Translational control of TWIST1 expression in MCF-10A cell lines

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SHORTCOMMUNICATION

TranslationalcontrolofTWIST1expressioninMCF-10Acelllinesrecapitulatingbreastcancerprogression

M-LNairisma¨gi1,4,5,AVislovukh2,QMeng3,GKratassiouk4,5,CBeldiman4,5,MPetretich4,5,RGroisman4,5,E-MFu¨chtbauer1,AHarel-Bellan4,5andIGroisman4,5TWIST1isahighlyconservedbasichelix-loop-helixtranscriptionfactorthatpromotesepithelial--mesenchymaltransition(EMT).Itsmisregulationhasbeenobservedinvarioustypesoftumors.UsingtheMCF-10A-seriesofcelllinesthatrecapitulatetheearlystagesofbreastcancerformationandEMT,wefoundTWIST1tobeupregulatedduringEMTanddownregulatedearlyincarcinogenesis.TheTWIST130UTRcontainsputativeregulatoryelements,includingmiRNAtargetsitesandtwocytoplasmic

polyadenylationelements(CPE).WefoundthatmiR-580,CPEB1,andCPEB2actasnegativeregulatorsofTWIST1expressioninasequence-specificandadditive/cooperativemanner.

Oncogeneadvanceonlinepublication,23January2012;doi:10.1038/onc.2011.650Keywords:breastcancer;CPEB;microRNA;translationalregulation;TWIST1

INTRODUCTION

TWIST1isabasichelix-loop-helixtranscriptionfactor,foundthroughoutthemetazoananimalkingdom.TWIST1hasamajorroleduringdevelopmentinmammalsandisanimportantfactorintumorigenesis.1Itcontributestometastasisbypromotingepithelial--mesenchymaltransition(EMT),2andismisregulatedinavarietyofhumancancers.3TWIST1isregulatedbothtranscriptionallyandpost-transcriptionally.Post-transcriptionalregulationwasfirstobservedinmouseembryos,inwhichTwist1mRNApreceedsproteinexpressioninepithelialsomites.4,5Similarobservationsweremadeinzebrafish.6Here,weanalyzepost-transcriptionalregulationofTWIST1earlyintumorigenesisandEMT,usingtheMCF-10A-basedseriesofcelllines.SpontaneouslyimmortalizedMCF-10Amammaryepithelialcells7havebeentransformedandfurtherselected,basedontheirabilitytoinducetumorsand/ormetastases,resultinginaseriesofcelllinesthatrepresentearlystepsoftumorigenesis,8-10providingavaluabletoolforanalyzingbreastcancerdevelopment.

Post-transcriptionalregulationistypicallymediatedbyregula-torysequencesinthe30UTR,includingmiRNA-bindingsites.miRNAsareendogenoussmallnon-codingRNAsthatregulategeneexpression,primarilybyinhibitingproteinsynthesis,eitherbyrepressingtranslationand/orbydeadenylation,andsubse-quentdegradationofmRNA.11Cytoplasmicpolyadenylationwasinitiallydescribedasamechanismthatregulatestheexpressionofgenesinvolvedincell-cyclecontrolduringearlyembryogenesis.12Thismechanismhasalsobeenshowntocontrolpoly(A)taillengthinmammaliancells.13,14Cytoplasmicpolyadenylationismediatedbycytoplasmicpolyadenylationbindingprotein(CPEB),whichbindstothehighlyconservedcytoplasmicpolyadenylationelement(CPE)inthe30UTRofmRNA.15Here,weshowthatCPEB1andCPEB2playaroleinthetranslationalcontrolofTWIST1inaCPE-dependentmanner.

1Moreover,miR-580targetsthe30UTRofTWIST1,controlsTWIST1expression,andaffectscellmigration.CloselylocatedCPE-IandmiR-580siteshaveadditiveandcooperativeeffectsonTWIST1expression.

RESULTSANDDISCUSSION

TWIST1expressionduringbreastcancerdevelopment

MCF-10A(termedA10inthefigures)cellsoriginatefromspontaneouslyimmortalizednon-malignanthumanmammaryepithelialcellsafterlong-termculturesofmortalhumandiploidmammaryepithelialcellsfromapatientwithextensivefibrocysticdisease.7TransformationofMCF-10AcellswithanactivatedH-RasoncogeneresultedinpremalignantMCF-10AT/MCF-10ANeoTcells(termedNeoTinthefigures)16thatneverformtumors,butdoformpreneoplasticlesionsinimmunodeficientmice.8,17Subse-quentserialxenograftinginmicecreatedMCF-10CA1acells(termedCA1ainthefigures).8Thesecellsyieldeda100%incidenceoftumorswhentransplantedintonudemice.10StabletransductionofMCF-10ANeoTcellswithY-boxbindingprotein1YB-1(MCF-10ANeoT-YB-1,termedYB-1infigures)inducedEMT.9YB-1isaknowntranscriptionalandtranslationalregulator,18andisadownstreamtargetofTWIST1.19Figure1ashowsaschematicsummaryofthecelllinesused.

WeconfirmedTWIST1mRNAandproteinupregulationduringEMT,9butobserveddownregulationincelllinesrepresentingearlystagesofcancerdevelopment(Figures1bandc;SupplementaryFigure1a).Thiswasunexpected,astheupregulationofTWIST1hasbeendemonstratedinavarietyofhumancancers,3includingbreastcancer.20TWIST1upregulation,however,hasmainlybeencorrelatedwithaggressivephenotypes,metastasisformationandpoorprognosis.3TWIST1isrequiredforcellmigrationduringbothdevelopment21andoncogenesis.22Thus,TWIST1downregulationmighthelpprimarytumorcellstoremainlocalizedearlyinbreastcancerprogression.

DepartmentofMolecularBiologyandGenetics,AarhusUniversity,Aarhus,Denmark;2DepartmentofTranslationMechanisms,InstituteofMolecularBiologyandGenetics,NationalAcademyofSciencesofUkraine,Kiev,Ukraine;3TheBreastDepartmentoftheThirdAffiliatedHospitalofHarbinMedicalUniversity,Harbin,Chinaand4CNRSFRE3377,

´ne´tiqueetCancer,CNRSFRE3377,CEACEASaclay,Gif-sur-Yvette,France5Currentaddress:UnivParis-Sud,FRE3377,Gif-sur-Yvette,France.Correspondence:DrIGroisman,Epige

Saclay,91191Gif-sur-Yvette,France.E-mail:irina.groisman@gmail.com

Received24April2011;revised7December2011;accepted21December2011

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TumorigenesisMCF10A-A10H-RasMCF10A-NeoTXenograftsMCF10A-CA1aEMTMCF10A-NeoTTransductionMSCVEpithelialYB-1Mesenchymal28 kDa –40 kDa –1009080706050403020100A10A10NeoTNeoTCA1aCA1aMSCVMSCVYB-1TWIST1ActinYB-1Normalized TWIST1 mRNAexpression (TWIST1/GAPDH)11101153343403563446906686695AATAAApA1TTTTTATCATTCTCAACPE-ImiR-5803.5AATAAAApA2TTTTTATCPE-IIAATAAATAAApA3/4Normalized TWIST1 3'UTR expression (TWIST1/GAPDH)504035302520151050A10160140120100806040200NeoTCA1aMSCVTWIST1 3'UTR mRNA[pA3/(pA2-pA3)]pA2pA33.02.52.01.51.00.50.0A10NeoTCA1aMSCVYB-1YB-1TWIST1 protein level;R-luc/F-lucTWIST1 protein levelpA1pA2pA31.21.00.80.60.40.20.0pA3/pA2pA3/pA1A10NeoTCA1aMSCVYB-1A10NeoTCA1aMSCVYB-1Figure1.TWIST1expressioninMCF-10A-derivedcelllines.Utilizationofpolyadenylation(pA)signalsinTWIST130UTR.(a)Schematic

representationofearlytumorigenesisandEMTmodels.(b)QuantificationofTWIST1mRNAinMCF-10AcelllinesbyqPCR,normalizedtoGAPDH.(c)WesternblotshowingTWIST1proteinexpression;quantificationshowninSupplementaryFigure1A.(d)SchematicrepresentationofTwist130UTRandtheregulatoryelementsinit.Numberscorrespondtothesequenceofregulatoryelementsspecifiedbelow,arrowsindicatethelocationofthepAswheretheshorteningofthe30UTRoccurs.(e)TWIST130UTRmRNAsegments’expressioninindicatedcelllines.Leftpanel:quantificationbyqPCRusing30primersannealedtothe50-endofthecorrespondingnuclearpAsignal,normalizedtoGAPDH.Rightpanel:pA3usagerelativetopA2.AspA2primersalsodetecttheuseofpA3,pA3valuesweresubtractedfromthepA2valuestocalculatetheactualquantitiesoftheshorterformpresentinthesamples.(f)InfluenceofTwist130UTRonproteinexpressiontestedbyluciferasereporterassay.Leftpanel:RenillaenzymaticactivityfromTwist130UTRreporternormalizedtoFirefly(control)inMCF-10Acells.Rightpanel:luciferaseexpressionfromthepA3constructrelativetopA1orpA2,asindicated.F-luc,Fireflyluciferase;miR,microRNA;pA,polyadenylationsignal;R-luc,Renillaluciferase;UTR,untranslatedregion.

Importantly,unliketheearlycancerformationmodel,inwhichdownregulationofTWIST1atthetranscriptionallevelwasquitesignificant,TWIST1mRNAlevelswerebarelymodifiedduringEMT,whereastheTWIST1proteinwasdramaticallyinduced(compareMSCVandYB-1inFigures1bandc).Thus,TWIST1upregulationcannotbecompletelyaccountedforbytheupregulationofTWIST1mRNA,indicatingpost-transcriptionalregulation.TWIST1regulationvia30UTRduringbreastcancerdevelopmentPost-transcriptionalregulationcommonlyinvolvesproteinorRNAbindingtothe30UTRofmRNA.AnalysisoftheTWIST130UTRbyseveralalgorithmsdemonstratedahighdegreeofconservation

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¨gietal.,(87%)betweenmouseandhumansequences(Nairisma

submitted),includingfournuclearpolyadenylation(pA)signals,thelasttwoofwhichoverlap(pA1-4),twoCPEs,andapredictedtargetsiteformiR-580(Figure1d;SupplementaryFigure2).

Comparedwithsimilarlyproliferatingnon-transformedcells,cancercellsoftenexpresssubstantialquantitiesofmRNAisoformswithshorter30UTRsthatresultfromtheuseofalternativepAsignals.23ByusingquantitativePCR(qPCR)primerssurroundingthepA2andpA3/4signals(nt4and686fromthestopcodon,respectively),wefoundthattheTWIST130UTRisprogressivlyshortenedincelllinesrepresentingbreastcancerdevelopment(Figure1e,leftpanel).PrimersdesignedforpA1didnotresultinthesameqPCRefficiencyasthepA2andpA3/4primers,andwere

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Figure2.RepressionofTWIST1bymiR-580.(a)Expressionof13miRNAspredictedtotargetthe30UTRofTWIST1inindicatedMCF-10A-derived

celllines,normalizedtoU6smallnuclearRNA(snRNA).AmultiplicationfactorisindicatedabovetherespectivecolumnstocompareallmiRNAsonthesamehistogram.(b)miRNA-mediatedknockdownoftheWTTwist130UTRreporterinMCF-10Acellsco-transfectedwithamiRNAprecursorandfull-lengthWT30UTRRenillareporter.First,normalizationwasdonetoFireflyluciferaseandthentotheRenillaluciferasevalueobtainedfromtransfectionoftheTwist130UTRreporteralone.(c)MutationofthemiR-580targetsiteorinhibitionofendogenousmiR-580increasesTwist130UTRreporteractivity.MCF-10AcellsweretransfectedwithWTormutantTwist130UTRreporterindividuallyorwithmiR-580precursororanantisenseinhibitor,asindicated.(d)WesternblotshowingdownregulationofendogenousTWIST1proteinbymiR-580precursorandupregulationbymiR-580inhibitorinMCF-10Acells.TransfectionofscrambledmiRNAprecursorandinhibitorwereusedascontrols.(e)MCF-10ANeoT-MSCVcellsmigration.CellsweretreatedeitherwithmiR-580precursororsiRNAagainstTWIST1.After18hofcellchambermigration,cellswerefixed,stainedwithDAPI,andcountedinfiverandommicroscopefields.Statisticalsignificance(Student’st-test)wascalculatedincomparisonwiththefull-length30UTRreporter(b)ornon-treatedcontrol(e,(*)).F-luc,Fireflyluciferase;miR,microRNA;R-luc,Renillaluciferase;siRNA,shortinterferingRNA;UTR,untranslatedregion.

thereforeexcludedfromfurtheranalysis.WefoundthatthepA3/4signalisutilizedlessefficientlyduringcancerprogression(compareA10andCA1acelllinesinFigure1e,rightpanel)andmetastatictransformation(compareMSCVandYB-1celllines)thanthepA2signal(67%and57%,respectively).ShorterformsoftheTWIST130UTRlackanumberofpotentialregulatoryelements(oneCPEsiteandseveralmiRNAtargetsites),whichmightleadtoderegulatedTWIST1expressionduringcancerprogression.

ToinvestigatetheinfluenceoftheTWIST130UTRontranslationefficiency,weusedTwist130UTRluciferasereporterconstructsendingatthepA1,pA2orpA3/4signals.Fortechnicalreasons,the30UTRsequenceusedwasfrommouse,whichhasahighhomologytothehumanone(87%).Theshorterconstructs(endingeitheratpA1orpA2)weremoreefficientlyexpressedcomparedwiththelongestone(pA3)inallcelllines(Figure1f,leftpanel),suggestingtheexistenceofnegativeregulatoryelementsatthe30-endoftheTwist130UTR.Moreimportantly,theratioofluciferaseexpressionbetweenthelongest(pA3)andshorter(pA1

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andpA2)constructsdecreasedduringcancerprogression,butnotduringEMT(Figure1f,rightpanel).ThissuggeststhattheTWIST130UTRhasanincreasedinhibitoryroleduringcancerprogression,thatiscounteractedbythedecreaseinitsexpression,whereasthisphenomenonisnotobservedduringthemetastaticprocess.WethereforepostulatedthatinhibitoryfactorsregulatingTWIST1translationwouldbeupregulatedincelllinesthatrepresentearlystagesofcancerdevelopment,butdownregulatedinacelllinethatresemblesEMT.

miR-580controlsTWIST1expression

miRNAsarewell-knownnegativeregulatorsofgeneexpression.ToidentifycandidatesthatareupregulatedincelllinesthatrecapitulatebreastcancerformationanddownregulatedinacelllinewithEMTcharacteristics,wefirstanalyzedtheexpressionlevelsofseveralmiRNAsintheMCF-10A-seriesofcelllines,namelymiR-15b*,miR-33a,miR-137,miR-145,miR-151-5p,miR-214*,

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miR-337-3p,miR-361-5p,miR-450b-5p,miR-508-3p,miR-576-5p,

miR-580andmiR-591.UsingmiRNA-specificqPCRanalysis,weidentifiedfivemiRNAsthatfulfilledthisrequirementtosomeextent:miR-15b*,miR-137,miR-145,miR-151-5pandmiR-580(Figure2a).ToaddressthequestionofwhetherthesemiRNAsareinvolvedinthepost-transcriptionalregulationofTWIST1,wetestedtheminreporterassaysinMCF-10Acellsbyco-transfectingafull-lengthTwist130UTR(pA3)luciferasereporteralongwithamiRNAprecursor.TheeffectofamiRNAwasdeterminedrelativetofull-length30UTRvalues,whennomiRNAwasadded(Figure2b).Co-transfectionwithmiR-580resultedina36%(Po0.003)downregulationofreporterexpression,whereasmiR-137andmiR-151-5pyieldedanon-significantreduction(24%and20%,respectively).

ToconfirmthattheinhibitionwasduetomiR-580binding,wemutateditspredictedtargetsitetoarestrictionenzymerecognitionsite.Thisresultedinincreasedluciferaseactivitycomparedwiththewild-type(WT)reporterintheabsenceofexogenousmiRNA(Figure2c),confirmingthepredictedtargetsiteformiR-580.Moreover,co-transfectionoftheWT30UTRreporter,andanantisenseinhibitorofmiR-580alsoresultedinincreasedluciferaselevels,whereastheinhibitorhadnoeffectonthemutatedreporterlevel,demonstratingthatendogenousmiR-580isabletotargettheTwist130UTR.Importantly,transfectionofmiR-580precursortoMCF-10AcellsdownregulatedthelevelofendogenousTWIST1protein,whiletransfectionofanti-miR-580upregulateditsexpression(Figure2d).

TWIST1isknownforitsroleinEMTandmetastasisformation,2processesthatrequirecellmigration.TotesttheinfluenceofmiR-580onTWIST1functionality,wecarriedoutcellmigrationassays.DepletionofTWIST1mRNAinMCF-10ANeoT-MSCVcellsbyshortinterferingRNA(siRNA)ledtoa23%decreaseincellmotility(Figure2e).ThisreductionwasevengreaterwhenmiR-580precursorwasintroducedintothecells(57%),indicatingthat

Figure3.CPEB1andCPEB2controlTWIST1expression.(a)QuantificationofCPEB1-4mRNAbyqPCRinindicatedcells,normalizedtoGAPDH.TocompareallCPEBsinthesamediagram,expressionlevelsweremultipliedbytheindicatedfactors.(b)WesternblotanalysisofCPEB1-4proteinexpression;quantificationshowninSupplementaryFigure1B.(c,d)MCF-10Acellswereco-transfectedwithCPEB1-4expressionconstructs(c)orsiRNAsagainstCPEB1-4(d)alongwithWT(leftpanels)ordouble-CPEmutant(rightpanels)30UTRRenillareporter.RenillaexpressionnormalizedtoFireflyluciferase.ThedepletionefficiencyofseveralsiRNAsisshowninSupplementaryFigure1C.Statisticalsignificance(Student’st-test)wascalculatedincomparisonwiththemock-(c)orsiControl-(d)treatedsample(*).(e)WesternblotofendogenousTWIST1proteininMCF-10AcellstransfectedwithCPEB1-4expressionconstructsorsiRNAsagainstCPEB1-4.TransfectionofscrambledsiRNAandGFPexpressionconstructwereusedascontrols.(f)QuantificationofTWIST1mRNAboundtoindividualFlag-taggedCPEBproteinsimmunoprecipitatedwithanti-Flagantibody.CPEB1,2and4interactwithTWIST1mRNA.F-luc,Fireflyluciferase;GFP,Greenfluorescentprotein;NTC,nontransfectedcontrol;R-luc,Renillaluciferase;siRNA,shortinterferingRNA;UTR,untranslatedregion.

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miR-580isanegativeregulatorofcellmotilityandmightatleast

partiallyactviaregulationofTWIST1.

Interestingly,miR-580hasbeenidentifiedtobeamongthe20mostabundantmiRNAsinbreastmilk,24underliningitsprobableimportanceinnon-pathologicalmammarycells.WeshowherethatmiR-580isupregulatedinMCF-10ANeoTandMCF-10CA1acellsanddownregulatedinMCF-10ANeoT-YB1cells,whichhaveundergoneEMT.Thisexpressionpattern,whichisoppositetothatofTWIST1,iscompatiblewiththeideathatmiR-580regulatesTWIST1duringbreastcancerformationandEMT.Indeed,thefactthatoverexpressionofmiR-580anddownregulationofTWIST1bothinhibitcellmotilitysuggeststhatmiR-580functionsduringmetastatictransformation.

CPEBscontrolTWIST1expressionthroughCPEsinits30UTR

The30UTRofTWIST1alsocontainstwoCPEsignalslocatedatnt334(CPE-I)and0(CPE-II)fromthestopcodon.WehypothesizedthatCPEBscouldbeinvolvedinTWIST1regulation.TheCPEBgenefamilyconsistsoffourmembers,CPEB1-4.UsingqPCRandwesternblotanalyses,wefirstmeasuredthemRNAandproteinlevelsofallfourCPEBsinMCF-10A-derivedcelllines.WefoundthattheCPEBfamilymemberswereregulatedindifferentways:CPEB1andCPEB3mRNA(Figure3a)andprotein(Figure3b;SupplementaryFigure1B)weredownregulatedduringcancerprogressionandEMT,whereasCPEB2andCPEB4wereupregulated,comparedwithGAPDHmRNAandTubulinprotein,whichwereusedasinternalstandards.ThelatterwerethusinverselycorrelatedwithTWIST1expressionincelllinesthatrecapitulatetheearlystagesofbreastcancerformation,whereasCPEB1andCPEB3wereinverselycorrelatedwithTWIST1duringametastaticprocess.

TodeterminewhetheranyoftheCPEBsregulateTWIST1expression,wecarriedoutgain-andloss-of-functionexperiments.Thefull-lengthTwist130UTRreporterwasco-transfectedwithexpressionvectorsencodingCPEB1-4orsiRNAsagainstCPEBs.TheefficiencyofthevarioussiRNAswastestedbyqPCRanalysis

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Figure4.NecessityofCPE-IandCPE-IIforCPEB1-andCPEB2-mediatedTWIST1regulationandcooperativeeffectofCPEB1andmiR-580.(a)RenillaexpressionfromWTandmutantTwist130UTRreporterstransfectedintoMCF-10Acells.CPE-Iand-IIsitesintheTwist130UTRweremutatedeitherindividuallyorsimultaneously,asindicated.(b)MCF-10Acellswereco-transfectedwithaTwist130UTRmutantreporterandsiRNAsagainstCPEB1-4.CPE-IisnecessaryforCPEB1-mediatedTwist1regulation(rightpanel),whereasCPE-IIisimportantforbothCPEB1andCPEB2activity(leftpanel).(c)NormalizedRenillaexpressionfromWTTwist130UTRreporterinMCF-10Acellsco-transfectedwith

CPEB1andmiR-580,asindicated.Statisticalsignificance(Student’st-test)wascalculatedincomparisonwiththeWTfull-length30UTRreporter(a),siControl-(b),ormock-(c)treatedsample(*).(d)WesternblotofendogenousTWIST1proteininMCF-10Acellsco-transfectedwithCPEB1expressionconstruct,siRNAagainstCPEB1,miR-580precursorormiR-580inhibitor,asindicated.TransfectionofGFPexpressionconstruct,scrambledsiRNA,scrambledmiRNAprecursorandinhibitorwereusedasrespectivecontrols.F-luc,Fireflyluciferase;GFP,Greenfluorescentprotein;miR,microRNA;R-luc,Renillaluciferase;UTR,untranslatedregion.

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thetwoCPEsweremutated(mut-CPE-IþII),wasusedasacontrol.OverexpressionofCPEB1andCPEB2resultedindecreasedluciferaseactivity,whereasCPEB3andCPEB4hadnoeffect(Figure3c,leftpanel).NoneoftheCPEBshadanyeffectontheCPEmutantreporter(Figure3c,rightpanel).KnockdownofendogenousCPEB1andCPEB2withsiRNAsledtoincreasedluciferaseexpressionbytheWT30UTRvector(Figure3d,leftpanel).Consistentwiththegain-of-functionexperiments,deple-tionofCPEB30andCPEB4hadnoeffectontheexpressionoftheWTTwist13UTRreporter.NoneoftheCPEBsiRNAsaffectedtheexpressionofthemut-CPE-IþIIreporter(Figure3d,rightpanel).ThissuggeststhatonlyCPEB1andCPEB2areabletomediateTwist1regulationinaCPE-dependentmanner.Importantly,overexpressionofCPEB1andCPEB2downregulatedtheendo-genousTWIST1proteinlevelinMCF-10Acells,whereassiRNAknockdownofCPEB1andCPEB2upregulatedTWIST1expression(Figure3e).

ToverifywhetherCPEB1andCPEB2wereabletobindtoTWIST1mRNA,wecarriedoutCPEBRNAimmunoprecipitationassays.AftertransfectingHeLacellswithCPEB1-4constructsfusedtoFlagtag,weimmunoprecipitatedtheCPEBproteinswithanti-FlagantibodiesandelutedthemwithFlagpeptide.TWIST1mRNAwasdetectedbyqPCR.TWIST1mRNAwasassociatedwithCPEB2,andtoalesserextent,withCPEB1andCPEB3,butapparentlynotwithCPEB4(Figure3f).

ToidentifywhichofthetwoCPEswasimportantforCPEB-mediatedTWIST1regulation,weanalyzedtheexpressionofTwist130UTRmutantreportersthatlacktheCPEs(eitherindividuallyorincombination).AsshowninFigure4a,deletionofeachindividualCPEincreasedluciferaseactivityapproximatelytwofoldcomparedwiththeWTreporter,whereasamutationofbothsitesresultedinalmosttenfoldhigherluciferaseexpression,indicatingthatthebothCPEsareinvolvedinTwist1regulation,possiblywithsynergisticeffects.

TofindoutwhichoftheCPEswasresponsibleforCPEB1andCPEB2binding,weusedeachindividualCPEmutantincombina-tionwithCPEBdownregulation.TheconstructthatincludedonlyCPE-II(mut-CPE-I)wasinfluencedbysiCPEB1andsiCPEB2(Figure4b,leftpanel),whereastheconstructthatincludedonlyCPE-I(mut-CPE-II)wassensitiveonlytosiCPEB1(Figure4b,rightpanel).ThissuggeststhatCPE-Iinthe30UTRofTwist1isatargetonlyforCPEB1-mediatedregulation,whereasCPE-IIisatargetforbothCPEB1andCPEB2.

CPEB1andCPEB2havebeenshowntobindtothe30UTRofhypoxia-induciblefactor1aandareinvolvedinpositiveregulationofhypoxia-induciblefactor1afollowinginsulinstimulation.25TWIST1isaknownhypoxia-induciblefactor1atargetgene,andbotharehighlyrelevantinEMTandmetastasisformationinheadandneck27squamouscellcarcinoma26andnon-small-celllungcancer.ItwouldthereforebeofinteresttotestCPEBlevelsunderhypoxicconditions.15Nevertheless,ourobservations,aswellasthoseofothers,indicatethatCPEBproteinsarenotfunctionallyidentical.

BecauseofthecloseproximityofCPE-IandmiR-580targetsitesinthe30UTRofTWIST1,wehypothesizedthattheseregulatorymechanismsmightactcooperatively.Totestthis,wetransfectedMCF-10AcellswithCPEB1andmiR-580bothindividuallyandincombination.IndividualtransfectionofCPEBledto46%andmiR-580to32%downregulationoftheWT30UTRreporter,whereasco-transfectionofbothfactorsledtoa90%decreaseinluciferaseactivity(Figure4c),indicatingthattheyhaveanadditive,orpossiblyacooperative,effect.Importantly,co-transfectionofpre-miR-580alongwithCPEB1downregulatedtheendogeneousTWIST1proteinlevelmorethantransfectionofeachfactorseparately,andco-transfectionofanti-miR-580andsiCPEB1upregulatedTWIST1expressiontoahigherlevelthaneitherofthemalone(Figure4d).

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Insum,wehaveshownthatTWIST1isdownregulatedearlyincarcinogenesisandupregulatedinmetastaticcells,asdemon-strated0inthisseriesofMCF-10A-derivedcelllines.TheTWIST13UTRisshortenedduringcancerprogressionandEMT.WeidentifiedmiR-580,CPEB1andCPEB2asnegativeregulatorsofTWIST1anddemonstratedcooperativeeffectsbetweentheCPE-Iand0miR-580sites.Moreover,miR-580isnotonlyabletotargetthe3UTRofTWIST1andtocontrolTWIST1expression,italsoaffectscellmigration.Inaddition,wehaveshownthatCPEB2bindsonlytoCPE-II,whichisnotpresentintheshortformofTWIST1mRNAexpressedinmetastaticcells.ThiscorrelateswithhighTWIST1expressionandindicatesthatCPEB2isimportantinregulatingTWIST1expressionduringoncogenesis.CONFLICTOFINTEREST

Theauthorsdeclarenoconflictofinterest.

ACKNOWLEDGEMENTS

WethankValentinaEvdokimovaforprovidinguswiththeMCF-10ANeoT-MSCVandMCF-10ANeoT-YB-1celllines,LindaLouisePritchardforeditorialandscientificadvice,andLeslie-AnnLargittefortechnicalassistance.Thisworkwassupportedinpartbygrantn1LSHG-CT-2006-037900fromtheEuropeanCommissionSixthFrameworkProgrammetoAHBandbygrantn1DP08036fromTheDanishCancerSocietytoEMF.

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