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by the tumor microenvironmental molecule tenascin-C
Anja Schwenzer
To cite this version:
Anja Schwenzer. Mechanism and consequences of DKK1 downregulation by the tumor microenvi-ronmental molecule tenascin-C. Molecular biology. Université de Strasbourg, 2013. English. �NNT : 2013STRAJ086�. �tel-01148418�
UNIVERSITÉ
DESTRASBOURG
ÉCOLE
DOCTORALEdesSCIENCESDELAVIEETDELASANTE
Inserm
U1109–MN3Tteam
THÈSE
présentéepar:
AnjaSCHWENZER
soutenuele:30Septembre2013
pourobtenirlegradede:
Docteur
del’universitédeStrasbourg
Discipline/Spécialité
:Scienceduvivant/Aspectsmoléculairesetcellulairesdelabiologie
Mécanisme
etconséquencesdelarépressiondeDKK1
par
laténascineͲC,unemoléculedu
microenvironnement
tumoral
THÈSEdirigéepar:Dr.GertraudOREND UniversitédeStrasbourg,InsermU1109,France
RAPPORTEURS:
Dr.FlorenceRUGGIERO ENSdeLyon,UMRCNRS5242,France
Dr.AnnaͲKarinOLSSON UppsalaUniversity,Sweden
AUTRESMEMBRESDUJURY:
Prof.MaximeLEHMANN UniversitédeStrasbourg,UMR7213CNRSͲLBP,France
Mechanism
andconsequencesofDKK1downregulationbythetumor
microenvironmental
moleculetenascinͲC
Background: The tumor microenvironment plays a central role in driving cancer progression.
TenascinͲCisamajorcomponentofthetumorspecificextracellularmatrix(ECM)anditsexpression
hasbeenlinkedtotumorangiogenesis,metastasisandisamarkerofbadprognosis.However,itis
notwellunderstoodhowTNCpromotestumorprogression.
BasedonamicroarrayapproachofT98GglioblastomacellsculturedonFNorFN/TNCcoateddishes
the Wnt signalling inhibitor DKK1 had been identified to be strongly downregulated by TNC,
suggestingDKK1/WntsignallingasapotentialmechanismforTNCinducedtumorprogression.
Objectives:TheobjectiveofthisthesiswastoelucidatetheeffectofDKK1downregulationbyTNCon
Wnt signalling in tumor cells. Furthermore, this study was extended to the analysis of DKK1
expressionandWntsignallingactivityinstromalcellsinthepresenceofTNC.AdditionallyIaimedin
identifyingthemechanism(s)implicatedinDKK1downregulationbyTNC.
Results:IdemonstratedthatTNCdownregulatesDKK1inseveraltumorcellsinvitroandthatDKK1
downregulationwaslinkedtoenhancedWnt/EͲcateninsignalling.Inaddition,stromalcells,including
endothelialcells,pericytesandcancerͲassociatedfibroblasts,exhibitedreducedDKK1levelsinthe
presence of TNC. While TNC induced expression of the Wnt/EͲcatenin target gene Axin2 in
endothelialcells,Axin2remainedunchangedinpericytes.
Second,IdemonstratedthatTNCreducesDKK1promoteractivity.Reducedstressfibreformationin
the presence of TNC was identified as a major mechanism contributing to DKK1 downregulation.
DKK1geneexpressionwasinhibiteduponstressfibredisruptionandinduceduponenforcementof
stressfibres.Theactivity oftheactinͲregulated SRFcoͲtranscriptionfactor MKL1wasfound to be
reducedinthepresenceofTNC.MyresultsindicatethatTNCregulatedMKL1functionmaybeone,
but not the major mechanism of DKK1 regulation by the actin status and that other factors
presumablyregulatedbyactinstressfibresareinvolved.
Conclusion: Enhanced Wnt signalling activity downstream of TNCͲinduced DKK1 downregulation
might be a major mechanism by which TNC promotes tumor progression. In addition, for further
studiesitwillbeinterestingtoanalysewhethertheTNCͲinducedDKK1downregulationinstromal
cells impacts onEͲcateninͲdependent and/or EͲcateninͲindependent pathways. A strong DKK1
downregulationbyTNCintumorandstromalcellsmaycreateanenvironmentintumortissuethat
renders cells responsive for Wnt and, other DKK1 repressed signalling. Furthermore, this study
discovered a novel mechanism of regulating the Wnt inhibitor DKK1 by the integrity of the actin
RESUMÉDETHÈSEENFRANÇAIS
M
ECANISMEETCONSEQUENCESDELAREPRESSIONDEDKK1
PARLATENASCINEͲC,
UNEMOLECULEDUMICROENVIRONNEMENTTUMORAL
I.
Introduction
Les interactions entre les cellules tumorales et leur microenvironnement jouent des rôles instrumentaux durant la progression tumorale. Le microenvironnement tumoral est constitué de cellules stromales associées à la tumeur, de facteurs solubles tels que des cytokinesetdesfacteursdecroissanceainsiquedeprotéinesdelamatriceextracellulaire, cellesͲci incluant la ténascineͲC (TNC) (1, 2). La TNC est exprimée au cours de l’embryogenèsemaissonexpressionestréduitevoireabsentedanslestissusadultessains. SaréͲexpressionseproduitdansdessituationspathologiquestellesquelecancer.Uneforte expressiondelaTNCestcorréléeavecunpronosticpéjoratifdansdifférentstypestumoraux et son expression est par ailleurs liée à la progression tumorale, l’angiogenèse et la formation de métastases (3Ͳ5). La TNC constitue donc un acteur déterminant du stroma tumoralmaislesmécanismesmoléculairessousͲjacentssonttoujoursincompris.
Notre laboratoire a précédemment démontréque la TNCrégule l’adhésion cellulaire ainsi que la dynamique du cytosquelette d’actine en bloquant le complexe intégrine ɲ5ɴ1/syndecanͲ4(6Ͳ8)etquelaTNCconduitàlasousͲexpressiondeDKK1(8),uninhibiteur delasignalisationdépendantedesligandsWnt(9)etdel’angiogenèse(10).Laparticipation delaTNCalavoiedesignalisationWntaégalementétédémontréepard’autres(11).
NotrelaboratoireaétablidesmodèlesmurinstumorigéniqueRipTag2(RT2)surͲexprimantla TNC ou présentant une invalidation de la TNC. En bref, ces modèles nous ont permis de démontrerquelaTNCpromeutlaprogressiontumoraleenfavorisantl’angiogenèseetpar ailleurs que l’expression de DKK1 est corrélée de manière inverse à la TNC. De plus la surexpressiondelaTNCconduitàuneexpressionaccruedel’Axine2,ungènecibledelavoie designalisationWnt.Nousavonsdeplusmontréenutilisantunmodèledexénogreffeque DKK1 constitue un inhibiteur majeur de l’angiogenèse et de la croissance tumorale. Ces résultatssontcohérentsavecunepublicationrécenteidentifiantDKK1commeuninhibiteur puissantdel’angiogenèsesuiteàl’ischémie(10).
L’ensemble de ces résultats suggère que la TNC induit une activation de la voie de signalisationWntsuiteàl’inhibitiondel’expressiondeDKK1,uninhibiteurdesligandsWnts. Ainsi, un de buts de ma thèse était d’utiliser des modèles in vitro afin de déterminer le niveau d’activité de la voie Wnt en fonction de la TNC dans des cellules tumorales, et égalementd’évaluersilasousͲexpressiondeDKK1induiteparlaTNCpouvaitêtrelacause del’activationdelavoieWnt.Deplusjemesuisintéresséàlacontributionpotentielledes cellules stromales présentes dans le microenvironnement tumoral dans la diminution de l’expressiondeDKK1enprésencedelaTNC.Surlabasededonnéesprécédemmentpubliées parlelaboratoiremontrantquelaTNCinduituneperturbationdesfibresdestressd’actine (6, 7, 12), j’ai émis l’hypothèse qu’une forte expression de DKK1 dépend de la présence d’actinefilamenteusestabiliséeauseindefibresdestress.
RESUMÉDETHÈSEENFRANÇAIS
II.
Résultatsetdiscussion
1.RégulationdelavoiedesignalisationWntdansdescellulestumorales
J’aimontréquelaTNCconduitàlasousͲexpressiondel’inhibiteurdelavoieWntDKK1dans plusieurs lignées cellulaires tumorales d’origines différentes en analysant les niveaux d’expressiondeDKK1auniveaudel’ARNmparRTͲqPCRetauniveauprotéiqueparWestern Blot. J’ai confirmé que la TNC augmente l’activation de la voie de signalisation Wnt en utilisantuntestrapporteur(TOPflash)ainsiqu’enmesurantleniveaud’expressiondel’Axin2 dansdescellulesKRIBstimuléesparleligandWntͲ3A.
IlestprobablequelasousͲexpressiondeDKK1estliéedemanièrecausaleàl’inductiondela voie de signalisation Wnt par la TNC. Afin de tester cette hypothèse, j’ai déterminé si l’induction de la voie Wnt par la TNC est régulée de manière dépendante de DKK1 en réalisantdesexpériencesdesurexpressionoude«knockdown»deDKK1dansdescellules KRIB.J’aiétablideslignéescellulairesquisurexprimentmDKK1demanièrestable,cequia été confirmé par Western blot. La surexpression de DKK1 conduit à une répression de l’inductiondelavoiedesignalisationWntinduiteparWntͲ3Adansdescellulescultivéessur unsubstratTNC.J’aiégalementétablideslignéesdontl’expressiondeDKK1estinvalidéede façonstablepardesshRNAsciblantDKK1.AprèsconfirmationduknockͲdowndeDKK1par RTͲqPCR et Western blot, des tests rapporteurs ont permis de mettre en évidence Que l’invalidation de DKK1 conduit à une activation accrue de la voie de signalisation Wnt. A
contrario, sur un substrat contenant la TNC, le knockdown de DKK1 n’entraine plus d’
augmentationdel’activitéWnt.
En conclusion, ces résultats suggèrent que la stimulation de la voie de signalisation Wnt médiéeparlaTNCdansdescellulestumoralesestprincipalementdueàlasousͲexpression del’inhibiteurdesligandsWntDKK1. 2.RégulationdelavoiedesignalisationWntdanslescellulesstromales LescellulesstromalesdumicroenvironnementtumoralsécrètentégalementdelaTNCet/ou sontencontactaveclaTNC(13).J’aidoncanalysési,enparallèledescellulestumorales,des cellules stromales telles que des cellules endothéliales, des péricytes et des fibroblastes associésaucancer(CAF)cultivéessurunsubstratTNCensousͲexprimantl’inhibiteurdeWnt DKK1. J’ai pu montrer que les péricytes, deux lignées cellulaires de CAF et des cellules
Dans les cellules endothéliales, j’ai montré que la TNC conduit également à une augmentation de l’expression du gène Axin2, indiquant une activation de la voie de signalisationWnt.IlresteàdéterminersilaTNCstimulelavoiedesignalisationWntdans d’autrestypesdecellulesstromales,etsicelaestliédemanièrecausaleàlasousͲexpression deDKK1.DesniveauxfaiblesdeDKK1pourraientégalementexercerd’autresfonctionsdans cescellulesetcontribueràl’inductiondesignauxWntnoncanoniquesouencored’autres voiesdanscecontexte(10,14,15). 3.DéterminationdesmécanismesimpliquésdanslasousͲepxressiondeDKK1induitepar laTNC 3.1LaTNCinhibel’activitédupromoteurdeDKK1 PuisquelaTNCinduituneforteetrapidesousͲexpressiondeDKK1,j’aiémisl’hypothèseque l’expressiondugèneDKK1étaitsoitréguléeauniveaudel’activitédesonpromoteursoitau niveaudelastabilitédel’ARNm.J’aistimulédescellulesT98Gavecdel’ActinomycineD,un inhibiteurdelatranscription.CetraitementaabrogélasousͲexpressiondeDKK1induitepar un substrat de FN (Fibronectine)/TNC comparé à un substrat de FN seul, suggérant que l’expressiondeDKK1estréguléeauniveautranscriptionnel.J’aidoncclonéunfragmentde 3kb du promoteur de DKK1 d’origine humaine, en amont de la séquence codant pour la luciférase de luciole et analysé l’activité de ce plasmide rapporteur. J’ai observé qu’un substratcontenantdelaTNCinhibel’activitédecepromoteursynthétiquedeDKK1.
Comme la régulation de DKK1 par la TNC se produit de manière rapide et que les expériencesprécédemmentdécritesontpermisdemontrerquecetterégulations’effectue auniveautranscriptionnel,j’aiémisl’hypothèsequelarégulationdeDKK1pourraitêtredue àdeschangementsducytosqueletteinduitsparlaTNC,carceschangementsreprésenteun effet immédiat de la TNC. J’ai donc recherché un facteur de transcription qui serait directementréguléparlecytosqueletteetseschangements.
3.2LaTNCrégulelavoieSRF/MKL1
LaTNCréduitfortementl’adhésioncellulaireeninhibantlaformationdesfibresdestress d’actineendéstabilisantRhoA,enavalducomplexedel’intégrineɲ5ɴ1/syndecanͲ4(7,16).
RESUMÉDETHÈSEENFRANÇAIS
Apartird’uneanalyseprécédentedulaboratoire(8),jemesuisrenducomptequelaTNC conduitàlasousͲexpressiond’uncertainnombredegènesciblesdufacteurdetranscription SRF(facteurderéponseausérum)etdesoncoͲfacteurMKL1(«megakaryoblasticleukemia 1»)Letransportverslenoyauetl’activitédeMKL1estréguléparl’activitédeRhoAetla dynamique de l’actine (17). Lorsqu’il est nucléaire, MKL1 agit comme un coͲactivateur du facteurdetranscriptionSRFetactivelatranscriptiondesgènescontenantdesélémentsde réponse au sérum («boîte CARG») dans leurs promoteurs (17, 18). Ses gènes cibles sont impliqués dans la régulation du cytosquelette et des adhésions focales. J’ai confirmé la régulationdeplusieursgènesciblesdeSRF/MKL1dans3lignéescellulairescancéreusesen présencedeTNC.Deplus,j’aimontréquel’activationdecesgènesciblesinduiteparlaTNC dépendbiendeMKL1enutilisantuneapprochedeknockdownciblantMKL1. 3.3LasousͲexpressiondeDKK1surunsubstratdeTNCestindépendantedesvoiesRhoAet MKL1 Pourdéterminersil’expressiondeDKK1estcontrôléeparMKL1,j’airéaliséunknockdown deMKL1médiépardesshRNAsetmontréquesonexpressionetl’activitédesonpromoteur étaient inhibées. L’activation de la voie RhoA/MKL1 peut notamment s’effectuer en stimulantlescellulesparunlefacteurdecroissance,l’acidelysophosphatidique(LPA).Un traitementparduLPAstimulel’expressiondeDKK1demanièredoseͲdépendanteetsurun substrat de FN/TNC restaure l’expression à un niveau comparable de celui de cellules ensemencéessurunsubstratdeFN.Cecis’accompagneégalementparunerestaurationde l’«étalement»descellules.Toutefois,lasurexpressiond’uneformeconstitutionnellement activéedeRhoAoudeMKL1n’induitpasl’expressiondeDKK1,etnerestaurepasleniveau d’expressiondeDKK1surunsubstratdeTNC. Ainsi,lasousͲexpressiondeDKK1induiteparlaTNCestindépendantedeRhoAetdeMKL1. LeLPApourraitactiverunevoieindépendantedelasignalisationparRhoA/MKL1etstimuler l’expressiondeDKK1.LeknockdowndeMKL1dansdescellulestumoralesmimeleblocage des fibres de stress d’actine induit par la TNC et ainsi indirectement cause une sousͲ expressiondeDKK1.
3.4L’expressiondeDKK1estréguléeparladynamiqueducytosqueletted’actine Afindedéterminersilestatutdepolymérisationdel’actineréguleleniveaud’expressionde DKK1,j’aianalysésonexpressionaprèssurexpressiondusyndesmosoudelatropmyosineͲ1 (TPMͲ1),etenutilisantdesdroguespermettantdemanipulerlestatutdepolymérisationde l’actine,quidéstabilisent(LatrunculineB,CytochalasineD)oustabilise(Jasplakinolide)laFͲ actine. LaTNCaffectenotammentlastabilitédesfibresdestressd’actinevialasousͲexpressionde TPMͲ1(8).LasurexpresssiondeTPMͲ1restaurel’étalementdescellulesetlaformationde fibresdestresssurunsubstratdedeFN/TNC(8).J’aiobservéquelasurexpressiondeTPMͲ1 induisaitfortementl’expressiondeDKK1alorsqueleknockdowndeTPMͲ1conduisaitàune réduction de son niveau d’expression. La TNC est en compétition avec l’activation du syndecanͲ4 par la Fibronectine (6). Le syndesmos permet de contourner la nécessité du syndecanͲ4pourl’étalementdescellulessurunsubstratdeFN/TNCetderestaurerlesfibres de stress d’actine et la signalisation par les adhésions focales (12). En surexprimant le syndesmos, l’expression de DKK1 était fortement augmentée. Ces résultats soutiennent l‘hypothèsequelesfibresdestressd’actinerégulentlatranscriptiondeDKK1.
Lejasplakinolideinduitlapolymérisationd’actineetlaformationd’actinefilamenteuse(FͲ actine) stable. Mais des traitements de longue durée ou des concentrations fortes de jasplakinolideinduisentunepertedesfibresdestress(19,20).Lesfibresdestresssontdes faisceauxdefilamentsinterͲreliésd’actine.Lejasplakinolideréprimel’expressiondeDKK1, suggérantquel’expressiondeDKK1estdépendantedelaprésencedefibresdestress. La cytochalasine D inhibe l’élongation des polymères d’actine (22). A des concentrations fortes,lacytochalasineDréprimel’expressiondeDKK1.LalatrunculineBselieàlaGͲactine monomérique et inhibe la formation de FͲactine (23). J’ai observé que la latrunculine B réduitl’expressiondeDKK1.
Cesdonnéessuggèrentqueleniveaud’expressiondeDKK1estfortdansdescellulesayant del’actinefilamenteusestableetdesfibresdestress,maisquelaperturbationdecesfibres destressetladépolymérisationdel’actinefilamenteuseconduisentàlarépressiondeDKK1. Il sera intéressant de déterminer les mécanismes moléculaires impliqués dans cette régulationdeDKK1,etenparticulierd’identifierle(s)facteur(s)detranscriptionimpliqué(s).
RESUMÉDETHÈSEENFRANÇAIS cellules tumorales péricytes CAFs cellules endothéliales peu de peu de DKK1 DKK1 TNC voie de signalisation Wnt/E-caténine voie de signalisation dépendant et indépendant de Wnt/E-caténine ? cellules tumorales péricytes CAFs cellules endothéliales peu de peu de DKK1 DKK1 TNC voie de signalisation Wnt/E-caténine voie de signalisation dépendant et indépendant de Wnt/E-caténine ?
DKK1
GͲactine FͲactine FibresdestressLatrunculinB CytochalasinD Jasplakinolide LatrunculinB CytochalasinD TPM1 LPA Jasplakinolide TPM1 LPA
DKK1
GͲactine FͲactineFͲactine FibresdestressFibresdestressLatrunculinB CytochalasinD Jasplakinolide LatrunculinB CytochalasinD TPM1 LPA Jasplakinolide TPM1 LPA Figure:RésumédesprincipauxrésultatsconcernantlemécanismederégulationdeDikkopfͲ1(DKK1)parla ténascineͲC(TNC)etsesconséquences.
(A) La TNC réduit l’expression du gène DKK1 aussi bien dans les cellules tumorales que stromales. Dans les cellules tumorales, la répression de DKK1 permet de stimuler la voie Wnt/ɴͲcatenine. Dans les cellules stromales,l’effetdelarépressiondeDKK1surlavoieWnt/ɴͲcateninedoitencoreêtreanalyséplusendétail. (B)ModèlederégulationdeDKK1parlestatutdepolymérisationdel’actinedanslacellule.Larupturedes fibresdestressetladépolymérisationdel’actine(LatrunculineB,CytochalasineD)décroitl’expressiondugène DKK1,tandisquel’inductiondelapolymérisationdel’actineoudesfibresdestress(LPA,TPM1)augmentent l’expressiondeDKK1.LeJasplakinolide,quiinduitlapolymérisationdel’actinemaiségalementlarupturedes fibresdestress,décroitl’expressiondeDKK1. A B
IV.
Conclusion
L’activation de la voie de signalisation Wnt dans les cellules tumorales contribue à la progression tumorale (24). Mes résultats soutiennent l’hypothèse selon laquelle la TNC activelavoiedesignalisationWntdansdescellulestumoralesenconduisantàlarépression de DKK1, ce qui pourrait contribuer à l’effet promoteur de la TNC sur la progression tumorale. La TNC conduit à la répression de DKK1 non seulement dans des cellules tumorales mais aussi dans des cellules stromales associées aux tumeurs, telles que des cellulesendothéliales,despéricytesetdesCAF.IlresteàdéterminersilarépressiondeDKK1 dansdescellulesstromalesparlaTNCestégalementassociéeàl’activationdelavoieWnt oud’autresvoies.Deplus,dansuncontextetumoral,oùlaTNCestfortementexprimée,des niveauxfaiblesdeDKK1pourraientcontribueràuneangiogenèseaccrue.
La TNC induit des changements du cytosquelette qui semblent impliqué dans la sousͲ expressiondeDKK1observéeenprésencedeTNC.Mesexpériencesontdémontréquela restauration des fibres de stress par le LPA sur un substrat de FN/TNC, ou par la surexpressiondeTPMͲ1ousyndesmosinduitl’expressiondeDKK1.Deplus,larestauration du niveau d’expression de DKK1 sur FN/TNC est indépendante de la voie RhoA/MKL1. Finalementj’aimontréquedesfacteurs/voiesquidépendentdefibresdestressstables,et probablement pas uniquement du ratio global FͲactine/GͲactine, pourraient être cruciaux pourl’expressiondeDKK1.
RESUMÉDETHÈSEENFRANÇAIS
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Manuscript
andotherscientificcontribution
M
ANUSCRIPT1
PartoftheresultsofthisthesiscontributedtoamanuscriptwhichisacceptedinCellReports. MymajorcontributioncomprisestheanalysisoftheimpactofTNConDKK1regulationand Wntsignallingintumorandstromalcellsinvitro,andelucidationofthemechanismbywhich TNCmediatesDKK1downregulation.
TenascinͲC promotes tumor angiogenesis and progression in a neuroendocrine tumor modelbydownregulationofWntinhibitorDickkopfͲ1.
FalkSaupe*,AnjaSchwenzer*,YundanJia*,IsabelleGasser,CarolineSpenlé,BenoitLanglois, MartialKammerer,OlivierLefebvre,RuslanHlushchuk,TristanRupp,MarijaMarko,Michael van der Heyden, Gérard Cremel, Christiane Arnold, Annick Klein, Patricia SimonͲAssmann, ValentinDjonov,AgnèsNeuvilleͲMéchine,IreneEsposito,JuliaSlottaͲHuspenina,KlausͲPeter Janssen,OlivierdeWever,GerhardChristofori,ThomasHussenetandGertraudOrend (*equalcontribution)
M
ANUSCRIPT2,
INPREPARATIONReducedMKL1targetgeneexpressionbytenascinͲC. AnjaSchwenzer,AnnickKlein,ThomasHussenetandGertraudOrend
C
ONTRIBUTIONTOABOOKIn an exhaustive analysis, I summarized the current knowledge about the role of TNC in angiogenesis,stemcellbiologyandinmetastasisformation.Thisanalysiscontributedtothe book(section2.3,2.4and3): Theextracellularmatrixandcancer:regulationoftumorcellbiologybytenascinͲC. GertraudOrend*,FalkSaupe*,AnjaSchwenzer*andKimMidwood* (*equalcontribution) iConceptPress provisionallyaccepted Partsofthisbookhavebeenusedfortheintroductionofthisthesis(section1.2.3and1.2.4).
TABLEOFCONTENTS
Table
ofContents
1
Introduction _________________________________________________ 1
1.1 Thetumormicroenvironment _________________________________________ 1 1.1.1 Cellularcomponents ... 1 1.1.2 Matrixcomponents... 3 1.2 TNC ______________________________________________________________ 5 1.2.1 RegulationofTNCexpression ... 6 1.2.2 TNCpromotesproliferationandsurvivaloftumorcells... 7 1.2.3 TNCpromotescancercellmigration,invasionandepithelialͲmesenchymal transition(EMT)... 7 1.2.4 RoleofTNCinregulationoftumorangiogenesis ... 9 1.3 Thecellcytoskeleton _______________________________________________ 13 1.3.1 Extracellularsignalsregulatingactindynamics... 13 1.3.2 Actinpolymerisationandstressfibreformation ... 14 1.3.3 Thecytoskeletonasaregulatoroftranscription... 18 1.3.4 Actinbindingdrugs... 20 1.3.5 TNCregulatesactindynamics ... 20 1.4 WntSignalling_____________________________________________________ 23 1.4.1 CanonicalandNonͲcanonicalWntSignalling... 23 1.4.2 InhibitorsofWntsignalling ... 25 1.4.3 RegulationofDKK1expression ... 27 1.4.4 RoleofWnt/EͲcateninsignallingintumorigenesis,angiogenesisand metastasis... 28 1.4.5 RoleofDKK1inangiogenesisandmetastasis... 292
Aims_______________________________________________________ 32
3
Material
andMethods ________________________________________ 33
3.1 Molecularbiologymethods __________________________________________ 33 3.1.1 Transformationofcells ... 33 3.1.2 PlasmidDNApreparation... 33 3.1.3 PCRamplificationofthepromoterfragment ... 33 3.1.4 RestrictiondigestofDNAandcloning... 34 3.2 Cellbiologymethods _______________________________________________ 35 3.2.1 Cultureofcelllines... 35 3.2.2 Collectionofconditionedmedium... 37 3.2.3 Growthfactoranddrugtreatment ... 37 3.2.4 Retroviralparticleproductionandcelltransduction... 37 3.2.5 Lentiviraltransductionofcells ... 38 3.2.6 Transientandstabletransfectionofcells ... 38 3.2.7 Immunofluorescencestaining... 39 3.3 Biochemicalassays _________________________________________________ 39 3.3.1 Luciferasereporterassays... 39 3.3.2 WesternBlotting ... 40 3.3.3 RNAisolationandquantitativeRTͲPCR... 41 3.4 Proteinpurification ________________________________________________ 43 3.4.1 FNproteinpurification... 433.4.3 CoatingwithFNandTNC ... 44 3.5 Heterotopicxenograftmodel_________________________________________ 45 3.6 Statisticalanalysis__________________________________________________ 45
4
Results _____________________________________________________ 46
4.1 TNCinducesWntsignallingintumorcellsbydownregulationofDKK1 _______ 46 4.1.1 TNCdownregulatesDKK1intumorcells... 46 4.1.2 TNCinducesWntsignalling... 50 4.1.3 IsenhancedWntsignallingactivityinthepresenceofTNCmediatedbyTNC inducedDKK1downregulation?... 52 4.2 RegulationofDKK1expressionbyTNCinstromalcells ____________________ 55 4.2.1 TNCdownregulatesDKK1instromalcells ... 55 4.2.2 DoesTNCinduceWntsignallinginstromalcells? ... 57 4.3 DKK1asregulatoroftumorangiogenesis _______________________________ 59 4.4 MechanismofDKK1expressionregulationbyTNC _______________________ 63 4.4.1 TNCregulatesDKK1promoteractivity ... 63 4.4.2 DKK1geneexpressioncanbeinducedbystabilizationofstressfibres ... 64 4.4.3 RhoAaloneisnotimplicatedinDKK1downregulationonaFN/TNC substratum ... 69 4.4.4 TNCreducesSRF/MKL1activity ... 71 4.4.5 RegulationofDKK1expressionbyMKL1 ... 74 4.4.6 RegulationofDKK1expressionbyactinbindingdrugs... 755
Summary ___________________________________________________ 80
6
Discussion
andPerspectives____________________________________ 82
6.1 ConsequencesofdownregulationofDKK1byTNCintumorandstromalcells _ 82 6.1.1 TNCenhancesWntsignallingintumorcells... 82 6.1.2 RoleofTNConDKK1expressionandWntsignallinginpericytesandCAFs.... 85 6.1.3 PotentialImpactofTNContumorangiogenesisthroughDKK1... 87 6.2 RegulationofDKK1geneexpressionbytheactincytoskeleton _____________ 93 6.2.1 InductionofDKK1geneexpressionbystressfibres ... 93 6.2.2 IsDKK1geneexpressionregulatedbyRhoGTPasesactivity?... 95 6.2.3 TranscriptionsfactorspotentiallyinvolvedintheregulationofDKK1gene expressionbystressfibres ... 977
References_________________________________________________ 102
8
Annex_____________________________________________________ 112
LISTOFFIGURES
List
ofFigures
Figure 1. Thecellsofthetumormicroenvironment.
Figure 2. RoleoftheECMinthetumormicroenvironment.
Figure 3. DomainstructureofTNC.
Figure 4. Aselectionofproteinsbindingactinandtheirfunction.
Figure 5. RegulationofactindynamicsbyRhoGTPases.
Figure 6. Regulationofstressfibrecontractility.
Figure 7. RegulationofactindepolymerisationbyTNC.
Figure 8. Wnt/EͲcateninsignallingandEͲcateninͲindependentWntsignalling.
Figure 9. MechanismofWntsignallinginhibition.
Figure 10. TNCreducesDKK1geneandproteinexpressionintumorcellsinvitro.
Figure 11. TNCcopynumbernegativelycorrelateswithDkk1geneexpressioninvivointhe RipTag2(RT2)tumormodel.
Figure 12. TNCenhancesTOPFlashactivityandAxin2expressioninWnt3AtreatedKRIBcells andenhancesAxin2expressionintumorsofRT2/TNCmice.
Figure 13. EctopicexpressionofmDKK1reducesWntsignallingactivitywhileknockdownof DKK1enhancesWntsignallinginKRIBcells.
Figure 14. RegulationofTNCmediatedWntsignallingactivitybyDKK1.
Figure 15 TNCdownregulatesDKK1inprimaryendothelialcells(HUVEC),pericytesand
CRCͲderivedCAF.
Figure 16. TNCinducesAxin2expressioninHUVEC,butnotinpericytes.
Figure 17. TNCenhancesthenumberofangiogenicisletsintheRT2modeloftumor
progressionandincreasestheamountofCD31positiveendothelialcells.
Figure 18. EctopicexpressionofmDKK1inKRIBinhibitstumorangiogenesisinaxenograft
model.
Figure 19. EstablishmentofHEK293andKRIBcellsoverexpressingTNCandmDKK1.
Figure 20. TNCreducesDKK1PromoteractivityinT98Gcells.
Figure 21. LPAinducesstressfibreformationandDKK1geneexpression.
Figure 22. Ectopicexpressionofchicken(ch)SyndesmosinT98GenhancesDKK1gene
expression.
Figure 23. Tropomyosin1(TPM1)enhancesDKK1expressioninT98Gcells.
Figure 24. LPArestorescellspreadingandDKK1expressiononFN/TNCinT98Gcells.
Figure 25. RhoAsignallingdoesnotinduceDKK1geneexpressionanddoesnotrestorecell spreadingandDKK1geneexpressiononFN/TNCinT98Gcells.
Figure 26. TNCregulatestranscriptionalactivityofSRF/MKL1.
Figure 27. ShͲmediatedMKL1andMKL2knockdowninT98Gcells.
Figure 28. StableknockdownofMKL1inT98GcellsreducesDKK1geneexpression,protein levelsandpromoteractivity.
Figure 29. MKL1overexpressiondoesnotrestoreDKK1expressiononaFN/TNC
substratum.
Figure 30. EffectofLatrunculinBandCytochalasinDonactinpolymerisationandSRF/MKL1
targetgeneexpression.
Figure 31. RegulationofDKK1expressionbyFͲactindepolymerisingdrugs:CytochalasinD inducesDKK1expressionatlowandinhibitsDKK1expressionathigh
concentration,whileLatrunculinBreducesDKK1expression.
Figure 32. TreatmentofT98GcellswithJasplakinolidereducesDKK1geneexpressionwhile itenhancesSRFactivity.
Figure 33. SummaryofthemajorresultsontheconsequencesandmechanismofDKK1
downregulationbyTNC.
Figure 34. Summaryandnewworkingmodel–ConsequencesofDKK1downregulationon
Abbreviations
ADF actindepolymerisationfactor APC adenomatouspolyposiscoli APS ammoniumperoxodisulfate ARP2/3 actinrelatedprotein aSMA alphaͲsmoothmuscleactin BAEC bovineaorticendothelialcells bFGF basicfibroblastgrowthfactor CA constitutivelyactive
CAF cancerͲassociatedfibroblasts
CAMK calcium/calmodulinͲdependentproteinkinase CD clusterofdifferentiation
cdcͲHMEC humandermalmicrovascularendothelialcells CDM cellͲderivedmatrix cDNA complementaryDNA CM conditionedmedium CNS centralnervoussystem CRC colorectalcancer CTGF connectivetissuegrowthfactor CTR control DKK dickkopf Dll4 deltalikeligand4 DMSO dimethylsulfoxide DN dominantnegative DNA desoxyribonucleicacid DvlorDsh dishevelled
ECM extracellularmatrix
EDNRA/B endothelinreceptortypeA/B EGF epidermalgrowthfactor
EMT epithelialͲmesenchymaltransition ERK extracellularsignalregulatedkinase ETͲ1 endothelin1
FͲactin filamentousactin FAK focaladhesionkinase FCS foetalcalfserum FN fibronectin FzorFzd frizzled GͲactin globularactin
GBM glioblastomamultiforme
GEF guaninenucleotideexchangefactor GPCR GͲproteincoupledreceptor
GSK3 glycogensynthasekinase3 h hours
HEK humanembryonickidney HepII heparinII
His histidine
HMVEC humandermalmicrovascularendothelialcells HSPG heparinsulfateproteoglycans
HUVEC humanumbilicalveinendothelialcells IF immunofluorescence
IHC immunohistochemistry ILK integrinlinkedkinase
JMY junctionmediatingandregulatoryprotein JNK cͲjunnͲterminalkinase
ABBREVIATIONS kb kilobase kd knockdown KO knockout LB luriabroth LIMK LIMdomainkinase LPA lysophosphatidicacid
LRP5/6 lipoproteinreceptorͲrelatedprotein MAL megakaryocyticacuteleukaemia MAPK mitogenͲactivatedproteinkinase MEF mouseembryonicfibroblast miR microRNA
MKL1 megakaryoblasticleukaemia1 MLC myosinlightchain
MLCK MLCkinase
MMP matrixmetalloproteinase
MRCK myotonicdystrophykinaseͲrelatedCdc42Ͳbindingkinase mRNA messengerRNA
MRTF myocardinͲrelatedtranscriptionfactor NFʃB nuclearfactorkappaB OD opticaldensity PBS phosphateͲbufferedsaline PCR polymerasechainreaction PDGFͲBB plateletͲderivedgrowthfactor PKC proteinkinaseC POSTN periostin qRTͲPCR quantitativerealͲtimePCR REF52 ratembryonicfibroblasts RNA ribonucleicacid
ROCK rhoͲkinase RT2 rip1Ͳtag2
RTK receptortyrosinekinase RTͲPCR reverseͲtranscribedPCR SDS sodiumdodecylsulfate
sFRP secretedfrizzledrelatedprotein shRNA shorthairpinRNA
SRF serumresponsefactor
TAZ transcriptionalcoͲactivatorwithPDZͲbindingmotif TCF ternarycomplexfactors
TCF/LEF TͲcellfactor/lymphoidenhancerfactor TEAD TEAdomainfamilymember
TGFE transforminggrowthfactorbeta TNC tenascinͲC
TNIII fibronectintypeIIIͲlikerepeatsintenascinͲC TPM tropomyosin
VEGFA vascularendothelialgrowthfactorA VEGFR2 VEGFAreceptor
vWF vonWillebrandfactor WHO worldhealthorganisation WIF1 wntinhibitoryfactor1 wt wildtype
YAP yesͲassociatedprotein
1 Introduction
A tumor arises from cells that transform progressively into malignant cancerous cells by activationofoncogenesorlossoffunctionoftumorsuppressorgenes.Duringthisprocess thesecellsacquirehallmarkcapabilitiesasdefinedbyHanahanandWeinberg(Hanahanand Weinberg, 2000; Hanahan and Weinberg, 2011) allowing them to sustain proliferative signalling,evadegrowthsuppressors,resistcelldeath,enablereplicativeimmortality,induce angiogenesisandactivateinvasionandmetastasis.Atumorcanbeseenasacomplexand heterogeneous organ, composed of both cancer and stromal cells intermingled in a 3Ͳ dimensional extracellular matrix (ECM) that regulate cellular fates and reciprocal interactions.BothstromalcellsandthetumorECMcontributetothehallmarksofcancer,as willbediscussedbelow.
1.1 The
tumormicroenvironment
Thetumormicroenvironmentcomprisesstromalcells,secretedECMmolecules,andsoluble signallingmoleculessuchascytokines,growthfactorsand matrixremodellingenzymesas well as blood and lymphatic vessels, nerves and inflammatory cells. The tumor microenvironmenthasmanycharacteristicsnotfoundinnormaltissue,includingenhanced ECM deposition, increased numberof fibroblasts and enhanced capillary density. Multiple crosstalks exist between the tumor cells, stromal cells and the ECM, which fuel tumor progressionbysupportingtumorgrowth,angiogenesis,invasionandmetastasis(Kalluriand Zeisberg,2006;HanahanandWeinberg,2011).InthefollowingchapterIwilldetailwhich stromal cells are encompassed in the tumor microenvironment (Figure 1) as well as describingtheimportanceofthematricellularcomponentsfortumorprogression.
1.1.1 Cellularcomponents
Endothelial cells (EC) line the interior surface of the tumorͲassociated vasculature. The growth of a tumor strongly depends on its supply of nutrients and oxygen. Along tumor progression, the angiogenic switch is considered a crucial and early event (Hanahan and Weinberg,2011).Theangiogenicswitchischaracterizedbysprouting,newvesselformation, vessel maturation, and the recruitment of perivascular cells (Bergers and Benjamin, 2003;
INTRODUCTION
HanahanandWeinberg,2011).Thevasculatureofatumoroftendiffersfromthevasculature in healthy tissues and is characterized by abnormal vessels with a disorganized basement membrane,incompletepericytecoverageandleakiness(DeBocketal.,2011).
Pericytesareaspecializedmesenchymalcelltypethatwraparoundtheendothelialtubing of blood vessels. Pericytes and vascular endothelial cells are anchored to the vascular basement membrane, which is produced by both cell types. In tumors, pericytes are commonlylooselyassociatedwiththevasculature.Ithasbeendemonstratedthatpericytes arecrucialforthefunctionofthetumorvasculature.Severalstudiessuggesthoweverthata reduced coverage with pericytes might favour cancer cell dissemination (Hanahan and Weinberg,2011;PietrasandOstman,2010).
TumorͲpromoting immune inflammatory cells as macrophages, mast cells, neutrophils, T andBlymphocyteshavebeenshowntosecretetumorͲpromotingmoleculesaswellasto induceandpromotecancercellproliferation,invasion,metastasisandtumorangiogenesis (HanahanandWeinberg,2011).
CancerͲassociated fibroblasts (CAFs) are a major cell population in the stroma of most tumors.Onedistinguishesatleasttwodifferentcelltypes:(1)cellsthatexhibitsimilaritiesto fibroblastsfoundinnormalepithelialtissue(tissueͲderived)(2)andmyofibroblaststhathave distinctpropertiesandbiologicalrolesthantissueͲderivedfibroblasts(boneͲmarrowderived frommesenchymalstemcells).Thosecellsarealsooftentermed“activated”fibroblastsas they secrete more ECM, proliferate more than their nonͲactivated counterparts and have features more typical of smooth muscle cells. They are mainly characterized by the expressionofDͲsmoothmuscleactin(DͲSMA),amarkeroftheiractivation.CAFshavebeen shown to promote cancer cell proliferation, invasion, metastasis and tumor angiogenesis (KalluriandZeisberg,2006;HanahanandWeinberg,2011).
Figure 1. The cells of the tumor microenvironment (Hanahan and Weinberg, 2011). A solid tumor is
constitutedofdistinctcelltypesthatcontributetotumorgrowthandprogression.ECslinetheinnersideof bloodvesselsenablingthesupplyofthetumorwithnutrientsandoxygen.Pericyteswraparoundbloodvessels andplayanimportantroleinstabilizingbloodvessels;howevertheyarealsoconsideredtolimitdissemination of cancer cells. In addition, CAFs have been demonstrated to favour cancer cell proliferation, invasion and metastasis as well as angiogenesis. Immune inflammatory cells can both promote or inhibit tumor growth dependingontheirsubclass.
1.1.2 Matrixcomponents
The ECM is a complex meshwork of highly crossͲlinked fibrous proteins. The “core matrisome” as defined by Naba et al. (Naba et al., 2011) is composed of collagens, ECM glycoproteins and proteoglycans, encoded by approximately 300 different genes. The completematrisomefurthercomprisesECMͲaffiliatedproteins,ECMregulatorsandsecreted factors(Nabaetal.,2011;approximately1100genesintotal).DifferentformsoftheECM exist.The interstitial matrix contains collagens, proteoglycans and glycoproteins such as tenascinͲC (TNC), periostin (POSTN) and fibronectin (FN) and is characterized by an unorganized,looseassemblyofthesecomponents;itcontributestothetensilestrengthof tissues(Luetal.,2012).ThebasementmembranepresentsaspecializedformoftheECMat the basoͲlateral side of cells, which separates an epithelium or an endothelium from the adjacenttissue.ItisamorecompactECMformandlessporousthaninterstitialmatrix,and contains two networks of type IV collagen and laminins that are interconnected by moleculesofnidogenandperlecan(Luetal.,2012).Ascomparedtoahealthytissue,the ECM within a tumor is markedly different in terms of composition, abundance of certain componentsandstructure.
INTRODUCTION
TheECMexecutesdifferentfunctions(Figure2).Firstofall,theECMservesasastructural support for tissue architecture and integrity. Depending on the ECM structure and composition,cellscanusetheECMasananchoragesite,astracksfortheirmigration,but theECMcanalsofunctionasmigrationbarrier.TheECMprovidesmechanicalcues,ascells can directly sense the mechanical properties, e.g. stiffness, of the surrounding ECM. In addition to ECM binding to integrin cell adhesion receptors, the ECM has been shown to modulategrowthfactorsignalling.DifferentECMproteinscanbindgrowthfactorsandmay actasreservoirsforgrowthfactorsoraidinestablishingastablegradientofgrowthfactors; thosegrowthfactorscanlaterbereleaseduponECMdegradation.Inaddition,theECMcan function as coͲreceptor for growth factor binding or even intrinsic domains within ECM proteinsmightfunctionasligandsforreceptors,ashasbeenshownforEGFͲlikerepeatsof lamininsandTNC(reviewedinHynes,2009;Luetal.,2012). InsummarytheECMcanthereforetightlyregulatecellbehaviourbynumerousmechanisms andisactivelyinvolvedinpromotingtumorgrowth,angiogenesis,invasionandmetastasis. Figure2.RoleoftheECMinthetumormicroenvironment(Luetal.,2012).CellscananchortotheECM(e.g.
the basement membrane) through cell surface receptors (1). For migrating cells the ECM can function as a barrier(2)orfacilitatemigrationbyservingasmigrationtrack(3).ECMcomponentshavebeendemonstrated tobindgrowthfactorsthereforeactingasareservoirforgrowthfactors(4)orenablingtheestablishmentofa growth factorgradient. In addition, theECMcan function ascoͲreceptor for growth factorbinding(5)or in presentinggrowthfactors(6).IntrinsicdomainsoftheECMcanbinditselftocellsurfacereceptors(7).Cells sensethebiomechanicalproperties,includingstiffness,ofthesurroundingECMandreactbychangingactin stressfibrecontractilitythatresultsinalteredtargetgeneexpression(8).
1.2 TNC
TNCisasecreted190Ͳ300kDaglycosylatedprotein(Figure3)andthefoundingmemberof thetenascinproteinfamily,includingTNͲX,TNͲWandTNͲR.TheNͲterminalstretchof110 residuesisuniquetothetenascinproteinfamilyandisfollowedbyashortheptadrepeat region and 14.5 EGFͲlike repeat domains. TNC contains up to 17 FN type III like domains (constitutive and alternatively spliced) and a cͲterminal fibrinogen like globular domain (Figure3A).TNCmonomersareassembledintoahexamericform(Figure3B)(Orendetal., 2013).
TNCisexpressedduringdevelopmentbutnearlyabsentinadulttissue.However,TNCgets reͲexpressed under pathological conditions, such as wound healing, inflammation, fibrosis andcancer.Especiallyinsolidtumorsofthebreast,brainandcolonahighTNCexpression has been reported. TNC expression correlates with low survival rates and promotes lung metastagenicityofbreastcancercells(Orendetal.,2013).
In the following chapter I will summarize what is known about the regulation of TNC expression in tumors and which effects TNC has been shown to exert on proliferation, migration, invasion and angiogenesis. Furthermore, TNC has been described to be an adhesionͲmodulatingECMmolecule,studiesdescribingthepossibleunderlyingmechanisms Iwillsummarizeinchapter1.3.5. Figure3.DomainstructureofTNC.(A)OrganizationofTNCintodifferentproteindomains.(B)Theappearance ofpurifiedTNCproteinasahexameruponelectronmicroscopy(Orendetal.,2013;VanObberghenͲSchillinget al.,2011). A B
INTRODUCTION 1.2.1 RegulationofTNCexpression
Depending on the tumor type and stage TNC can be expressed by either the tumor cells, stromal cells or both cell types, which has been addressed in several studies by immunohistochemicalanalysisandinsituhybridization.
Inepithelialtumorssuchasfromthebreastandcolon,TNCismainlyexpressedbyCAFsand formsafibrousnetworkthatenclosesunstainedtumornests(Degenetal.,2007;Degenet
al., 2008; ChiquetͲEhrismann and Tucker, 2011). In contrast, in glioblastomas and
melanomasthecancercellsthemselvessecreteTNC(Natalietal.,1990;Herlynetal.,1991; Sivasankaranetal.,2009).Incolorectaladenomaandcarcinomas(Hanamuraetal.,1997)as well as fibroadenomatosus tumors (Lightner et al., 1989) and breast ductal carcinoma (Yoshida et al., 1997) TNC is expressed both by stromal and cancer cells. In gliomas TNC stainingoverlapswithstainingfordesminͲpositivecells,indicatingthatratherpericytesthan endothelialcellsareasourceforTNCinthesetumors(Martinaetal.,2010).Asanalysedbya proteomic approach from human nonͲmetastatic and metastatic melanoma cell lines xenografted in nude mice, TNC was both expressed by tumor and stromal cells in these tumors;however,inmetastaticmelanomacellstherewasahighercontributionbystromal cells(Nabaetal.,2011).
Oskarssonetal.(2011)demonstratedthatbreastcancercellͲderivedTNCisimportantfor breastcancerlungmetastasisoutgrowth,butlateronthestromalcompartmenttakesover asasourceofTNC(Oskarssonetal.,2011).O’Connelletal.showedthatTNCderivedfrom S100A4Ͳpositive cells promotes metastatic colonization of breast cancer cells in the lung (O’Connelletal.,2011).
TNCexpressioncanbeinducedbymechanicalstretch,whichdependsontheactivationof theRhoA/MKL1pathwayandTNChasbeen identifiedasadirecttranscriptionaltargetof MKL1(Asparuhovaetal.,2011).Furthermore,ithasbeenshownthatTNCisupregulatedby hypoxiaorinthepresenceofreactiveoxygenspecies(OrendandChiquetͲEhrismann,2006). TNCexpressioncanbeinducedbyTGFE,andothergrowthfactorssuchasEGF,bFGF,PDGFͲ BB and CTGF, which are mostly secreted by stromal cells (Orend and ChiquetͲEhrismann, 2006).
2009) are involved in regulation of TNC expression. A number of regulatory transcription factorbindingsiteshavebeenidentifiedandarefunctionalintheTNCpromoter,includingcͲ jun, NFkB, sox4, Prx1 (paired related homeobox 1) and Smad2/3. In contrast, GATA6 has been identified as a transcriptional repressor of TNC gene expression (reviewed in Tucker andChiquetͲEhrismann,2009;ChiquetͲEhrismannandTucker,2011).Furthermore,miRͲ335 canregulateTNCmRNA(Tavazoieetal.,2008).
1.2.2 TNCpromotesproliferationandsurvivaloftumorcells
ChiquetͲEhrismann et al. (1986) demonstrated that TNC increases proliferation in primary mammary tumor cellsafter serum depletion. In T98G and MDAMBͲ435 cells TNC induced proliferationbyinhibitionofintegrinD5E1/SyndecanͲ4Ͳdependentcelladhesion(Huanget
al., 2001). However, TNC inhibited DNA replication in normal human fibroblasts (MRCͲ5),
immortalratembryonicfibroblasts(REF52)andswiss3T3fibroblasts(NIH3T3)(Orendetal., 2003).
WhengrowninreconstitutedbasementmembraneMCFͲ10AcellsformthreeͲdimensional polarized, growthͲattenuated, multicellular acini, enveloped by a basement membrane. AdditionofexogenousTNCinthissettingpromotedcellproliferationandluminalfillingby increasingtheexpressionanddownstreamsignallingofcͲmet(Taraseviciuteetal.,2010). In breast cancerͲderived lung metastasis TNC did not affect proliferation but promoted survival by decreasing apoptosis of metastatic breast tumor cells (Oskarsson et al., 2011). Also upon intravenous injection of 4T1 cells into mice, the metastatic burden (area) was significantly reduced in TNC knockout (TNC KO) mice compared with control mice, supportingthenotionthatTNCexpressedbystromalcellsplaysaroleinstimulatingsurvival ofmetastasizingtumorcellsorprotectingthemfromapoptosis(O’Connelletal.,2011). 1.2.3 TNCpromotescancercellmigration,invasionandepithelialͲmesenchymal transition(EMT) IthasbeendemonstratedthatTNCincreasesmigrationandinvasionusingseveralinvitro tumor cellular models (breast, colon cancer, glioma, chondrosarcoma, squamous cell carcinoma)(forsummaryseetable7inOrendetal.,2013).
INTRODUCTION
Tumors derived from GBM cells knocked down for TNC and implanted in the striatum of nude mice consisted of less infiltrating tumor cells and less tumor cell clusters in the surrounding brain tissue, despite the fact that no difference in tumor growth and proliferationwereobserved(Hirataetal.,2009).
TNCalsoappearstoplayaroleintheintricatecrossͲtalkbetweenmyofibroblastsandtumor cells. Human primary myofibroblasts derived from colorectal tumor tissue stimulated the invasivebehaviourofcolorectalcancer(CRC)cellsinaTNCͲdependentmanner(DeWeveret
al., 2004). CRC cells coͲcultured with myofibroblasts rapidly invaded a collagen gel, which
could be blocked by treatment with an antibody directed against the TNC EGFR repeats, suggestingthatdepositionofTNCbythemyofibroblastsinthegeldrivestheinvasionofthe cancer cells. The authors linked this TNCͲstimulated proͲinvasive behaviour of carcinoma cellstothedownͲregulationofRhoAsignalling(DeWeveretal.,2004).SimilarlyGaggioliand colleagues (2007) observed that squamous carcinoma cells invade an organotypic matrix uponcoͲculturewithstromalfibroblaststhatwerederivedfromoralorvulvalsquamouscell carcinoma. Stromal fibroblasts invaded and strongly modified the matrix by promoting degradationanddepositionofmatrixincludingTNCandFN.Thecollagenmatrixwithinthese tracks was organized into thick bundles (Gaggioli et al., 2007). The authors demonstrated that those tracks promoted carcinoma cell invasion. However, TNC and FN seemed not necessary for tumor cell invasion, as a either single or combined knockdown of these moleculesinfibroblastsdidnotchangethenumberofinvadingcarcinomacells(Gaggioliet
al.,2007).
An overͲrepresentation of specific TNC domains mostly encompassing the alternatively splicedTNIIIrepeatsbutnottheEGFͲlikerepeatshadbeendemonstratedinseveralcancers. InparticularinbreastcancertheexpressionofTNCisoforms,containingTNIIID,TNIIIBDor TNIII BAD1D domains, were associated with increased tumor cell invasion (Adams et al., 2002; Gutteryetal.,2010;Hancoxetal.,2009).Theexpositionofcertaindomainswithin TNC could arise from cleavage of the molecule by matrix metalloproteinases (MMPs) and otherproteases.CleavageofTNCbymeprinͲbeta1wasshowntoabrogatethesyndecanͲ4 inhibitoryactivityofTNConamixedFN/TNCsubstratum(Ambortetal.,2010).
TNCitselfmightalsoincreasetheexpressionandactivityofMMPsstimulatingtheinvasive behaviourofcancercells.Ilungaetal.(2004)showedthatMDAMB231invasionintomatrigel
wasstimulatedbytheadditionofbothTNCandTGFͲE1,whichcouldbeblockedbyaMMPͲ inhibitor(Ilungaetal.,2004).Similarly,TNCalsostimulatedtheinvasionofglioblastomacells byincreasingproteinkinaseCGactivityandMMP12expression.Migrationwasrepressedby MMPͲinhibitors or an antibody specific for MMP12 (Sarkar et al., 2006; Sarkar and Yong, 2010).
In breast carcinoma TNC expression correlates with the expression of the mesenchymal marker vimentin and, in several cancer cell lines TNC and vimentin are found to be coͲ expressed(Dandachietal.,2001).Moreover,TNCishighlyexpressedattheinvasivefrontof colorectaltumorsatsiteswithnuclearEͲcateninintumorcells(Beiteretal.,2005).Nuclear EͲcateninhasbeendemonstratedtobeanEMTmarker(Kimetal.,2002).KnockͲdownofEͲ catenininCRCcelllinesindeedresultsinreducedTNCexpressionandtheauthorsprovided evidencesthatEͲcateninisdirectlyregulatingtheactivityoftheTNCpromoter(Beiteretal., 2005).Nagaharuandcolleagues(2011)demonstratedthatuponacombinedtreatmentwith TNC and TGFEͲ1 MCF7 and T47ͲD breast cancer cells acquire an EMTͲlike phenotype, characterized by loss of membranous EͲcadherin and EͲcatenin, which was linked to increasedcellmigration(Nagaharuetal.,2011).Furthermore,TNCseemedtobeinvolvedin regulatingtheEMTprocessinthemouselenseepitheliumuponinjury(Tanakaetal.,2010). InanotherstudyTNCpromotedapartialEMTinMCF7cellsthatchangedtheircobblestone epithelial morphology into a fibroblastoid phenotype upon growth on a TNC substratum. This was linked to an altered expression of the adaptor protein 14.3.3tau (Martin et al., 2003). These observations suggest that TNC promotes EMT but may also be regulated by EMT,asitwasshowntobeaEͲcatenintargetgene.ThushighlyexpressedTNCatthetumor invasionfrontmayenhancecancercellmigrationandinvasion.
1.2.4 RoleofTNCinregulationoftumorangiogenesis 1.2.4.1 TNCasamarkerfortumorigenicbloodvessels
In several studies TNC has been found to be preferentially localized around tumor blood vessels.Analysisof86gliomas,whereTNCexpressionincreasedalong tumorprogression, revealedthatTNCwasstronglyexpressedaroundtumorbloodvesselsingliomasofWHO grade IV (glioblastoma multiforme Ͳ GBM). Although this was different in grade II and III gliomas with an overall reduced frequency of TNC lined blood vessels, perivascular TNC
INTRODUCTION
staining significantly correlated with a shorter diseaseͲfree time in these grade II and III glioma (HeroldͲMende et al., 2002). Behrem et al. (2005) observed that GBM with strong perivascular TNC staining contained more newly formed blood vessels than tumors with moderateorweakTNCexpression,asassessedbystainingofCD105positivemicrovessels (Behrem et al., 2005). CD105 is a cell membrane glycoprotein overexpressed on tumorͲ associatedvascularendothelium(Fonsattietal.,2003).Furthermore,intissuesamplesfrom 63 patients with nonͲsmall cell lung cancer, Ishiwata et al. (Ishiwata et al., 2005) found a correlation between TNC concentration in the serum and intraͲtumoral vessel density. In juvenilenasopharyngealangiofibromaTNCexpressionwasalsofoundaroundbloodvessels and its expression correlated with vessel density, tumor stage and endothelial cͲkit expression(Renkonenetal.,2012).
Berndt et al. (2010) showed that in CD31Ͳpositive blood vessels of clear cell renal cell carcinomaandatypicalcarcinoidsofthelungTNCislocalizedontheextraͲluminalsideof the basement membrane (Berndt et al., 2010). By in situ hybridization TNC mRNA was detected in hyperplastic capillaries of astrocytoma tumor tissues. Staining was observed liningthevascularlumen,indicatingthepresenceofTNCinendothelialcells.Butothercells could also be a source of TNC, as additional staining was observed in the walls of the vascularstructures.ImmunostainingconfirmedTNCexpressionwithinandaroundthewalls of hyperplastic blood vessels and staining was also detected adjacent to vascular sprouts (Zagzagetal.,1996).Indeed,Martinaetal.(2010)showedthatTNCisexpressedbypericytes inGBMbutnotbyendothelialcells(Martinaetal.,2010).
Recently three studies using proteomic approaches have identified TNC as a marker preferentiallyexpressedinthevasculatureoftumorsoratthemetastaticsite.Borgiaetal. (2009)usedinvivoperfusionofbiotinforlabellingofvascularproteins.FourdifferentTNC isoforms(containingTNIIIdomainsA1,A2,A4,B)havebeenidentifiedasmoleculeswhich wereexpressedinthevasculatureoflivermetastasesinasyngeneicheterotopicmodelof colon cancer (Borgia et al., 2009). Hill et al. (2011) used laser capture microͲdissection of microvessels of invasive ductal carcinoma and identified TNC as one of the proteins overexpressedintumorvesselsincomparisontovesselsfromadjacenthealthytissue(Hillet
al.,2011).Similarly,byusinglasercapturemicroͲdissectionandproteinexpressionprofiling
Insummary,thesedatashowthatinanumberoftumortypesTNCspecificallymarkstumor bloodvessels.TNCexpressionoftencorrelateswithhighertumorstageandincreasedblood vesseldensitywhicharguesforaroleofTNCinthetumorvasculature.
1.2.4.2 TNCimpactsonendothelialcellbehaviourinvitro
TNC supported the switch from a nonͲangiogenic (resting) cobblestone phenotype to an angiogenic sprouting cordͲforming phenotype in bovine aortic endothelial cells (BAEC) (CanfieldandSchor,1995).Schenketal.(1999)showedthatTNCisexclusivelyexpressedin the sprouts and cords of the sprouting but not in the resting BAEC. For the induction of sprouting by TNC the basic fibroblast growth factor (bFGF) seems to be required. Furthermore,theauthorsshowedthatthefibrinogenglobedomainofTNCwasresponsible fortheBAECsprouting(Schenketal.,1999).TheauthorsarguedthatthesproutͲsupporting effectofTNCmightbeexplainedwiththeantiͲadhesiveeffectofTNC. Moreover,TNCincreasessproutingofHUVECspheroidsembeddedincollagengels(Martina etal.,2010).WhenseededonaTNCͲcoatedbasementmembraneorwhenTNCwasadded totheculturemediumendothelialcellshadenhancedtubeformationability(Castellonetal., 2002). AnantiͲadhesiveeffectofTNConendothelialcellsofdifferentoriginswasshown(Ballardet
al., 2006; Sriramarao et al., 1993), however, others reported that TNC significantly
stimulatedendothelialcelladhesion(Delaneyetal.,2006;Zagzagetal.,2002).
TheantiͲadhesiveTNCeffectreportedhasbeenlinkedtoareductioninfocaladhesionsin endothelial cells (MurphyͲUllrich et al., 1991). These authors showed that the TNIII AͲD domain mediates the antiͲadhesive effect, which can be reversed by blocking cell surface annexinII,aTNCreceptor(Chungetal.,1996).
DespiteitsantiͲadhesiveandantiͲspreadingeffectearlyuponcellplating,mostendothelial cellseventuallydoattachandspreadonTNCaftercultureforlongerperiodsoftime.This cellattachmentcanbeblockedwithanRGDpeptide.TheRGDpeptideisthebindingmotif of integrin ligands and therefore inhibits integrinͲligand interaction. Therefore cell attachmenttoTNCmightbeintegrinͲdependent(BourdonandRuoslahti,1989).Endothelial cellattachmentandspreadingonTNCismediatedbydifferentTNCcellsurfacereceptors
INTRODUCTION
including annexin II (Chung and Erickson, 1994),D2E1andDvE3integrins(Delaneyetal., 2006; Joshi et al., 1993; Sriramarao and Bourdon, 1993). But in longer term assays endothelial cells secrete other ECM molecules such as FN, and adhesion to FN would be blocked with RGD peptides. Thus the described observation may be due to inhibiting adhesion to FN with the RGD peptide rather than blocking the interaction with TNC. Altogether these studies show that through different cell surface receptors adhesion and spreading of endothelial cells can be modulated by TNC, which could be crucial in tumor angiogenesis.
TNCwasshowntoenhanceendothelialcellproliferation(Castellonetal.,2002;Chungetal., 1996;Delaneyetal.,2006)andmigration(Ballardetal.,2006;Castellonetal.,2002;Chung
et al., 1996; Ishiwata et al., 2005; Martina et al., 2010; Zagzag et al., 2002). This was
demonstratedusingdifferentinvitroassaysandwithendothelialcellsofdifferentorigins. 1.2.4.3 TNCsupportsangiogenesis SeveralstudiesprovideevidenceforacorrelationbetweenTNCexpressionandexpression ofthevascularendothelialgrowthfactorA(VEGFA).TheinitialstudybyTanakaetal.(2004) suggeststhatTNCsupportsmelanomaangiogenesisbyregulatingtheexpressionofVEGFA. Afterinjectionofmelanomacellsinimmunecompromisedwildtype(wt)andTNCknockout (KO)micetumorsinKOmiceweresmallerandwerelessvascularized.MeasuringtheVEGFA contentofthetumorsbyELISAtheauthorsobservedalowerVEGFAcontentintumorsfrom theKOmicethanthosegrowninwtmice.AlsoincoͲcultureswithmelanomacellsandthe mesenchymederivedfromeitherwtorTNCKOmicetheauthorsmeasuredahigherVEGFA levelwhenthemiceexpressedTNC(Tanakaetal.,2004).Otherstudiessupporttheauthor’s observationthatTNCexpressioncorrelateswithVEGFAlevelsase.g.inGBM(Behremetal., 2005).InserafromnonͲsmallcelllungcancerpatientsTNClevelscorrelatedwiththeVEGFA levels(Ishiwataetal.,2005).MoreoverSumiokaetal.(2011)showedthatoccularfibroblasts derivedfromTNCKOmiceexpressedlessVEGFAthanwtfibroblastswhichwasassociated withlessneovascularisationinTNCKOmiceinthecornealstroma(Sumiokaetal.,2011).But howTNCimpactsontheexpressionofVEGFAisunknown.
