Epithelialmesenchymaltransitionfocuson metastaticcascadealternativesplicing noncodingRNAsandmodulatingcompounds TimurRSamatov 1 AlexanderGTonevitsky 23 andUdoSchumacher 4 Abstract Epithelial ID: 345318
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REVIEWOpenAccess Epithelial-mesenchymaltransition:focuson metastaticcascade,alternativesplicing, non-codingRNAsandmodulatingcompounds TimurRSamatov 1* ,AlexanderGTonevitsky 2,3 andUdoSchumacher 4* Abstract Epithelial-mesenchymaltransition(EMT)isakeyprocessinembryonicdevelopmentandmetastasesformation duringmalignantprogression.Thisreviewfocusesontranscriptionalregulation,non-codingRNAs,alternative splicingeventsandcelladhesionmoleculesregulationduringEMT.Additionally,wesummarizetheknowledge withregardtothesmallpotentiallydruggablemoleculescapableofmodulatingEMTforcancertherapy. Keywords: Alternativesplicing,Celladhesionmolecules,Epithelial-mesenchymaltransition,Metastaticcascade, Non-codingRNAs,Smallmoleculecompounds,Transcriptionfactors Introduction Epithelial-mesenchymaltransition(EMT)isamulti-step morphogeneticprocessduringwhichepithelialcells downregulatetheirepithelialpropertiesandupregulate mesenchymalcharacteristics(Figure1).Namely,static quencetheyloseapico-basalpolaritytobecomemigratory mesenchymal-likecells.Thisprocessofdown-regulation oftheepithelialphenotypemimicsthenormaldevelop- mentalprocessofgastrulation,inwhichcellsfromthe epithelialsheetoftheectodermstarttoformthethird germinallayer,themesoderm,whosemigratorycellsare calledmesenchymalcells.Thisprocessisthereforeaptly calledtheepithelial-mesenchymaltransition,whichiscur- rentlyclassifiedintothreesubtypes[1]. Type1EMTisassociatedwiththeoriginalembryonic developmentandalsooccursduringpostnatalgrowth. ThestepsofthisEMTtypearespecificandwell-defined. Epithelialcellsarecuboidaltocylindricalinshapeandare incontactwitheachotherviaadherentandtightjunc- tions.Primarymigratorymesenchymalcellsgeneratedthis waymaypotentiallygothroughareversesteptobecome epitheliaagain.Thisstepiscalledthemesenchymal- epithelialtransition(MET)andgeneratessecondary epitheliainthedevelopingembryo[2].Differentiated cellsinalmostallorgansinadultsdevelopedasare- sultofEMT-MET. Type2EMTisinitiatedbyinjuryandresultsingener- ationoffibroblaststorebuildwoundedtissues[3].Dur- inginflammationfibroblastsandimmunecellsrelease cytokinesandotherpro-inflammatoryfactorsaswellas extracellularmatrixproteinswhichresultsinstimulation ofcellstoundergoEMT.Ifinflammationpathologically persists,continuousEMTofnormalepithelialcellscan resultinfibrosisandorgandamage[4]. Oncogenictype3EMTenablesepithelialcellstoac- quireinvasivemesenchymalphenotypecharacteristics whichareessentialinmetastaticspread[5].Typicalde- velopmentalEMTfeaturesarerecapitulatedinonco- genicEMT[6],however,theyarelessorderedand coordinated.AsaresultofthisdisorderedEMT,hybrid epithelialandmesenchymalcelltypes[7]. TranscriptionfactorsregulatingEMT Thereareanumberoftranscriptionfactorsknownto beinvolvedintheregulationofEMT.Themostchar- acterizedareZEB1andZEB2,snail,slugandtwist (Figure2). *Correspondence: t.samatov@bioclinicum.com ; uschumac@uke.de 1 SRCBioclinicum,Ugreshskayastr2/85,Moscow115088,Russia 4 DepartmentofAnatomyandExperimentalMorphology,UniversityCancer Center,UniversityMedicalCenterHamburg-Eppendorf,Martinistr.52, HamburgD-20246,Germany Fulllistofauthorinformationisavailableattheendofthearticle ©2013Samatovetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Samatov etal.MolecularCancer 2013, 12 :107 http://www.molecular-cancer.com/content/12/1/107 ZEB1andZEB2arehighlyconservedzincfingerpro- teinswhichcandirectlybindtothepromoterregionsof targetgenesandthusrepresstheexpressionofE-cadherin andsomeotherepithelialmarkers[8]andinducetheex- pressionofvimentinandanumberofothermesenchymal markers[9].ZEB1and2areinducedbyTGF ,hypoxic conditionsandinflammatorycytokines,factorswhich allinitiateEMT.ZEBsplayanimportantroleinnor- malembryonicdevelopmentandtheyarereportedto beupregulatedinmanytumors[10]. Snailandslugbelongtothesnailfamilyoftranscription factors,withC-terminalzincfingerbindingtoE-boxesof theregulatoryregionsoftargetgenes[11].Snailfactors repressE-cadherinexpressionbydirectbindingtoits promoterandcanalsorepressotherepithelialproteinsin- cludingdesmoplakinandclaudins.Atthesametimesnail proteinsactivateexpressionof pro-invasivegenes(vimentin, fibronectin,MMPs)promotingcellmigration[12].Likethe twoZEBtranscriptionfactors,snailandslugcanbeinduced byTGF ,hypoxicconditionsandotherEMT-relatedsignal- ingpathways[13].Snailtranscriptionfactorsarenotpresent innormalepithelialcells,howevertheyarefoundinthein- vasivefrontoftumorsandconsideredtobeprognosticfac- torsforpoorsurvivalinanumberofcarcinomas[11]. EMT MET Figure1 Epithelial-mesenchymaltransition. VariousmesenchymalcelltypescanbederivedviaEM T.Thereversemesenchyma l-epithelialtransition cangeneratesecondaryepithelia. III b III c 12345161718 12345161718 v8 v9 v10 FGFR2 CD44 ESRP1 ESRP2 N-cadherin vimentin MMPs miR-200 miR-10b miR-21 E M T ZEB1 ZEB2 snail slug twist inflammatory cytokines hypoxia TGF E-cadherin desmoplakin claudins Figure2 MarkersandregulatorsofEMT. DuringEMTcomplexchangesofmRNAexpressionlevelandalternativesplicingofnumerousgenes occur.Thesechangesareinfluencedbythetumormicroenvironment,transcriptionandsplicingfactorsandnon-codingRNAs. Samatov etal.MolecularCancer 2013, 12 :107 Page2of12 http://www.molecular-cancer.com/content/12/1/107 Thetwistproteincontainsabasic/helix-loop-helixdo-mainwhichprovidesforbindingtoDNAanddimerization.ItsC-terminalendcontainsatwistboxresponsibleforbothtranscriptionalactivation(e.g.forN-cadherin)andrepression(E-cadherin)[14].Regulationofgenesbytwistdependsonitsbindingtoothertranscriptionalfactors,post-translationalmodifications,andchoiceofpartnerfordimerization.Twistisupregulatedinhumancancersanditsabundancyincreasesduringtumorprogression.Itsexpressionalsocorrelateswithhighertumorgrade,invasiveness,andmetastasis,cellularprocessesbeingcon-sideredasprognosticfactorsforenhancedtumoraggres-siveness,tumorrecurrence,andpoorersurvival[11].Remarkably,thereisasignificantoverlapintheregu-latorysignalsofthesetranscriptionfactors.Namely,ex-pressionofZEBfactorsisregulatedbysnail[15,16].Snailinturnalsoincreasesthestabilityoftwistwhichthenactivatesthetranscriptionofslug[15,17].Thisinteractionnetworkmayplayaroleinspatialandtem-poralregulationofEMT.EMTandmetastaticcascadeOneoftheclassicalmodelsforcancermetastasisisStephenPagetsseedandsoilhypothesisinwhichthetumorcellistheseedandtheorganinwhichthemetasta-sisgrowsisthesoil[18].Thismodelimpliesthatcertaintumorcellshaveanaffinitytotheparticularorganwhichprovidesagrowthadvantagetothem.Thusthesiteofme-tastasisisdependentontheaffinityofthetumorforthegivenmicroenvironment,whichelegantlyexplainswhysomeorgans(lung,liver,bonemarrow)areparticularlypronetoharbourmetastaseswhileothersarenot(intes-tine,skeletalmuscle,skin).Afterpassingtheendothelialbarrier,additionalfactorssuchaslocalgrowthfactorpro-ductionplayaroleinstimulatingthegrowthoftheseeva-sivetumorcells[19].Accordingtothislaterexpandedmodel,metastasisformationstartswhentheprimaryma-lignantcelldividesandoncethecellmasshasreachedthesizeofafewdozenscells,itsendsoutangiogenicsignals,thusleadingtotheingrowthofbloodvesselsintothenewlyformedtumor.Asanextstep,futuremetastaticcellshavetofreethemselvesfromtheprimarytumormass,havetodegradethesurroundingextracellularmatrixincludingthebasementmembrane,mustenterthebloodvesselsandsurvivewithinthecirculation(=theseed).Oncetheyhavereachedthetargetorganofthefu-turemetastasis(=thesoil),thetumorcellhastoattachtotheendotheliuminthisorganandhastomigratethroughit.Whenthisprocessisaccomplished,themetastaticcan-cercellhas-probablyundertheinfluenceoflocalgrowthfactorstostarttodivideagaininordertoformaclinic-allydetectablemetastasis.Onceproliferationhasstarted,thismetatsaticcycleresumesinordertospawnfurthermetastasesoriginatingfromametastasis.Differentcelladhesionmolecules(CAMs)playvitalandopposingrolesduringthisprocess.Duetotheirveryepithelialnaturecancercellsformmoreorlesstighthomologousepithelialcelltoepithelialcellcontactsatthesiteoftheprimarytumors.Molecularlythisencom-passesoftenhomologousCAMswhicharepartofdesmo-somes,tightjunctionsandgapjunctions(seeTable1).Inaddition,celltobasallaminacontacts(focaladhesions,hemidesmosomes)areformedfromthosecellsdirectlyadjacenttoabasallamina.Inordertoescapefromthepri-marytumor,theproteinsformingthesejunctionshavetobedown-regulatedinordertoallowcellmigration.Thecontactofepithelialcellstothebasallaminanotonlyhin-dersmigrationbutalsopreventscelldeath.Ifanormalepithelialcellloosesthecontacttothebasallamina,aspecialformofdetachment-inducedapoptosis,termedanoikisbyStevenFrisch[20],istriggeredasthecellintegrinsaredetachedfromtheirligandsinthebasallamina.Asmesenchymalcellsdonotnecessarilyhaveadirectcontacttothebasallamina,theyarenotsub-jecttoanoikisandtheEMTwouldthereforeaidsur-vivaloftheloosenedcancercells.AfterEMThasenabledthetumorcellstomigrateoutoftheprimarytumor,theyhavetoentercirculationandsurvivewithinit(Figure3).Later,theymustadheretothemicrovascularendothelialcellsatthesiteofthetar-getorganandbythisadhesiontheyhavetocommuni-catetotheendothelialcellstoopentheircelljunctions.Thisallowsthepassageofthecancercellthroughtheendotheliumtotheconnectivetissuespaceofthehostorgan.Again,CAMsmediatethisprocess,however,theseCAMsaredifferentfromthoseformingtheintra-epithelialcelladhesion.Here,heterologousCAMsmediatingcellad-hesionbetweendifferentcelltypestumorcellsandendo-thelialcells-areimportant.SimilarlytothemimicryoftheEMT,cancercellsevadingcirculationmimictheleukocyteadhesioncascade(seeTable2).TheCAMsandtheirli-gandsusedinthisadhesionareselectinglycoconjugateligands,integrinsandtheirextracellularmatrixligands,ALCAMandICAMs.IncontrasttotheepithelialCAMs,whichweredown-regulatedduringEMT,theseCAMswereup-regulatedaspartofthemesenchymalphenotypeduringEMT.Thesedown-andup-regulationsofcelladhe-sionmoleculeexpressionaregovernedbytranscriptionfactorswhichareimportantduringgastrulationincludingtwist,snail,slug,brachyuryandZEB1andZEB2.Circulatingtumorcells(CTCs)arecellswhichhavealreadyseparatedfromthetumorandenteredthebloodstream.IthasbeendemonstratedthatthenumberofCTCsinbloodisanimportantprognosticmarkerforbreast[33],prostate[34],lung[35],bladder[36]andcolon[37]cancerpatients.CTCsareaheterogeneouspopulationoftumorcells,someofthempresumablyunderwentEMTandhencepossessmesenchymalfeatures,et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Table1HomologousCAMsTypeofTypeofproteinProteinGenenameFunctionDesmosomeCadherin(calcium-dependent)Desmoglein1,Desmoglein2,Desmoglein3,Desmoglein4DSG1DSG2,DSG3,Playimportantrolesincelladhesion,ensuringthatcellswithintissuesareboundtogether.Cadherinsbehaveasbothreceptorsandligands.Desmocollin1,Desmocollin2,Desmocollin3,Desmocollin4DSC1DSC2DSC3CateninJunctionplakoglobinJUPcanbindtothedesmogleinI.ClaudinsClaudin1ThemaincomponentofthetightjunctionsOccludinsOccludinThemaincomponentofthetightjunctionsCadherinE-cadherinLossofE-cadherinfunctionorexpressionhasbeenimplicatedincancerprogressionandmetastasis.E-cadherindownregulationdecreasesthestrengthofcellularadhesionwithinatissue,resultinginanincreaseofcellularmotility.Thisinturnmayallowcancercellstocrossthebasementmembraneandinvadesurroundingtissues[F11receptor(JCAM)TheligandfortheintegrinLFA1,aplateletreceptor-(E,N,T),-catenin(orJunctionplakoglobinCTNNA1(CAP102),CTNNA2(CAPR),CTNNA3(VR22),CTNNB1,CTNND1,CTNND2,JUPCateninsbelongtoafamilyofproteinsfoundincomplexeswithcadherincelladhesionmolecules.Theprimarymechanicalroleofcateninsisconnectingcadherinstoactinfilaments,specificallyintheseadhesionjunctionsofepithelialcells[mayplayaroleintellingthecelltostopproliferating,asthereisnoroomformorecellsinthearea.TheroleofcatenininEMThasalsoreceivedalotofrecentattentionforitscontributionstocancerdevelopment.IthasbeenshownthatHIF-1inducetheEMTpathway,aswellastheWnt/signalingpathway,thusenhancingtheinvasivepotentialofLNCaPcells(humanprostatecancercells)cells)23].Asaresult,itispossiblethattheEMTassociatedwithupregulatedHIF-1iscontrolledbysignalsfromthisWnt/-cateninpathway[].CateninandEMTinteractionsmayalsoplayaroleinhepatocellularcarcinoma.VEGF-Btreatmentofhepatomacarcinomacellscancause-catenintomovefromitsnormallocationonthemembraneintothenucleusandE-cadherinexpressiontodecrease,thuspromotingEMTandtumorinvasiveness[JUPproteinistheonlyknownconstituentcommontosubmembranousplaquesofbothdesmosomesandintermediatejunctions.JUPalsoassociateswithclassicalcadherinssuchasE-cadherin;inthatcontext.PlakoglobinisO-glycosylated.CingulinCingulinCingulinisspecificallylocalizedattightjunctionsinepithelialcells,unlikeZO-1,whichisalsodetectedatadherens-typejunctionsinnon-epithelialcells.CingulininteractswithZO-1andseveralothertightjunctionproteins,inadditiontointeractingwithactinandmyosin[ACTA1,ACTA2,ACTB,ACTG1,ACTG2Participatesinmanyimportantcellularprocesses,includingcellmotility,celldivisionandcytokinesis,vesicleandorganellemovement,cellsignalling,andtheestablishmentandmaintenanceofcelljunctionsandcellshape.(orhemichannel)ConnexinsGJC1,GJB4etc.Connexinsareassembledingroupsofsixtoformhemichannels,orconnexons,andtwohemichannelsthencombinetoformagapjunction.Theconnexingenefamilyisdiverse,with21identifiedmembersinthesequencedhumangenome.Themoleculesforminghomologousepithelialcelltoepithelialcelltightcontacts.etal.MolecularCancerPage4of12http://www.molecular-cancer.com/content/12/1/107 whileothershavenotandstillrepresentwithamoreepi- thelialphenotype.Ithasbeendemonstratedongroupsof patientswithdistinctbreastcancerstagesthatCTCswith mesenchymalmarkersaremoretypicalforthelatemeta- staticstage[38]andprovideforthereliableprognosisof recurrence[39].Anotherrecentlyreportedobservationis thatmesenchymalCTCsinpatientswithadvancedcancer comprisemulticellularclustersratherthansinglecells,in contrasttoepithelialones[40].Theauthorsexplainedthis observationwiththeproliferationofthemesenchymalcell thathasundergoneEMTandafterproliferationdifferenti- atedbackintoamoreepitheliallydifferentiatedcellcluster which,however,seemscontradictorytothetypicalindivid- ualmesenchymalphenotype.Alternatively,theauthours hypothesizedsimultaneousEMTofapre-existingcluster ofCTCsinthebloodstreammediatedbyTGF- released fromplatelets. Despitethemanyefforts,thedetectionofCTCsstill suffersfromtechnicalcomplexitiesandnon-reliabilityof theirisolation.Theseproblemsareduetothelowabun- danceandheterogeneityofCTCs.TheCellSearchand AdnaTestsystemsapprovedbytheFDAinUSAandby EUauthorities,respectively,arebasedonthedetection ofepithelialmarkers.However,ifthecellsintheblood- streamaremoreofthemesenchymalphenotype,some importantcellpopulationmightbemissedbyusing theseisolationtechniques.Currentlythereisnoreliable methodandnodefinedlistofmarkersforthedetection ofdedifferentiatedEMT-derivedCTCs[41]. EMTandalternativesplicing Morethan88%ofhumanpre-mRNAsarealternatively spliced,thusgeneratingproteindiversityinanorganism [42].Alternativesplicingeventsareregulatedinacell- andtissuetype-specificmanner,atdifferentdevelop- mentalstagesorinresponsetoextra-cellularstimuliand activationofspecificsignallingpathways[43,44].As manyoftheseprocessesoccurduringEMT,alternative splicingisofimportanceinEMTaswell(Figure2).Ex- amplesofthebestcharacterizedEMT-dependentalter- nativelysplicedgenesareFGFR2,CD44,p120-catenin andMena. Thefibroblastgrowthfactorreceptor2(FGFR2)en- codesforafibroblastgrowthfactor-activatedtransmem- branereceptortyrosinekinaseandisthefirstdiscovered exampleofEMT-relatedalternativesplicing[45].The PRIMARYTUMOR EMT INTRAVASATION EXTRAVASATION MET METASTASIS Figure3 Themetastaticcascade. InearlystageofthemetastaticcascadeEMTenablesmigrat ionandintravasationoftumorcells.Afterextravasation followedbyMETmetastasisisgenerated. Samatov etal.MolecularCancer 2013, 12 :107 Page5of12 http://www.molecular-cancer.com/content/12/1/107 Table2HeterologousCAMsAdhesionmoleculeGenenameLocalizationandotherinformationLigandGenenameoftheligandLocalizationoftheligandandotherinformationIntegrinalpha(CD11A,p180)Integrinalphacombineswiththebeta2chain(ITGB2)toformtheintegrinlymphocytefunction-associatedantigen-1(LFA-1).LFA-1playsacentralroleinleukocyteintercellularadhesionthroughinteractionswithitsligands,ICAMs13(intercellularadhesionmolecules1through3),asarollingandsignalingmolecule[andalsofunctionsinlymphocytecostimulatorysignaling.ICAM1(CD54)ICAM1Amemberoftheimmunoglobulinsuperfamily.Aglycoproteinwhichistypicallyexpressedonendothelialcellsandcellsoftheimmunesystem.Integrinbeta-2ITGB2ICAM-1canbeinducedby(IL-1)and(TNF)andisexpressedbythevascularendothelium,macrophages,andlymphocytes.ICAM-1isaligandforLFA-1(integrin),areceptorfoundonleukocytes.IntegrinalphaM(ITGAM)(CD11B,CR3A)IntegrinalphaMisoneproteinsubunitthatformstheheterodimericintegrinalpha-Mbeta-2()molecule,alsoknownasmacrophage-1antigen(Mac-1)orcomplementreceptor3(CR3).isexpressedonthesurfaceofmanyleukocytesinvolvedintheinnateimmunesystem.Itmediatesleukocyteadhesionandmigration.Integrinalpha4(CD49d)VLA4(-integrin)isfoundonleukocytesandendothelialcells.VCAM1[VCAM1(CD106)VLA4-interectionssupportlymphocyterollinginvenulesofthecentralnervoussysteminconjunctionwithP-selectinorcandirectlymediaterapidadhesionindependentofP-selectinengagement[Integrinbeta-1ITGB1FibronectinFibronectinisahigh-molecularweightglycoproteinoftheextracellularmatrix[].Insolublecellularfibronectinisamajorcomponentoftheextracellularmatrix.Itissecretedbyvariouscells.Fibronectinplaysamajorroleincelladhesion,growth,migration,anddifferentiation.Alteredfibronectinexpression,degradation,andorganizationareassociatedwithanumberofpathologies,includingcancerandfibrosis[-integrinMADCAM-1MADCAM1MADCAM-1isacelladhesionleukocytereceptorexpressedbymucosalvenules.Ithelpstodirectlymphocytetrafficintomucosaltissues.Itcanbindbothintegrinalpha-4/beta-7andL-selectinregulatingboththepassageandretentionofleukocytes.Isoform2lackingthemucin-likedomainmaybespecializedinsupportingintegrinalpha-4/beta-7-dependentadhesionstrengthening,independentofL-selectinSelectinsP-selectinP-selectinisexpressedonactivatedendothelialcellsandplatelets.SynthesisofP-selectincanbeinducedbythrombin,leukotrieneB4,complementfragmentC5a,histamine,TNForLPS.PSGL-1(P-selectinglycoproteinSELPLG(CD16)PSGL-1isfoundonwhitebloodcellsandendothelialcells.PSGL-1canbindtoallthreemembersoftheselectinfamilyhoweveritbindstoP-selectinwiththehighestaffinity.P-selectinplaysanactiveroleintherollingofleukocytes[seeaboveand:PSGL-1wasshowncontributetoE-selectin-mediatedinitialleukocytecaptureandrollinginvivo[E-selectin(CD62E,ELAM-1)(CD62E,ELAM-1)E-selectinisexpressedonactivatedendothelialcells.E-selectinisnotstoredwithinthecellandhastobeSELPLG(CD16)etal.MolecularCancerPage6of12http://www.molecular-cancer.com/content/12/1/107 Table2HeterologousCAMstransportedtothecellsurface.SynthesisofE-selectinfollowsshortlyafterP-selectinsynthesis,inducedbycytokinessuchasIL-1,andlipopolysaccharide(LPS).ShearforcescanalsoaffectE-selectinexpression.E-selectinmayinteractindiscriminatelywithmanyglycoproteinsandglycolipids[ESL-1(golgiglycoprotein1)ESL-1isaglycoproteinandavariantofareceptorforfibroblastgrowthfactor.ESL-1isamajorE-selectinligandonleukocytes[CD44CD44isexpressedinalargenumberofmammaliancelltypes.Thisproteinparticipatesinavarietyofcellularfunctionsincludinglymphocyteactivation,recirculationandhoming,hematopoiesis,andtumormetastasis.E-selectinwasshowntoplayapivotalroleinmediatingcellinteractionsbetweenbreastcancercellsandendothelialmonolayersduringmetastasis[E-selectinplaysanactiveroleintherollingofleukocytes[ThecontributionofCD44issignificantonlyatthelaterstagesoftheleukocyterecruitmentcascade[GlyCAM-1GLYCAM1Inbreastcancerthesplicevariant4ofCD44wasshownasamajorE-selectinligandinfacilitatingtumorcellmigrationacrossendothelialmonolayersolayers32].L-selectin(CD62L)(CD62L,LAM1)L-selectinfoundonlymphocytesandpreimplantationembryo.Itplaysimportantrolesinlymphocyte-endothelialcellinteractions.GlyCAM-1isaproteoglycanligandexpressedoncellsofthehighendothelialvenulesinlymphnodes.CD34Acellsurfaceglycoproteinwhichfunctionsasacell-celladhesionfactor.Itmayalsomediatetheattachmentofstemcellstobonemarrowextracellularmatrixordirectlytostromalcells.CellsexpressingCD34arenormallyfoundintheumbilicalcordandbonemarrowashematopoieticcells,asubsetofmesenchymalstemcells,endothelialprogenitorcells,endothelialcellsofbloodvesselsbutnotlymphatics(exceptpleurallymphatics).CD34isalsoanimportantadhesionmoleculeandisrequiredforTcellstoenterlymphnodes.ItisexpressedonlymphnodeendotheliawhereastheL-selectintowhichitbindsisontheTcell.MADCAM-1MADCAM1MADCAM-1isacelladhesionleukocytereceptorexpressedbymucosalvenules.Ithelpstodirectlymphocytetrafficintomucosaltissues.Itcanbindbothintegrinalpha-4/beta-7andL-selectin,regulatingboththepassageandretentionofleukocytes.PSGL-1(CD16)SeeaboveThemoleculeswhichareresponsiblefortheleukocyteadhesioncascadeinvolvedintheinflammatoryresponse.etal.MolecularCancerPage7of12http://www.molecular-cancer.com/content/12/1/107 secondhalfofthethirdextra-cellularimmunoglobulin-likedomainoftheFGFR2isencodedbyoneoftwomutu-allyexclusiveexonsIIIb(expressedinepithelialcells)orIIIc(characteristicformesenchymalcells).ThefunctionalmodelsuggeststhatepithelialcellsexpressingtheFGFR2-IIIbformspecificallyinteractwithfibroblastgrowthfactorsproducedbymesenchymalcells.Accordingly,thefactorsexpressedbyepithelialcellsinteractwithFGFR2-IIIc[46].Theseinteractionshavebeendemonstratedtobeimport-antduringembryonicdevelopmentandlimboutgrowthandlung-branchingmorphogenesis.Remarkably,targeteddown-regulationofmesenchymal-specificFGFR2-IIIciso-formwasshowntodecreasemetastaticabilityofTSU-PrIbladdercancercellsandtoincreasesurvivalfollowinginvivoinoculationinmice[47].Interestingly,alternativesplicingofasimilarprotein,namelyFGFR3,isregulatedbysnoRNAHBII-180C[48].Thisfindingimpliesthatnon-codingRNAsregulateEMTthroughmodulationofalter-nativesplicing.TheCD44geneencodesforatransmembraneproteinwhichmaintainstissuestructurebymediatingcell-celladhesion[49].TheN-terminaldomainofCD44isextra-cellularandinteractswiththeextracellularmatrixgly-cosaminoglycanhyaluronicacid(HA)facilitatingthebindingofanumberofextracellularligands.Theformedcomplexinitiatesadownstreamsignalingcascadeviatheinteractionoftheintracellulardomainwithbindingpart-ners.TheCD44pre-mRNAcomprisesexons15attheendandexons1620atthe3endthataresplicedto-getherintothestandardisoformCD44s.Thisisoformisthesmallestandispresentonthemembraneofmostvertebratecells.Betweenexons5and16aretenalterna-tivelysplicedvariableexons(v1v10).ThesealternativelysplicedvariantsarelongerthanthestandardisoformofCD44andtheproteinsencodedbythesevariantsshowextendedextracellularmembrane-proximalregionswhichformaglycosylatedstalk-likestructureprovidinginteractionsitesforadditionalmolecules[50].TheCD44Eisoformcontainingexonsv810ispredominantlyexpressedinepithelialcellscorrelatingwiththeexpressionofE-cadherin[51].Remarkably,inductionofEMTinculturedcellsresultedinaswitchfromCD44Etostandardisoform,andexpressionofthelatterwasupregulatedinhumanbreastcancersandwascorrelatedwiththemesenchymalmarkerN-cadherininthesetumors[52].SplicingisnotaparticularfeatureofCD44,indeed,CAMsingeneralarealternativelyspliced.ThemostremarkableexampleisDSCAM(DownSyndromeCellAdhesionMolecule)whichhasupto18,000spliceisoforms[53].ThisIg-likereceptorisinvolvedinin-nateimmunityandneuralwiringanditsgeneislocatedon21chromosome.p120-CateninregulatescadherinstabilityandmodulatesRhoGTPaseactivity[54,55].Theisoformscontainingexons2and3areexpressedinmesenchymalcells.Epithelialcellsskiptheseexonsproducingashorterproteinisoform.Con-sistently,EMTinducestheexpressionofmesenchymalp120-cateninisoform[56].RhoGTPasesareknowntoregulateactincytoskeletonandcellmotility[57].Thefull-lengthmesenchymalisoformofp120-catenincanbindRhoAGTPase,reducingitsactivity,andpromotemigra-tionandinvasivenessofthecells[58].Mena(alsoknownasEnah,mammalianenabledhomologofDrosophilaproteinEna)isexpressedinvari-ouscelltypesandregulatesthebranchingactinfila-ments[59].Theisoformwhichcontainstheexon11aischaracteristicforepithelialcellsandisnotfoundinmes-enchymalcells.Remarkably,ithasbeenalsofoundtobeexpressedinprimarytumorcellsbutnotininvasivetumorcells[60].Sofaritisnotclearwhatkindoffunc-tionalimplicationsthisproteinhasonEMT.Recentlygenome-wideapproacheswereusedtodeter-mineEMT-relatedalternativesplicingsignatures[61].ItwasshownthatEMT-relatedextensivechangesinalternativesplicingareregulatedbyepithelialsplicingregu-latoryproteins1and2(ESRP1andESRP2)[62].Thesepro-teinsarepresentinepithelialcells.TheirsiRNA-mediatedknockdownresultedinasplicingswitchofFGFR2,CD44,p120andMenagenestomesenchymalphenotype.There-verseeffectwasobservedwhentheectopicexpressionofESRP1andESRP2wasperformedinmesenchymalcells.Thus,thereareclearlydistinctprofilesofalternativesplicingwhichallowdiscriminationbetweenepithelialandmesenchymalcelltypes.EMTandnon-codingRNAsMiRNAsareonefamilyofsmall(2022nucleotides)non-codingRNAs.Theirfunctionistoregulategeneex-pressionpost-transcriptionallythroughbindingtothesiteswhichareperfectlycomplementary,orwhichmaycontainmismatches(non-canonicalsites).Thesesitesarelocatedin3UTRs,howeverrecentreportsdemon-stratethatmiRNAscanalsofunctionthroughbindingtootherregionsoftargetmRNAs[63,64].BybindingtotargetmRNAs,miRNAsplayimportantrolesinregulat-ingdiversebiologicalprocesses[65].Theseprocessesin-cluderegulationoftheEMT,inwhichvariousmiRNAsareinvolved[66].Remarkably,theregulatorymiRNA-mRNAnetworkscanberapidlyregulated[67].Itshouldalsobementionedthatanotherrecentlyreportedinter-estingfunctionofmiRNAs,whichmayplayacertainroleinEMTregulation,isparacrine-modeintercellularsignaling[68].ThemiR200andthemiR205familieswereshowntobehighlyassociatedwithEMTandastrongcorrelationbetweentheexpressionofthemiR200familyandE-cadherinexpressionindifferentcelllinesandepithelialtissueshasbeendemonstrated[69,70].DuringEMT,etal.MolecularCancerPage8of12http://www.molecular-cancer.com/content/12/1/107 expressionofmiR-200familyisrepressedbyZEBtran-scriptionfactors.ThesefactorsinturnarethetargetsformiR-200familythuscomprisingadoublenegativefeed-backloop[71].ItwasshownrecentlythatmiR200calsoregulatesEMTthroughtargetingfibronectin,moesinandotherproteinsthatnormallysuppresscellmigrationandresistancetoanoikis[72].Moreover,thesamelabfoundthatmiR200ctargetsaNF-B-dependentneurotrophictyrosinereceptorkinase,whichalsosuppressesresistancetoanoikis,andthismiRNAisdown-regulatedinhighlyaggressivetriplenegativebreastcancers[73].OtherEMT-relateddownstreamtargetsofthemiR200familyaremiR141inhibitingTGF2[74]andmiR200asuppressing-catenin(CTNNB1)[75].TheEMT-relatedtranscriptionfactorshavebeende-scribedastranscriptionalregulatorsofmiRNAsaswell.Forexample,miR21isabundantinvarioustumorsandknowntoinducemetastasisthroughEMT.ThepromoterregionsofmiR21containconsensusE-boxsequencescom-prisingbindingsitesforZEB1[76].BindingofZEB1in-ducestranscriptionofmiR21[77].MiR10bisalsoknowntobeassociatedwithcellmigration,invasion,andmetasta-sisofbreastcancercells.Itwasshownthatthetranscrip-tionfactortwistcanbindtotheE-boxelementclosetothepredictedpromoterofmiR10bandactivateitstranscription,thuspromotingtwist-mediatedEMT[77].OverallregulationofmiR10biscomplexandcontextdependent:ZEB1increasestheexpressionofmiR10bincolorectalcancercellsbutdecreasesexpressioninbreastcancercells[74].Similarly,snailreducestheexpressionofmiR10binhumanbreastcancercells[77].Thesedatasug-gestthatmiRNAscanbeconsideredasmarkersforEMTthroughtheactivityofEMT-relatedtranscriptionfactors.MiRNAswereshowntobeassociatedwiththeTGFsignalingpathway.TheTGF-mediatedinductionofEMTinmammaryepithelialcellsresultsinlossoftightjunc-tionsandcellpolarityandup-regulatestheexpressionofmiR155[78].ThetargetofmiR155isRhoAwhichisim-portantforthecontrolofactincytoskeletonandcellinva-sion.RhoAcontainsthreeconservedregionswhicharepotentialbindingsitesformiR155[78].Down-regulationofRhoAleadstoactincytoskeletonrearrangementsandincreasedcellmotility[79].TheTGF-inducedEMTinmammaryepithelialcellsalsoleadstothehigherexpressionlevelsofmiR29aandmiR21[78,80].EctopicexpressionofmiR29asuppressestheexpressionoftristetraprolinandpromotestoEMTincooperationwiththeRaspathway[80].IthasbeendemonstratedthatmiR9regulatesthemRNAencodingforE-cadherin[81].Hencetheincreasedexpres-sionofmiR9inducedEMTinhumanmammaryepithelialcells[81].Remarkably,ithasbeendemonstratedrecentlythatcir-culatingmiRNAsinplasmaofmetastaticbreastcancerpatientscanindicatetheirCTCstatus[82].CirculatingmiRNAsareeasiertoisolateandhandlethanCTCs,whichwillprobablymakethemprognosticmarkersofchoiceinfuture.Longnon-codingRNAs(lncRNAs)areanemergingclassofRNAslongerthan200nt.Ourcurrentunder-standingoftheirfunctionalroleislimited,howevertherearereportsdescribingtheirinvolvementintheregulationofgeneexpression,chromatinremodeling,tran-scription,post-transcriptionalRNAprocessingandcancerprogression[83].Metastasis-associatedlncRNAsMALAT1(8000nt),HOTAIR(2200nt)andANRIL(3800nt)areup-regulatedinsometumorsandcanbepotentiallyconsideredasEMT-relatedastheyregulateEMTtranscription[84].Morespecifically,siRNA-mediatedMALAT1silencingresultedindown-regulationoftheEMT-associatedtranscriptionfactorsZEB1,ZEB2andslug,andup-regulationofE-cadherin[85].Moreover,MALAT-1promotedEMTbyactivatingtheWntsignal-ingpathway.ItalsohasbeendemonstratedthatMALAT-1levelsweresignificantlyincreasedinprimarytumorsthatsubsequentlymetastasizedcomparingtothosetu-morsthatdidnotmetastasize.SmallmoleculecompoundsmodulatingEMTTherearenumerouskinasesinvolvedinTGF-,Wnt,hedgehogandothersignallingpathwaysregulatingEMTandthusmalignantprogression.Thebasisofmodernmoleculartargetedcancertherapeuticsisthedevelop-mentofsmallmoleculeinhibitorscapableofbindingtotheATP-bindingsiteofthedysregulatedkinases.ThusthemajorityofthecompoundsaffectEMTtargetkinases.Forexample,gefitinibanderlotinib,whicharecompetitiveinhibitorsofEGFR,currentlyusedforthetreatmentofad-vancedcarcinomas,alsodemonstrateaprotectiveeffectagainstpulmonaryfibrosisandhepaticfibrosis/cirrhosis,whichsupportstheirEMT-inhibitingactivity[86,87].Otherwell-knowncompoundsareantiangiogenicdrugssorafenibandsunitinibthatinhibitVEGFRandPDGFR,exhibitantifibroticeffectsintheliverandhavebeendem-onstratedtoinhibitEMTininvitrocellculturemodels[88-90].CompoundsEW-7195andEW-7203targetTGF-typeIreceptorkinase/activinreceptorlikekinase-5(ALK5)inasimilarway,inhibitingTGF--inducedEMTofmammaryepithelialcellsandpreventingbreastcancermetastasistolung[91,92].ThedrugBI5700directlyinhibitskinaseIKK2,amemberofNF-BsignalingpathwaywhoseactivationcausesEMT,andwhichhasbeendemonstratedtorevertEMTinmetastasizingmousecoloncarcinoma[93].An-othercompoundSL0101targetsribosomalproteinS6kinase(RSK)-2whichisanimportantcomponentofRONandTGF-signalingpathways[94].BothpathwaysregulateEMT,andinhibitionofRSK-2resultsinetal.MolecularCancerPage9of12http://www.molecular-cancer.com/content/12/1/107 suppressionofEMT-associatedcellmigrationinaninvitroexperimentalsystem[94].Interestingly,thecompletereversalofEMTinvitrowasachievedwhenacombinationoftheinhibitorsofki-nasesTRI(inhibitorSB431542)andROCK(inhibitorY27632)wasused[28].SB431542down-regulateZEB1andZEB2levels,thusblockingmesenchymalgeneex-pressionofTGF--inducedmesenchymalrenaltubularepithelialcells.TheRhopathwayinhibitingY27632wasnecessarytofullyeliminatemesenchymalactinstressAnothertypeofsmallmoleculemodulator4Ei-1isanon-toxicnucleotideanaloguewhichpreventstheasso-ciationofeIF4EandthemRNAcap.Itinhibitedcap-dependenttranslationinadose-dependentmannerinzebrafishembryoswithoutcausingdevelopmentalab-normalitiesandpreventedeIF4EfromtriggeringEMTinzebrafishexplantmodel[95].Thiscompoundcanbeconsideredasapotentialanti-cancerdrugandinvestiga-tionofitseffectonthetumorswouldbeofagreatinterest.Recentlyahigh-throughputassaywasdevelopedtoscreenforsmallmoleculesinterferingwithEMTiniti-atedbygrowthfactorsignallingusingamodelcarcin-omareportercelllineNBT-II[96].Inthisassaybothcellgrowthandcellmigrationcanbeanalysedsimultan-eouslyviatime-courseimaginginmulti-wellplates.TheauthorshavevalidatedseveralcompoundstargetingALK5,MEK,andSRCkinasesasefficientEMTinhibi-tors.ThisworkhighlightsthegrowinginterestinthesmallmoleculecompoundsabletomodulateEMT.Me-tastasesareresponsiblefor�90%ofthecancerassoci-ateddeaths.ThereforenewstrategiestopreventEMTwhichleadstometastasesformationmightbeapromis-ingnovelapproachinoncology.ConclusionsEpithelial-mesenchymaltransitionremainsinthefocusofalargenumberofresearcherstodayduetoitsfunda-mentalnatureandimportantclinicalimplications.Non-codingRNAsandalternativesplicingswitchesdiscussedinthisreviewplayimportantrolesinEMTandcancerprogressionandcanserveasmarkersfordistinctepithe-lialormesenchymalstatesofcells.Also,thereareagrowingnumberofdiscoveredsmallmolecules,belong-ingmostlytokinaseinhibitors,whichmodulateEMTandhaveanti-cancereffect.Epithelial-mesenchymaltransition;MET:Mesenchymal-epithelialtransition;TGF:Transforminggrowthfactor;CAM:Celladhesionmolecule;CTC:Circulatingtumorcell;miRNA:MicroRNA;lncRNA:Longnon-codingCompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.TRSwrotethefirstdraftofthearticle,AGTandUSfinalizedthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.AcknowledgementsWewouldliketothankDrD.MaltsevaforthehelpwithpreparationofthetablesandMsE.Grundyforthecarefullycorrectingourmanuscript.ThisworkwassupportedbyfundsfromtheBundesministeriumfürBildungundForschung(BMBFNo.RUS10/022)andRussianMinistryofScienceContracts(No.14.512.11.0024and14.512.11.0083).AuthordetailsSRCBioclinicum,Ugreshskayastr2/85,Moscow115088,Russia.InstituteofGeneralPathologyandPathophysiology,RussianAcademyofMedicalSciences,Baltiiskayastr.8,Moscow125315,Russia.P.A.HertsenMoscowResearchOncologyInstitute,2ndBotkinskiip.3,Moscow125284,DepartmentofAnatomyandExperimentalMorphology,UniversityCancerCenter,UniversityMedicalCenterHamburg-Eppendorf,Martinistr.52,HamburgD-20246,Germany.Received:10April2013Accepted:16September2013Published:23September20131.KalluriR,WeinbergRA:Thebasicsofepithelial-mesenchymaltransition.JClinInvest2.ChafferCL,ThompsonEW,WilliamsED:Mesenchymaltoepithelialtransitionindevelopmentanddisease.CellsTissuesOrgans2007,185:19.3.ZeisbergEM,TarnavskiO,ZeisbergM,DorfmanAL,McMullenJR,GustafssonE,ChandrakerA,YuanX,PuWT,RobertsAB,NeilsonEG,SayeghMH,IzumoS,KalluriR:Endothelial-to-mesenchymaltransitioncontributestocardiacNatMed4.KimKK,KuglerMC,WoltersPJ,RobillardL,GalvezMG,BrumwellAN,SheppardD,ChapmanHA:Alveolarepithelialcellmesenchymaltransitiondevelopsinvivoduringpulmonaryfibrosisandisregulatedbytheextracellularmatrix.ProcNatlAcadSciUSA5.ThieryJP:Epithelial-mesenchymaltransitionsintumourprogression.NatRevCancer6.KopantzevEP,MonastyrskayaGS,VinogradovaTV,ZinovyevaMV,KostinaMB,FilyukovaOB,TonevitskyAG,SukhikhGT,SverdlovED:Differencesingeneexpressionlevelsbetweenearlyandlaterstagesofhumanlungdevelopmentareoppositetothosebetweennormallungtissueandnon-smalllungcellcarcinoma.LungCancer7.LeeJM,DedharS,KalluriR,ThompsonEW:Theepithelial-mesenchymaltransition:newinsightsinsignaling,development,anddisease.JCellBiol8.VandewalleC,VanRoyF,BerxG:TheroleoftheZEBfamilyoftranscriptionfactorsindevelopmentanddisease.CellMolLifeSci2009,66:773787.9.BindelsS,MestdagtM,VandewalleC,JacobsN,VoldersL,Noe¨lA,VanRoyF,BerxG,FoidartJM,GillesC:RegulationofvimentinbySIP1inhumanepithelialbreasttumorcells.10.DeCraeneB,BerxG:RegulatorynetworksdefiningEMTduringcancerinitiationandprogression.NatRevCancer11.Sánchez-TillóE,LiuY,deBarriosO,SilesL,FanloL,CuatrecasasM,DarlingDS,DeanDC,CastellsA,PostigoA:EMT-activatingtranscriptionfactorsincancer:beyondEMTandtumorinvasiveness.CellMolLifeSci12.PeinadoH,MarinF,CubilloE,StarkHJ,FusenigN,NietoMA,CanoA:SnailandE47repressorsofE-cadherininducedistinctinvasiveandangiogenicpropertiesinvivo.JCellSci13.ThieryJP,AcloqueH,HuangRY,NietoMA:Epithelialmesenchymaltransitionsindevelopmentanddisease.14.QinQ,XuY,HeT,QinC,XuJ:Normalanddiseaserelatedbiolo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