d0,6eande0).
BrdU(5-bromo-20-deoxyuridine)-incorporatingcellsinitiallywereconnedtothebottomofthecrypts,butlaterwerealsopresentalsoindistalsegmentsofthecrypts(datanotshown).
Inammatoryinltrateswereevidentatday15.
Inammationatrstwasmild,butrapidlybecamesevere,withinammatorycellseventuallyconstitutingthedominantcellpopulationofthemucosaandsubmucosa.
Polypswerenotobservedinanyoftheexaminedcolonsbeforemalignanttransformation,indicatingthatSt14ablation-associatedadenocarcinomas,likeinammatoryboweldisease-associatedcolorectalcancers,arisefromatlesionswithinhyperproliferativeandinamedmucosa.
Abnormalepithelialdifferentiationandactivationofinammatorypathwaysprecedeinammatoryboweldisease-likecolitisanddysregulationofcommoncoloncancer-associatedsignalingpathwaysToelucidatethemoleculareventsthatprecedetheearlydevelopmentofcolitisinmicewithmatriptase-ablatedSt14-St14-St14-Colon,H&EColon,keratinIHC**Colon,keratin&LYVE-1IHCN=6normalregenerativeatypia10080%ofanimalsN=23N=2N=16adenocarcinomaadenocarcinoma+lymphaticinvasion604020St14+St14-0Figure2RapidandspontaneousmalignanttransformationoftheSt14-ablatedcolonicepithelium.
(a)Representativeexampleofadenocarcinomainthelargeintestineofan8-week-oldSt14mouse.
Tumorcellsinvadingthemuscularisexterna(star)areshownwitharrows.
(b)Theepithelialoriginoftumorcellsinvadingthemuscularisexterna(star)isdemonstratedbyimmunohistochemicalstainingforkeratin(exampleswitharrows).
(c)Combinedimmunohistochemicalstainingforkeratininred(exampleswitharrows)andthelymphaticvesselmarkerLYVE-1inbrown(exampleswitharrowheads)showsinvasionofmalignantcellsintolymphaticvesselsofa7-week-oldSt14mouse.
Scalebarforpanelsa,bandc100mm.
(d)Enumerationofcoloniclesionsin4-to18-week-oldSt14(left)andlittermateSt14mice(right),showingadenocarcinomawithlymphaticinvasionin6,adenocarcinomawithoutlymphaticinvasionin2andregenerativeatypiaintheremaining16St14mice.
SeeTable1foradditionaldetails.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3682Oncogenecolonicepithelium,wenextperformedstage-specictranscriptomicanalysisusingwhole-genomearrays.
Weselectedtwotimepoints(days0and5)wherethematriptase-ablatedcolonicepitheliumwashistologicallynormal,andonetimepoint(day10)wherepathologicalchangeswereemerging(seeabove).
Theanalysiswas****St14+St14+St14+St14-St14-St14-Colon,-cateninIHCColon,-cateninIHCColon,lamininIHCColon,lamininIHCColon,MassonSt14-Colon,MassonColon,H&Ea′b′c′Figure3Characterizationofmatriptaseablation-associatedcolonadenocarcinoma.
(a,a0)Immunohistochemicalstainingof8-week-oldSt14(panela)andlittermateSt14(panela0)colonsforb-cateninshowsamembrane-associatedb-cateninlocalizationinSt14epithelialcells(arrowsinpanela),ascomparedwithcytoplasmicandnuclearlocalizationinadenocarcinomasofSt14colons(exampleswitharrowsinpanela0).
(b,b0)Immunohistochemicalstainingforthebasementmembranemarkerlamininin15-week-oldSt14(panelb)andlittermateSt14(panelb0)miceshowsthenormalappearanceofthebasementmembrane(examplewitharrowinpanelb)inSt14mice.
Lossofmatriptaseexpressionleadstoincreaseddepositionoflaminin(exampleswithstarsinpanelb0)andlossofnormalstructureofthebasementmembrane.
(c,c0)MassonTrichromestainingofthecolonof6-week-oldSt14(panelc)andlittermateSt14(panelc0)miceshowsconnectivetissueinthesubmucosaofanormalcolon(examplewitharrowinpanelc)andbrosisofboththemucosaandsubmucosaofSt14colon(exampleswithstarsinpanelc0).
(d)HighmagnicationshowsthecytologicalappearanceofadenocarcinomasofSt14mice.
Atypicalmitosisisshownbyarrows.
Scalebar200mm(panelsa,a0,b,b0,candc0)and20mm(paneld).
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3683OncogenerepeatedfourtimesforeachofthethreetimepointsbyanalyzingindividualSt14miceandtheirassociatedSt14littermates.
Genesthatweremorethantwofoldupregulatedordownregulatedineachofthefourseparateexperimentswereconsideredforanalysis.
NosignicantdifferencesinthetranscriptomesofSt14-ablatedandSt14-sufcientcolonswereapparentatday0(datanotshown).
Interestingly,however,dysregula-tionofepithelialdifferentiationwasapparentalreadyatday5andwaspronouncedatday10(Tables2and3).
Thiswasevidencedbytheconspicuousupregulationofgenestypicallyexpressedinthebasal,suprabasalorkeratinizinglayersofthestratiedsquamousepitheliumliningtheoralcavity,interfollicularepidermis,hairandnailsoffollicularepidermis,aswellasliformpapillaeofthetongue.
Theseincludedkeratin14(Krt14),keratin36(Krt36),keratin84(Krt84),smallprotein-richprotein1a(Sprr1a),smallprotein-richprotein2h(Sprr2h)andsecretedLy6/Plaurdomaincontaining1(Slurp1).
Abnormalcolonicepithelialdifferentiationatday5wasfurtherevidencedbythedownregulationoftheexpressionofPanethcell-specicalpha-defensin4(Defa4).
AlthoughSt14colonictissueswerehistologi-callynormalatday5,activationofinammatorypathwayswasevidencedbytheincreasedexpressionofseveralinammation-associatedgenes,includingchemokine(C–X–Cmotif)ligand-1(Cxcl1),matrixmetalloproteinase10(Mmp10),lymphocyteantigen6complex,locusC2(Ly6c2),tumornecrosisfactor(Tnf),myelinandlymphocyteprotein(Mal),serumamyloidSt14+St14+St14-St14+St14-Colon,BrdUColon,PASColon,BrdUColon,PASColon,T-cellsIHCColon,B-cellsIHC**Colon,T-cellsIHC**Colon,B-cellsIHCSt14-St14+St14-*a′c′b′d′Figure4Characterizationofmatriptaseablation-associatedcolonadenocarcinoma.
(a,a0)BrdUstainingof8-week-oldSt14(panela)andlittermateSt14(panela0)miceshowsproliferationrestrictedtothebottomofthecryptsofnormalcolons(exampleswitharrowsinpanela).
IntheSt14colon,proliferatingcellsarefoundbothinthebottom(exampleswitharrowsinpanela0)anddistalpartsofcrypts(exampleswitharrowheadsinpanela0).
(b,b0)Periodicacid-Schiff(PAS)stainingofmucopolysaccharidesproducedbydifferentiatedgobletcellsinthecolonof11-week-oldSt14(panelb)andlittermateSt14(panelb0)mice.
Redstainingshowsmucininthenormalcolon(arrowsinpanelb).
Absenceofredstainingin(panelb0)indicatescessationofmucinproductioninmatriptase-ablatedcolon.
(c,c0,d,d0)ImmunohistochemicalstainingforT-cells(panelscandc0)andB-cells(panelsdandd0)in,respectively,7-and15-weekoldSt14(panelscandd)andlittermateSt14(panelsc0andd0)colons.
BaselinelevelsofTandBcellsinthelaminapropriaoftheSt14colon(exampleswitharrowsinpanelsdandc)andabundanceofTandBcellsinboththemucosaandthesubmucosaofSt14colons(exampleswithstarsinpanelsc0andd0).
Scalebar100mm.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3684OncogeneA3(Saa3),lymphocyteantigen6complex,locusI(Ly6i),lymphocyteantigen6complex,locusD(Ly6d)andGPI-anchoredmolecule-likeprotein(Gml).
Inam-matorycircuitactivationwasmanifestatday10,withupregulatedexpressionofanumberofadditionalinammation-associatedgenes,includingreceptor-inter-actingserine-threoninekinase3(Ripk3),lipopolysac-charide-bindingprotein(Lbp),lactotransferrin(Ltf),TNFAIP3-interactingprotein3(Tnip3),secretoryleu-kocytepeptidaseinhibitor(Slpi),leucine-richalpha-2-glycoprotein1(Lrg1)andchemokine(C–X–Cmotif)ligand5(Cxcl5).
Severalepithelialproliferation-asso-ciatedgenesalsowereupregulatedatday10,includinggenesencodingthegrowthfactorsamphiregulin(Areg),heparin-bindingEGF-likegrowthfactor(Hbegf)andthep53-bindingproliferationinducer,tripartitemotifcontaining-29(Trim29).
Takentogether,thecombinedstage-specichistologicalandtranscriptomicanalysesshowthatcolonicepithelialablationofmatriptasecausesaberrantearlypostnatalepithelialdifferentiationthattriggersexpressionofpro-inammatorymediators,whichinturncausespersistentinammationandchronicepithelialhyperproliferation.
Colonictumorinitiationinhumansandanimalmodelsisfrequentlylinkedtodysregulatedbonemorphogeneticprotein(BMP),NotchandWntsignal-ing,leadingtotheexpansionofthecolonicstemcellpopulation(deLauetal.
,2007;Hardwicketal.
,2008;MedemaandVermeulen,2011;Zekietal.
,2011).
Therefore,weexaminedthelevelofexpressionofseveralputativeandvalidatedcolonicstemcellmarkers(Aldh1a1,Ascl2,Ets2,Lgr5,Phlda1),aswellasBMP(Cbfb,Dlx2,Hes1,Id1,Id2,Id3,Id4,Junb,Sox4,Stat1),Notch(Cdkn1a,Ccdn1,Cdk2,Hes1,Hes6,Klf4,Myc,Nfkb2)andWnt(Ascl2,Axin2,Cd44,Csnk1a1,Csnk1d,Csnk1e,Ctnnb1,Cryl1,Ephb2,Ephb3,G1,Hdac2,Id3,Ihh,Lef1,Myc,Nkd1,Nlk,Pascin2,Pcna,Plat,Rbbp4,Snai1,Sox4,Sox9,Spdef,Stra6,Tcf4,Yes1)targetgenesinSt14miceandtheirassociatedSt14littermates(SupplementaryTable2).
Thistranscrip-tomicanalysisprovidednoclearevidenceofstemcellexpansionordysregulationofeitherofthethreesignalingpathwaysatday0,atday5,whenabnormaldifferentiationandinnateimmuneactivationwereapparentorevenatday10,whenabnormalcolonicmorphologywasmanifest.
Inagreementwiththisanalysis,expressionoftheWnttarget,Sox9,inSt14micewasappropriatelyconnedtothecryptsatday10,andaberrantlocalizationofSox9wasnotdetecteduntilday15(SupplementaryFigure2).
Diminutionoftheintestinalmicrobiotaretardsdevelopmentofinammatoryboweldisease-likecolitisWehypothesizedthatthechronicinammatorymicro-environmentthatfacilitatedneoplasticprogressionoftheSt14-ablatedcolonicepitheliumwasgeneratedinpartbyincreasedexposureofthemucosalimmunesystemtotheresidentmicrobiotaduetoimpairedbarrierformationandaberrantdifferentiation.
Tochallengethishypothesis,wenexttreatedweaning-ageSt14+St14+St14-St14+St14+St14-*Colon,biotinColon,PBSdef*Colon,biotin*Smallintestine,biotinSmallintestine,PBSSmallintestine,biotinFigure5Matriptase-ablatedcolonisleaky.
Thelumenofthecolonandsmallintestineofweaning-ageSt14andlittermateSt14animalswasinjectedwithSulfo-NHS-LC-BiotininPBS(a,b,d,e)orPBS(c,f).
After3min,theintestinewasexcised,sectionedandstainedforbiotin(green).
Nucleiwerestainedwith4,6-diamino-2-phenylindol(blue).
Arrowsinpanelsa,dandeshowbiotinboundtothesurfaceofthemucosa.
Arrowheadsinpanelbandtheinsetinpaneleshowbiotinlabelingofthebasolateralmembraneofpolarizedepithelialcells.
Thediffusionofbiotinintotheintercellularspacewasnotobservedinthenormalcolonorsmallintestine(panelsaandd,alsocompareinsetsinpanelsdande).
Starsshowbiotinlabelingofconnectivetissueofbothmatriptase-ablatedcolon(panelb)andsmallintestine(panele).
Therewasnosignalforbiotininthecolonandsmallintestine(panelscandf)injectedwithPBS.
Scalebar20mm.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3685OncogeneSt14micewithacocktailofthefourantibiotics,ampicillin,neomycin,metronidazoleandvancomycinorwithvehiclefor2weeks,astandardprocedureforcleansingoftheintestinalmicrobiota(Rakoff-Nahoumetal.
,2004).
Owingtothefailureofsomeofthefourantibioticstobepresentinmilk,thetreatmentwasinitiatedatweaning,whensignicantpre-neoplasticprogressionwasalreadyapparent(Figures6eande0).
Asexpected,antibioticstreatmentreducedthecolonicbacterialloadbyB1500-fold,asjudgedbytheabundanceofbacterial16SribosomalDNAinfeces(Figure7a),withoutcompromisingbodyweight(Figure7b).
Helicobacteriscommonlyfoundinthecommensalmicrobiotaofmiceandpromotesneoplasticprogressioninvariousmodelsofcoloncarcinogenesis(Newmanetal.
,2001;Engleetal.
,2002;Erdmanetal.
,2003;Maggio-Priceetal.
,2006;Haleetal.
,2007).
Therefore,wespecicallydeterminedthepresenceofhelicobacterDNAinfecesofantibiotics-treatedandantibiotics-untreatedSt14micebyPCRanalysis(SupplementaryTable3).
Inall,9of17(53%),7of17(41%)and1of17(6%)controlmicetestedwerepositiveforHelicobactertyphlonius,Helicobacterroden-tiumandforanundeterminedHelicobacterspecies,respectively,whereas2of13(15%)oftheanalyzedantibiotics-treatedmicewerepositiveforH.
typhlonius.
Althoughinitiatedonlyafterweaning,antibioticstreatmentmarkedlybluntedabnormalepithelialdiffer-St14+a′b′c′d′e′St14-P0P5P10P15P20*Figure6Progressivepostnatallossofepithelialintegrityofmatriptase-ablatedcolonprecedesmalignanttransformation.
HistologicalappearanceofSt14(a–e)andlittermateSt14(a0-e0)colonsatpostnatalday0(panelsaanda0),5(panelsbandb0),10(panelscandc0),15(panelsdandd0)and20(panelseande0).
Nohistologicaldifferencescanbeobservedbetweennormalandmatriptase-ablatedcolonatdays0and5(comparepanelsaanda0,bandb0).
Atday10,St14colonsshowsporadicfociofdetachingandapoptoticcells(arrowheadsinpanelc0).
Thisphenotypeissignicantlystrongeratdays15and20withextensiveanoikis(arrowheadsinpaneld0),apoptoticcells(arrowsinpanelsd0ande0),ulcerations(arrowheadinpanele0)andinammatorycellinltrates(starinpanele0).
Scalebar100mm.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3686Oncogeneentiation,epithelialhyperproliferationandinamma-tionofthecolon.
Thiswasevidencedbyaquantitativereductionincolonicmucosalthickness(Figure7c),increasedmucinproduction(Figures7d,8aandb),decreasedepithelialproliferation(Figures7eand8c,d)anddecreasedinammatorycellinltration(Figures7f–hand8e–j),althoughthediminutionofT-cellandneutrophilabundancedidnotreachstatisticalsignicance.
Furthermore,b-cateninexpressionlevelswerenormalizedintwooffourantibiotics-treatedSt14micewhenanalyzedbyimmunoblot(Supplemen-taryFigure3a,comparelanes7–10with13and14),andSox9wasonlyinfrequentlyfoundinthedistalportionofthecoloniccryptsofantibiotics-treatedSt14mice(SupplementaryFigures2dande),suggest-ingadiminutionofWntsignaling.
AnalysisofBMP(phospho-SMAD1/5)andNotch1(Notch1intracellulardomain)signalingdidnotrevealatreatment-orgenotype-specicpattern,buttheabundanceofeachofthetwoproteinspeciesinintestinaltissueextractswasdifculttoassessaccurately(Supplemen-taryFigure3b).
Takentogether,thedataarecompatiblewithaprincipalroleofthecommensalmicrobiotainpre-neoplasticprogression.
Amechanisticmodelforma-triptaseablation-inducedcoloncancerbasedontheabovendingsisshowninFigure9.
WeproposethattheintrinsicdefectinbarrierfunctionassociatedwithTable2Genesdifferentlyregulatedin5-day-oldSt14miceAgilentTechnologiesprobeIDGenBankRegulationFoldchangeaP-valuebGenesymbolGenenameA_51_P124665NM_011474Up17.
860.
001Sprr2hSmallproline-richprotein2HA_51_P272066NM_025929Up9.
400.
001RIKENcDNA2010109I03geneA_51_P451966NM_001177524Up5.
390.
035GmlGPI-anchoredmolecule-likeproteinA_52_P151240NM_001195732Up4.
340.
024Fam150aPredictedgene,familywithsequencesimilarity150,memberAA_51_P343517NM_010742Up3.
870.
027Ly6dLymphocyteantigen6complex,locusDA_51_P363187NM_008176Up3.
130.
001Cxcl1Chemokine(C–X–Cmotif)ligand1A_51_P291950NM_010266Up2.
910.
037GdaGuaninedeaminaseA_52_P545650NM_001174099Up2.
830.
011Krt36Keratin36A_51_P187121NM_008127Up2.
630.
028Gjb4Gapjunctionprotein,beta4A_51_P411495XM_897643Up2.
500.
045RIKENcDNA4930465A12geneA_51_P420918NM_020498Up2.
390.
050Ly6iLymphocyteantigen6complex,locusIA_51_P367880NM_008474Up2.
350.
037Krt84Keratin84A_51_P120830NM_019471Up2.
290.
037Mmp10Matrixmetallopeptidase10A_51_P385639NM_010291Up2.
280.
011Gjb5Gapjunctionprotein,beta5A_51_P337308NM_011315Up2.
270.
019Saa3SerumamyloidA3A_51_P228574NM_146214Up2.
260.
043TatTyrosineaminotransferaseA_52_P562661NM_010762Up2.
170.
023MalMyelinandlymphocyteprotein,T-celldifferentiationproteinA_51_P499071NM_010762Up2.
140.
011MalMyelinandlymphocyteprotein,T-celldifferentiationproteinA_51_P503494NM_018790Up2.
110.
003ArcActivity-regulatedcytoskeletal-associatedproteinA_52_P299446U90654Up2.
090.
013Zinc-ngerdomain-containingproteinA_52_P220241AK164337Up2.
090.
032RIKENcDNAA430106P18gene,hypotheticalproline-richregioncontainingproteinA_51_P139678NM_009264Up2.
090.
021Sprr1aSmallproline-richprotein1AA_51_P214275NM_001174099Up2.
080.
023Krt36Keratin36A_51_P245090NM_016689Up2.
070.
020Aqp3Aquaporin3A_51_P385099NM_013693Up2.
060.
019TnfTumornecrosisfactorA_52_P26416AF106279Up2.
050.
045Lamc2Laminingamma2chainA_52_P445360NM_023256Up2.
040.
028Krt20Keratin20A_51_P197528NM_001099217Up2.
030.
050Ly6c2Lymphocyteantigen6complex,locusC2A_52_P421234NM_133832Up2.
020.
011Rdh10Retinoldehydrogenase10A_51_P323195NM_172613Down4.
650.
045Atp13a4ATPasetype13A4A_52_P453814NM_011242Down3.
090.
045Rasgrp2RAS,guanyl-releasingprotein2A_51_P394847NR_024599Down2.
860.
045Predictedgene11346A_51_P492940AK035376Down2.
630.
045RIKENfull-lengthenrichedlibrary,clone:9530027C22,unclassiableproductA_51_P394172NM_007954Down2.
440.
021Es1Esterase1A_51_P375969NM_053200Down2.
370.
036Ces3Carboxylesterase3A_52_P994399NM_010039Down2.
340.
048Defa4defensin,alpha,4A_52_P115950AK036853Down2.
280.
045RIKENfull-lengthenrichedlibrary,clone:9930018I23,hypotheticalproteinA_51_P391934NM_029706Down2.
170.
050Cpb1CarboxypeptidaseB1A_51_P358037NM_001014423Down2.
060.
045Abi3bpABIgenefamily,member3(NESH)-bindingproteinA_52_P819243AK049777Down2.
050.
045RIKENfull-lengthenrichedlibrary,clone:C530048O03,hypotheticalproteinA_51_P242967NM_021308Down2.
010.
011Piwil2Piwi-likehomolog2aComparedwithexpressioninthenormalmucosa.
bStudent'st-test(two-tailed,unpaired,asymptotic),Benjamini–Hochbergmultipletestingcorrection.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3687Oncogenethefailuretoformfunctionaltightjunctions(Listetal.
,2009;Buzzaetal.
,2010)causesincreasedexposureoftheimmunesystemtothecommensalmicrobiota.
Thisexposureelicitsvigorousinammatoryandrepairresponsesthatinvolveepithelialstemcellactiva-tionandarecontinuous,ratherthantransient,dueTable3Genesdifferentlyregulatedin10-day-oldSt14miceAgilentTechnologiesprobeIDGenBankRegulationFoldchangeaP-valuebGenesymbolGenenameA_52_P295432NM_009141Up32.
090.
044Cxcl5Chemokine(C–X–Cmotif)ligand5A_51_P124665NM_011474Up28.
950.
037Sprr2hSmallproline-richprotein2HA_51_P256827NM_013650Up9.
860.
044S100a8S100calcium-bindingproteinA8(calgranulinA)A_51_P346938NM_029796Up8.
300.
044Lrg1Leucine-richalpha-2-glycoprotein1A_51_P363187NM_008176Up5.
020.
044Cxcl1Chemokine(C–X–Cmotif)ligand1A_52_P487686NM_001082546Up4.
360.
044cDNAsequenceBC100530A_51_P279437NM_029662Up3.
980.
048Mfsd2aMajorfacilitatorsuperfamilydomaincontaining2AA_51_P359046NM_020519Up3.
780.
044Slurp1SecretedLy6/Plaurdomaincontaining1A_51_P303160NM_007482Up3.
710.
044Arg1ArginaseA_51_P451966NM_001177524Up3.
580.
044GmlGPI-anchoredmolecule-likeproteinA_52_P1172382Q8C9Z4cUp3.
440.
049PutativeuncharacterizedproteinA_52_P472324NM_011414Up3.
130.
044SlpiLeukocytepeptidaseinhibitorA_51_P200544NM_001001495Up3.
120.
044Tnip3TNFAIP3-interactingprotein3A_51_P214275NM_001174099Up3.
100.
037Krt36Keratin36A_51_P187461NM_009044Up3.
030.
044RelReticuloendotheliosisoncogeneA_51_P116609NM_025687Up2.
870.
044Tex12Testisexpressedgene12A_52_P531140NM_010416Up2.
810.
044Hemt1Hematopoieticcelltranscript1A_52_P545650NM_001174099Up2.
740.
038Krt36Keratin36A_52_P31510NM_008814Up2.
650.
037Pdx1Pancreaticandduodenalhomeobox1A_52_P273394AK137552Up2.
580.
013Igl-5Immunoglobulinlambdachain5A_51_P272066NM_025929Up2.
570.
044RIKENcDNA2010109I03geneA_52_P116006NM_010266Up2.
570.
044GdaGuaninedeaminaseA_51_P225634NM_027306Up2.
500.
044Zdhhc25Zincnger,DHHCdomaincontaining25A_52_P200286NM_001167746Up2.
460.
044Dnahc17Dynein,axonemal,heavychain17A_52_P482897NM_009704Up2.
400.
044AregAmphiregulinA_52_P15388NM_008522Up2.
330.
044LtfLactotransferrinA_51_P500082NM_001110517Up2.
270.
044predictedgene14446A_52_P884135AK085881Up2.
260.
049RIKENfull-lengthenrichedlibrary,clone:D830023G23,unclassiableproductA_51_P165182NM_028967Up2.
250.
044Batf2Basicleucinezippertranscriptionfactor,ATF-like2A_52_P338066NM_023137Up2.
240.
049UbdUbiquitinDA_51_P454008NM_008489Up2.
230.
037LbpLipopolysaccharide-bindingproteinA_51_P291950NM_010266Up2.
220.
044GdaGuaninedeaminaseA_52_P375047NM_009184Up2.
220.
039Ptk6PTK6proteintyrosinekinase6A_51_P409349NM_023655Up2.
210.
046Trim29Tripartitemotif-containing29A_51_P228971NM_023219Up2.
190.
044Slc5a4bSolutecarrierfamily5(neutralamino-acidtransporters,systemA),member4bA_51_P249989NM_145133Up2.
190.
047TifaTRAF-interactingproteinwithForkhead-associateddomainA_52_P299446U90654Up2.
180.
044Zinc-ngerdomain-containingproteinA_52_P569327AK045953Up2.
160.
044Usp53mKIAA1350,ubiquitin-specicpeptidase53A_52_P208213TC1638459Up2.
150.
044Kalirin-12a,partial(6%)A_51_P503494NM_018790Up2.
080.
049ArcActivity-regulatedcytoskeletal-associatedproteinA_52_P562661NM_010762Up2.
060.
044MalMyelinandlymphocyteprotein,T-celldifferentiationproteinA_51_P491987NM_019955Up2.
040.
044Ripk3Receptor-interactingserine-threoninekinase3A_51_P499071NM_010762Up2.
040.
044MalMyelinandlymphocyteprotein,T-celldifferentiationproteinA_51_P506417NM_016958Up2.
030.
044Krt14Keratin14A_51_P181565NM_010415Up2.
020.
044HbegfHeparin-bindingEGF-likegrowthfactorA_51_P371500BC049570Up2.
000.
044Atp8b3ATPase,classI,type8B,member3A_51_P426055AK048117Down3.
760.
013RIKENfull-lengthenrichedlibrary,clone:C130035O18,unclassiableproductA_52_P506984ENSMUST00000044976dDown3.
220.
044GlyatGlycine-N-acyltransferaseA_52_P707475AK053952Down2.
530.
044RIKENfull-lengthenrichedlibrary,clone:E230006P11,unclassiableproductA_52_P739568AK082480Down2.
170.
044RIKENfull-lengthenrichedlibrary,clone:C230053P15,unclassiableproductA_52_P101443NM_198111Down2.
140.
044Akap6Akinase(PRKA)anchorprotein6aComparedwithexpressioninthenormalmucosa.
bStudent'st-test(two-tailed,unpaired,asymptotic),Benjamini–Hochbergmultipletestingcorrection.
cUniProtKB.
dMouseGenomeInformatics.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3688Oncogenetotheinherentfailureofmatriptase-ablatedcolonicepithelialcellstoestablishafunctionalbarrier.
Thesustainedhyperproliferationofepithelialstemcellswithinagenotoxicchronicinammatorymicroenviron-mentinturninducesprogressivegenomicinstabilityandsubsequentrapidmalignantconversion.
DiscussionIthaslongbeensuspectedthatintrinsicalterationsintheparacellularintestinalpermeabilitybarrierinhu-manscouldbeaprimingfactorfordevelopmentoftheaberrantimmuneresponsetothecommensalmicrobiotaBodyweightBacteriainfecesP=NSP<0.
0001Bodyweight(g)16SrDNAexpression(AU)ControlAntibioticsControlMucinproductionThicknessofcolonmucosaP=0.
03P=0.
002AntibioticsThicknessofmucosa(AU)Alcianbluepositivecells/mm2ProliferationB-cellsP=0.
03P=0.
03Ki67positivecells/mm2CD3positivecells/mm2Kappalightchainpositivecells/mm2T-cellsNeutrophilsP=NSP=NSControlAntibioticsControlAntibioticsControlAntibioticsControlAntibioticsControlAntibioticsControlAntibioticsMyeloperoxidasepositivecells/mm2Figure7Theresidentmicrobiotacontributestopreneoplasticprogressionofthematriptase-ablatedcolon.
LittermateSt14micewerekeptonregularwater(controlina–h)ortreatedwithacombinationofampicillin,neomycin,metronidazoleandvancomycinindrinkingwater(antibioticsinpanelsa–h)for2weeksstartingimmediatelyafterweaning.
Animalswereeuthanized,fecessampleswereusedfortheisolationofbacterialDNAandthecolonictissuewassubjectedtoquantitativehistomorphometricanalysis.
(Panela)PCRquanticationof16SbacterialribosomalDNAshowsa1500-folddecreaseintheintestinalmicrobiotaofantibiotics-treated(N15)comparedwithcontrol(N13)mice.
(Panelb)Bodyweightsofantibiotics-treated(N7)andcontrol(N7)micearesimilar.
(c)Decreasedthicknessofthemucosaofantibiotics-treated(N6)comparedwithcontrol(N5)mice.
(Panelsd–f)Preservationofmucinproduction(paneld),decreasedproliferation(panele)anddecreasedinltrationofB-cells(panelf),T-cells(panelg)andneutrophils(panelh)inantibiotics-treated(N6)comparedwith(N5)mice.
StatisticalsignicancewascalculatedbyStudent'st-test(two-tailed)(Panelsa–c,e–h),andnon-parametricMann–WhitneyU-test(two-tailed)(d),NS,notsignicant.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3689OncogeneSt14-ControlMucinproductionProliferationB-cellsT-cellsNeutrophilsAntibioticsEpithelialbarrierdefect-inducedcarcinogenesisPKosaetal3690Oncogenethatunderliesinammatoryboweldiseaseanditsassociatedmalignancies.
Thisstudynowprovidesstrongexperimentalsupportforthisnotionbyshowingthatintestinalepithelial-specicablationofmatriptase—amembrane-anchoredserineproteasethatisessentialforintestinalepithelialtightjunctionformation—causesacommensalmicrobiota-dependentinammatoryboweldisease-likecolitisthatveryrapidlyprogressestoadenocarcinoma.
Furthermore,thespontaneousandrapidmalignanttransformationofthecolonicepithe-liumdemonstratesthatasimpleincreaseinintestinalparacellularpermeabilitysufcestobothinitiateanddriveinammation-associatedadenocarcinomaforma-tion.
Thisndingparallelstherecentidenticationofthepermeabilityoftheepidermalbarrierasamajordeterminantofthedevelopmentofotherchronicinammatorydiseases,includingichthyosisvulgaris,atopiceczemaandasthma(Smithetal.
,2006;Sandilandsetal.
,2009).
Previouslypublishedanimalmodelsofinammatoryboweldisease-associatedcolor-ectalcancerincludedextransodiumsulfate-inducedordextransodiumsulfatecombinedwithazoxymethane-inducedchemicalcoloncarcinogenesisininbredmousestrains(Okayasuetal.
,1996,2002),andgeneticmodels,includinggermlineIl10-ablated(Bergetal.
,1996),combinedgermlineTbx21-andRag2-ablated(Garrettetal.
,2009),myeloidlineageStat3-ablated(Dengetal.
,2010),myeloidlineageItgav-ablated(Lacy-Hulbertetal.
,2007),germlineGnai2-ablated(Rudolphetal.
,1995)andcombinedgermlineIl2-andb2m-ablatedmice(Shahetal.
,1998).
Inthesemodels,colitisandcoloncarcinomaoccurasaconsequenceofeitherasustainedchemicaldamagetothecolonicepitheliumorperturba-tionoftheimmunesystemthrougheliminationofkeyeffectorsofinnateoradaptiveimmunity.
Comparedwiththeabovemodels,coloncarcinogenesisinintestinalSt14-ablatedmiceappearstodisplaysomeuniquefeatures.
First,itisinitiatedbyalossofintestinalbarrierfunction,whichisassociatedwithaberrantdifferentiationandimmuneactivation,buttheseprim-ingeventsinitiallyoccurintheabsenceofdetectableperturbationofcommoncoloncancer-associatedsignal-ingpathways(Kaiseretal.
,2007).
Second,adenocarci-nomawithinvolvementoflymphoidtissuesisobservedeveninveryyounganimals.
Thisveryrapidneoplasticprogressionmaybeexplained,atleastinpart,bythepresenceofvariousHelicobacterspeciesintheintestinalmicrobiotaofSt14mice,ascoloncarcinogenesisinseveralmousemodelshasbeenshowntobeacceleratedbyortobedependentuponHelicobactercolonization(Newmanetal.
,2001;Engleetal.
,2002;Erdmanetal.
,2003;Maggio-Priceetal.
,2006;Haleetal.
,2007).
Inlightofthendingsinthisstudy,itistemptingtospeculatethatneoplasticprogressioninpreviouslydescribedmousemodelsofinammatoryboweldis-ease-associatedcolorectalcancer(andperhapscolitis-associatedhumancolorectalcancer)maybeacceleratedbyanimmuneactivation-induceddecreaseintheintestinalparacellularpermeabilitybarriercausedbydownregulatingtheactivityofmatriptaseorothermoleculeswithinamatriptase-dependentproteolyticpathwaythatfacilitatestightjunctionformation.
Conclusivelinkshavebeenforgedbetweenincreasedactivityofmultiplemembersofthecomplementofextracellularandpericellularproteasesandtheinitiationandprogressionofavidevarietyofhumanmalignancies(reviewedinthestudiesbyAndreasenetal.
,2000;Netzel-Arnettetal.
,2003;BorgonoandDiamandis,2004;MohamedandSloane,2006;Kessenbrocketal.
,2010).
Muchlessstudiedisthecapacityofextracellular/pericellularproteasestoactassuppressorsoftumorigen-Figure8Histologicalappearanceofantibiotics-treatedmatriptase-ablatedcolon.
(a,b)AlcianBluestainingofmucinproducedbydifferentiatedgobletcellsinuntreated(panela)andantibiotics-treated(panelb)St14colons.
Arrowheadspointtomucin(blue).
(c,d)ImmunohistochemicalstainingforKi67inuntreated(panelc)andantibiotics-treated(paneld)St14miceshowsignicantlydecreasedratesofproliferationofbothepithelialcells(arrowheadsinpanelscandd)andconnectivetissuecells(arrowsinpanelscandd).
(e–j)ImmunohistochemicalstainingforB-cells(panelseandf),T-cells(panelsgandh)andneutrophils(panelsiandj)inuntreated(panelse,gandi)andantibiotics-treated(panelsf,handj)St14colonsshowsreducedchronic(exampleswitharrowheadsinpanelse–h)andacute(exampleswitharrowheadsinpanelsiandj)inammatorycellinltration.
Scalebar50mm.
LossofmatriptaseDiminutionofepithelialbarrierfunctionCommensalmicrobiotaRegenerativeatypiaAdenocarcinomaRepairInflammation,hyperproliferationFigure9Modelformatriptaseablation-inducedcoloncarcino-genesis.
Lossofmatriptasefromtheintestinalepitheliumcompromisesepithelialbarrierfunction,therebycausingexposureofthecommensalmicrobiotatoresidentimmunecells.
Thistriggersarepairresponsethatincludesactivationoflocalinammatorycircuitsandcolonicstemcellactivation.
Thisresponseisperpetual,ratherthantransient,duetotheintrinsicinabilityofmatriptase-ablatedintestinalepitheliumtoformafunctionalbarrier.
PersistenthyperproliferationofcolonicstemcellswithinaDNA-damagingchronicinammatorymicroenviron-mentcausestheformationofadenocarcinoma.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3691Oncogeneesis(reviewedinthestudiesbyLopez-OtinandMatrisian,2007;Lopez-Otinetal.
,2009).
Inducedgermlineorlineage-specicgenedeletionstudiesinmiceandsponta-neoussomaticmutationanalysisofhumancancershaveprovideddirectevidenceforatumor-suppressivefunctionofmatrixmetalloproteinase3(MMP3)(McCawleyetal.
,2004,2008),MMP8(Balbinetal.
,2003;Palavallietal.
,2009),MMP12(Acuffetal.
,2006)andCathepsinL(Reinheckeletal.
,2005).
Furthermore,thefrequentepigeneticandgeneticsilencingofotherextracellularandpericellularproteasesinhumancancersandtheabilitytoinhibitdistinctstepsoftumorprogressioninexperimentalmodelsofcancerthroughmodulationoftheirlevelofexpressionsuggestthatthenumberofextracellularandpericellularproteaseswithtumor-suppressivefunctionmaybesubstantial(reviewedinthestudiesbyLopez-OtinandMatrisian(2007)andLopez-Otinetal.
(2009)).
Thisstudyaddsmatriptasetotheabovelistofpericellularproteaseswithtumor-suppressivefunctions.
Matriptase,however,sofarisuniqueamongpericellularproteasesinthesensethatitsabsencebyitselfsufcestocausemalignancy,whereasthetumor-suppressivefunctionofotherproteaseswasrevealedinchemicalandtransplanta-tionmodelsofcancer.
Previousstudieshaveshownthatoverexpressionofmatriptasecaninitiatecarcinogenesisandacceleratedisseminationofvariouscarcinomasindiversemodelsystems.
Thispropertyofmatriptaseisowedatleastinparttoitsabilitytoautoactivate(Oberstetal.
,2003)andsubsequentlyserveasaninitiatorofseveralintracellularsignalingandproteolyticcascadesthroughproteolyticmaturationofgrowthfactors,protease-activatedreceptoractivationandproteasezymogenconversion(Leeetal.
,2000;Takeuchietal.
,2000;Iharaetal.
,2002;Suzukietal.
,2004;Forbsetal.
,2005;Listetal.
,2005;Jinetal.
,2006;Kilpatricketal.
,2006;Netzel-Arnettetal.
,2006;Bhattetal.
,2007;Chengetal.
,2009;Owenetal.
,2010;Salesetal.
,2010;Ustachetal.
,2010;Szaboetal.
,2011).
Thedualabilityofaproteasetopromotecarcinogenesisinsomecontexts,whilesuppressingcarcinogenesisinothers,israre,butnotentirelyunprecedented.
Aclearexampleisgivenbythetumor-promotingeffectoftransgenicmisexpressionofthestromalprotease,MMP3,inthemammaryepithelialcompartment,asopposedtothestrongprotectionofmicefromchemicallyinducedsquamouscellcarcinogenesiscausedbygermlineablationoftheprotease(Sternlichtetal.
,1999,2000;McCawleyetal.
,2004,2008).
Additionalproteases,suchasMMP9,MMP11andMMP19mayalsopromoteorsuppress,respectively,malignantprogressioninastageortissue-dependentmanner(Lopez-OtinandMatrisian,2007).
Inconclusion,ourstudyhasuncoveredacriticalroleofthetransmembraneserineproteasematriptaseinpreservingimmunehomeostasisinthegastrointestinaltractandinsuppressingtheformationofcolitisandcolitis-associatedadenocarcinomaformation.
Further-more,thisstudysurprisinglyrevealsthatthesimpleperturbationoftheepithelialpermeabilitybarriersufcestorapidlyandefcientlyinducemalignanttransformationofthecolonicepithelium.
MaterialsandmethodsAnimalexperimentsAllproceduresinvolvingliveanimalswereperformedinanAssociationforAssessmentandAccreditationofLaboratoryAnimalsCareInternational-accreditedvivariumfollowinginstitutionalguidelinesandstandardoperatingprocedures.
Withinthestudyperiod,sentinelswithinthemouseholdingroomsporadicallytestedpositiveforHelicobacter,murinenorovirusandmouseparvovirus,andtestednegativeforectromeliavirus,mouserotavirus,Theiler'sencephalomyelitisvirus(GDVIIstrain),lymphocyticchoriomeningitisvirus,mycobacteria,mousehepatitisvirus,minutevirusofmice,mousepolyomavirus,pneumoniavirusofmice,reovirustype3,Sendaivirusandfecalendoparasitesandectoparasites.
TheNIDCRInstitutionalAnimalCareandUseCommitteeapprovedthisstudy.
Allstudieswerestrictlylittermatecontrolled.
St14knockout(St14/)andconditionalknockout(St14LoxP/LoxP)micehavebeendescribedpreviously(Listetal.
,2002,2009).
Villin-Cre/0(B6.
SJL-Tg(Vil-Cre)997Gum/J)mice(Madisonetal.
,2002)werepurchasedfromTheJacksonLaboratory(BarHarbor,ME,USA).
Allexperimentalanimalswereinamixed129/C57BL6/J/NIHBlackSwiss/FVB/NJbackground.
ThegenotypesofallmiceweredeterminedbyPCRofearortailbiopsyDNA.
St14andSt14LoxPallelesweredetectedusingtheprimers50-CAGTGCTGTTCAGCTTCCTCTT-30and50-GTGGAGGTGGAGTTCTCATACG-50,respectively.
ThepresenceoftheSt14knockoutallelewasdetectedusingprimers50-GTGGAGGTGGAGTTCTCATACG-30and50-GTGCGAGGCCAGAGGCCACTTGTGTAGCG-30.
TheCretransgenewasdetectedusingtheprimers50-GCATAACCAGTGAAACAGCATTGCTG-30and50-GGACATGTTCAGGGATCGCCAGGCG-30.
Forantibioticstreatment,micewereadministeredacombina-tionofampicillin(1g/l,Sigma-Aldrich,StLouis,MO,USA),neomycin(1g/l,Sigma-Aldrich),metronidazole(1g/l,Sigma-Aldrich)andvancomycin(0.
5g/l,Sigma-Aldrich)indrinkingwaterfor2weeks,startingimmediatelyafterweaning(P20).
QuantitativePCRanalysisRNAwaspreparedfrommouseorgansbyextractioninTRIzolreagent(Invitrogen,Carlsbad,CA,USA),asrecom-mendedbythemanufacturer.
First-strandcDNAsynthesiswasperformedusingoligo(dT)primerswiththeiScriptcDNAsynthesiskit(Bio-RadLaboratories,Hercules,CA,USA).
AniCycler,geneexpressionanalysissoftwareandIQSYBRGreenSupermix(allfromBio-RadLaboratories)wereusedforquantitativePCRanalysisinaccordancewiththemanufacturer'sinstructions,usingaprimercomplementarytosequenceofmatriptaseexon1,50-AACCATGGGTAGCAATCGGGGC-30,andmatriptaseexon2,50-AACTCCACACCCTCCTCAAAGC-30(annealingtemperature601C,dena-turationtemperature951C,40cycles).
Matriptaseexpressionlevelswerenormalizedtoglyceraldehyde-3-phosphatedehy-drogenase(Gapdh)levelsineachsample.
GapdhmRNAwasampliedwiththeprimers50-GTGAAGCAGGCATCTGAGG-30and50-CATCGAAGGTGGAAGAGTGG-30(annealingtemperature601C,denaturationtemperature951C,40cycles).
QuanticationofbacterialintestinalcolonizationBacterialDNAwasisolatedfromfecesusingQIAampDNAStoolMiniKit(Qiagen,Valencia,CA,USA)accordingtothemanufacturer'sinstructions.
BacterialDNAwasquantiedbyquantitativePCRanalysisofbacterial16SribosomalDNA,ampliedbyprimers:50-AGAGTTTGATCMTGGCTCAG-30Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3692Oncogeneand50-TTACCGCGGCKGCTGGCAC-30.
AniCycler,geneexpressionanalysissoftwareandIQSYBRGreenSupermixwereusedforreal-timePCRinaccordancewiththemanufacturer'sinstructions.
Thethermalcyclingprogramconsistedof951Cfor10min,followedby40cyclesof951Cfor30s,551Cfor30s,601Cfor45s,651Cfor15sand721Cfor15s.
Helicobactertestingwasperformedasdescribedpre-viously(Rileyetal.
,1996;Fengetal.
,2005).
IntestinaltightjunctionassayInall,50mlof10mg/mlEZ-LinkSulfo-NHSLC-Biotin(ThermoFisherScientic,Waltham,MA,USA)inphos-phate-bufferedsalinecontaining1mmol/lCaCl2wasinjectedintothelumenofthedistalcolonandjejunumof3-week-oldSt14andSt14littermatesthatwereanesthetizedbyintraperitonealinjectionofananestheticcombination(keta-mine(20mg/ml),xylazine(2mg/ml),50ml/10g).
After3minincubation,micewerekilled,andtheinjectedportionoftheintestinewasexcisedandxedin10%neutral-bufferedzincformalin(Z-x,Anatech,BattleCreek,MI,USA)for3h,processedintoparafnandsectioned.
Five1-mmsectionswereblockedfor30mininblockingsolution(5%bovineserumalbumininphosphate-bufferedsaline),andthenincubatedfor1hatroomtemperaturewithstreptavidinAlexaFluor448conjugate(Invitrogen)(5mg/ml)inblockingsolution.
Sectionswerewashedthreetimeswithphosphate-bufferedsalineandmountedwithVECTASHIELDHardSetMountingMediumwithDAPI(VectorLaboratories,Burlingame,CA,USA).
ImageswereacquiredonanAxioImager.
Z1microscopeusinganAxioCamHRc/MRmdigitalcamera(CarlZeissLtd,Jena,Germany).
HistopathologyMicewerekilledbyCO2inhalation.
Tissueswerexedfor24hinZ-x,processedintoparafn,cutinto5mmsectionsandstainedwithhematoxylinandeosin.
Forvisualizationofmucinproduction,sectionswerestainedusingthePeriodicacid-Schiffkit(Sigma-Aldrich)orAlcianBlue(1%AlcianBluein3%aceticacid).
CollagenwasdetectedusingMasson-trichromestaining.
ImmunohistochemistryTissuesectionswerepreparedandantigensretrievedbyheatinginepitope-retrievalbuffer(0.
01Msodiumcitrate,pH6.
5)orEpitopeRetrievalBuffer-ReducedpH(BethylLabora-tories,Montgomery,TX,USA)formatriptaseimmunohisto-chemistry(IHC).
Sectionswereblockedfor1hin5%bovineserumalbumin(Sigma-Aldrich)or10%horseserum(formatriptaseIHC)inphosphate-bufferedsalineandincubatedovernightat41Cwithprimaryantibody:matriptase(Sheep,Polyclonal,R&DSystems,Minneapolis,MN,USA),cytoker-atins(Rabbit,Polyclonal,DakoCytomation,Carpinteria,CA,USA),LYVE-1(Goat,Polyclonal,R&DSystems),b-catenin(Rabbit,Monoclonal,CellSignaling,Danvers,MA,USA),laminin(Rabbit,Polyclonal,Sigma-Aldrich),BrdU(Rat,Monoclonal,AccurateChemicalsandScientic,Westbury,NY,USA),CD3(Rabbit,Polyclonal,DakoCytomation),k-lightchain(Rabbit,Polyclonal,DakoCytomation),Ki67(Rabbit,Polyclonal,Novocastra,Westbury,NY,USA),myeloperoxidase(Rabbit,Polyclonal,DakoCytomation)andSox9(Rabbit,Polyclonal,Millipore,Temecula,CA,USA).
Boundantibodieswerevisualizedusingeitherbiotin-conju-gatedhorseanti-goat,goatanti-rabbit,goatanti-rat(VectorLaboratories),rabbitanti-sheep(ThermoScientic,Rockford,IL,USA)oralkalinephosphatase-conjugateddonkeyanti-rabbit(DakoCytomation)secondaryantibodiesandaVectastainABCkit(VectorLaboratories)using3,30-diaminobenzidinesubstrate(Sigma-Aldrich)orVulcanFastRedChromogenKit2(BiocareMedical,Concord,CA,USA).
Hematoxylinwasthecounterstain.
ImmunoblottingApproximately1cmofthedistalpartoflargeintestinewashomogenizedandlysedin25mMTris–HCl,pH7.
6,150mMNaCl,1%NP-40,1%sodiumdeoxycholate,0.
1%SDSandproteaseinhibitorcocktail(Sigma-Aldrich).
Boiledandreducedsamplesweresubjectedtoimmunoblottingusingprimaryantibodies:anti-b-catenin(Rabbit,Monoclonal,CellSignaling),anti-phospho-SMAD1/5(Rabbit,monoclonal,CellSignaling),anti-Notch1(Rabbit,monoclonal,CellSignaling)andanti-GAPDH(Rabbit,polyclonal,SantaCruzBiotech-nology,SantaCruz,CA,USA).
Thesignalwasdetectedwithsecondaryanti-rabbitantibodyconjugatedtoeitheralkalinephosphatase(DakoCytomation)orhorseradishperoxidase(ThermoScientic).
MicroarrayanalysisMicroarrayanalysisofgeneexpressionwasperformedusingMouseGE444Kv2Microarrayslides(AgilentTechnolo-gies,SantaClara,CA,USA)accordingtothemanufacturer'sinstructions.
Inbrief,thecolontissuewasdissectedfromfourpairsofSt14andlittermateSt14mice.
RNAwasisolatedusingTRIzolreagent,asrecommendedbythemanufacturer.
ThequalityandtheintegrityoftheRNAweredeterminedusingthe2100Bioanalyzerplatform(AgilentTechnologies).
IsolatedcolonRNAtogetherwithUniversalMouseReferenceRNA(AgilentTechnologies)werelabeledusingtheTwo-colorQuickAmpLabelingKit(AgilentTechnologies).
LabeledRNAwashybridizedtotheslidesovernight.
Afterwashing,theslideswerescannedusingahigh-resolutionmicroarrayscanner.
TherawmicroarrayimageleswerereadandprocessedusingtheFeatureExtractionSoftware(AgilentTechnologies).
TheanalysisofmicroarraydatawasperformedusingGeneSpringSoftware(AgilentTechnologies).
GeneexpressionwasnormalizedtotheuniversalreferenceRNA.
Fromtheoriginaldataset,onlyprobesaggedasdetectedatleastinonesamplewereselected.
ThesedatasetswerefurtherlteredforprobeswithtwoormorefoldchangeinexpressionbetweenSt14andSt14tissues.
StatisticalanalysisofthesedatasetsledtotheidenticationofprobeswithsignicantlydifferentchangeingeneexpressionusingunpairedStudent'st-testwithBenjamini–Hochbergmultipletestingcorrection.
ProbeswithPo0.
05wereconsideredsignicantandareshowninTables2and3.
HistomorphometricanalysisFivecontrolSt14andsixantibiotics-treatedSt14micewereanalyzed.
Todeterminemucosalthickness,2mmofthedistalcolonmucosaadjacenttothesquamousepitheliumoftherectumwasidentiedonahematoxylinandeosinsectionandtheareawascalculatedusingAperioImageScopesoftware(Aperio,Vista,CA,USA).
Forquanticationofproliferation,differentiationandinammatoryinltrates,sevenindividualareasofdistalcolononeachslidewereselectedforcounting.
Thenumberofcountswasnormalizedtothesurfaceoftheselectedareaandaveragedforeachindividualanimal.
ConictofinterestTheauthorsdeclarenoconictofinterest.
Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3693OncogeneAcknowledgementsWethankDrsJerroldMWard,RobertDCardiffandStephenMHewittforpathologyadvice,DrsVyomeshPatelandKantimaLeelahavanichkulforhelpwiththearrayanalysis,DrMyrnaMandelforhelicobactertesting,aswellasDrsSilvioGutkindandMaryJoDantonforcriticallyreviewingthismanuscript.
HistologywasperformedbyHistoservInc.
(Germantown,MD,USA).
ThisstudywassupportedbytheNIDCRIntramuralResearchProgram(THB).
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SupplementaryInformationaccompaniesthepaperontheOncogenewebsite(http://www.
nature.
com/onc)Epithelialbarrierdefect-inducedcarcinogenesisPKosaetal3695Oncogene