RU486 and FGF2 in mammaRy caRcinoGenesis 529

SHORT COMMUNICATION MEDICINA (Buenos Aires) 2010; 70: 529-532

ISSN 0025-7680

MIfEprIstoNE INhIBIts MpA- AND fGf2-INDuCED MAMMAry tuMor Growth But NotfGf2-INDuCED MAMMAry hypErplAsIA

JUAN P. CeRlIANI1, SebASTIAN GIUlIANellI1, ANA SAHOReS1, VICTORIA WARGON, ADRIAN GONGORA1, AlbeRTO bAlDI1, AlfReDO MOlINOlO2, CAROlINe A. lAMb1, ClAUDIA lANARI1

1Laboratorio de carcinogénesis Hormonal, instituto de Biología y medicina experimental (iByme)- consejo nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina,

2oral and Pharyngeal cancer Branch, nidcR, niH, Bethesda

Abstract Wehavepreviouslydemonstratedacrosstalkbetweenfibroblastgrowth factor2(FGF2)andpro- gestinsinducingexperimentalbreastcancergrowth.TheaimofthepresentstudywastocomparetheeffectsofFGF2andofmedroxyprogesteroneacetate(MPA)onthemousemammaryglandsandtoinvesti-gatewhethertheantiprogestinRU486wasabletoreversetheMPA-orFGF2-inducedeffectsonboth,mammaryglandandtumorgrowth.WedemonstratethatFGF2administeredlocallyinducedanintraductalhyperplasiathatwasnotrevertedbyRU486,suggestingthatFGF2-inducedeffectsareprogesteronereceptor(PR)-independent.However,MPA-inducedparaductalhyperplasiawasrevertedbyRU486andapartialagonisticeffectwasobservedinRU486-treatedglands.UsingC4-HDtumorswhichonlygrowinthepresenceofMPA,weshowedthatFGF2administered intratumorallywasabletostimulatetumorgrowthasMPA.ThehistologyofFGF2-treatedtumorsshoweddifferent degreesof glanddifferentiation.RU486 inhibitedboth,MPAorFGF2 induced tumor growth.However, only complete regressionwas observed inMPA-treated tumors.Our results support the hypothesisthatstromalFGF2activatesPRinducinghormoneindependenttumorgrowth.

Key words:breastcancer,mammarygland,mammarycarcinomas,FGF2,progestins,RU486

Resumen La mifepristona inhibe el crecimiento de carcinomas mamarios inducidos por MPA o por FGF2 pero no las hiperplasias mamarias inducidas por FGF2. hemos demostrado previamente quelavíadeseñalizacióndelfactordecrecimientofibroblástico2(FGF2)interactúaconlavíadelosreceptoresdeprogesterona(RP) induciendoelcrecimientodelcáncerdemamaexperimental,yhemospostuladoqueelFGF2estromalactivaríalosRPenlostumoreshormonoindependientes.ElobjetivodeestetrabajoescompararlosefectosdelFGF2ydelacetatodemedroxiprogesterona(MPA)enlaglándulamamariaderatóneinvestigarsielantiprogestágenoRU486 induce laregresióndel tumorhormonodependienteC4-HDquecrececonMPAoconlaadministraciónintratumoraldeFGF2.DemostramosquelaadministracióndiarialocaldeFGF2induceunahiperplasiaintraductalmamariaquenoesrevertidaporeltratamientoconRU486.Porotraparte,elRU486reviertelahiperplasiaparaductalinducidaporMPAysóloinduceunefectoagonistaparcial.Estosdatossugie-renqueelefectodelFGF2en laglándulamamariaesRP independiente.Demostramosqueel tumorC4-HDcrece in vivoconlaadministraciónintratumoraldeFGF2.Enestecaso,lahistologíarevelaunmayorgradodediferenciación,similaralobservadoeneltumorC4-HIquecrecesinelaporteexógenodehormonas.ElRU-486inhibió tanto laestimulación inducidaporMPAcomoporFGF2.Los resultadosapoyan lahipótesisdequeelFGF2estromalactivaalRPinduciendoelcrecimientohormonoindependientedetumoresmamarios.

Palabras clave:cáncerdemama,glándulamamaria,carcinomasmamarios,FGF2,progestágenos,RU486

Recibido:26-X-2010 Aceptado:15-XI-2010

Postal address:Dra.ClaudiaLanari,IBYME,VueltadeObligado2490,1428BuenosAires,ArgentinaFax:(54-11)4786-2564 e-mail:clanari@dna.uba.ar

Mostbreastcancersexpressestrogenreceptorsalpha(Era) andprogesterone receptors (PR) at the timeofdiagnosisandarethus,susceptibletoanendocrinetreat-ment,thatcurrentlyaimsateitherblockingtheERortosignificantlydecreasethelevelsofcirculatingestrogens1.However, there is increasing evidence indicating that

progesteronereceptors(PR)arealsoinvolvedinbreastcancergrowth2,suggestingthatPRsmayalsobevalidtargetsforbreastcancertreatment.

Eventhoughthesesteroidhormonepathwaysrequirethepresenceof thehormone to activate their cognatereceptors,neoplasticderegulationofothergrowthsignalsmaycontribute to theaberrantactivationof thesteroidreceptorsintheabsenceofsuchligand.Thismechanismmightexplainwhyhormone-dependentcellsexpressingsteroid receptors start to grow in the absence of hor-mones3.OurresultsindicatethatonesuchpathwaymaybethatofFibroblastgrowthfactor2(FGF2),asurvivalfac-

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torinvolvedinneoangiogenesisthatinducesmitogenicandchemotacticresponsesindifferentcelltypes(reviewedin4).ItsactivitiesandthoseofotherFGFsaremediatedbytheirbindingtoafamilyoffourreceptortyrosinkinases(RTK)designatedFGFreceptors (FGFRs)1-4,whichhaveanextracellularportioncontainingthreeimmunoglobulin-likedomainsandintracellulartyrosinkinasedomains.Heparinandheparinsulfateproteoglycansplayacriticalrolefacili-tatingFGFbindingtoFGFR(reviewedin4).

Even though thec-erbB familyandsteroid receptorsignalingremainthemoststudiedproliferationpathwaysinbreastcancer,thereiscompellingevidencesuggest-ing that FGFRsare also among the key regulators ofbreastcancergrowth5.Inthisregard,asinglenucleotidepolymorphism (sNp) in fGfr-2 was recently related to increased Er+ breast cancer risk6,7.

Usingahormonalcarcinogenesismurinebreastcancermodel8,wehave shown thatmifepristone (a progestinantagonist,RU486)inhibitedthegrowthofmammarycar-cinomasthatexpresshighlevelsofERandPRisoformAandareabletogrowwithoutexogenoushormonesupply.Weproposedthatthesetumors(C4-HI),recruitcarcinomaassociatedfibroblasts thatprovidegrowth factorssuchasFGF2that,bybindingtotheircognatereceptors,areableinturn,toactivatePRregardlessofthepresenceofthenaturalhormone.Asaproofofprinciple,we inves-tigatedwhetherFGF2wasabletostimulatethegrowthofahormonedependenttumor,C4-HD,intheabsenceofhormones,and toevaluate if theFGFR inhibitorPD173074decreasedthegrowthofthehormoneindependentvariant,C4-HI.Interestingly,FGF2stimulatedC4-HD,andPD173074decreasedC4-HItumorgrowth9.

Theaimofthepresentstudywastocomparetheef-fectsofFGF2andMPAonadultmousemammaryglandsandtoinvestigatewhethertheantiprogestinwasabletoreversetheFGF2-inducedeffectsonthemammaryglandandonC4-HDtumorgrowth.

Two-month-oldBALB/c orNOD/SCID female virginmicewereusedintheexperiments.Theanimalswerefedad libitum and kept in air-conditioned rooms at 20 ± 2 °C witha12hlight-darkperiod.Animalcareandmanipula-tionwasinagreementwithinstitutionalguidelines,whichareinaccordancewiththeGuidefortheCareandUseofLaboratoryAnimals.Twoexperimentswereperformedusing3miceineachgroup.Tostudythemammaryglands,BALB/cmiceweretreatedforoneweekeitherwith0.1mlofmedroxyprogesteroneacetate(MPA)depot(20mg;Medrosterona,LaboratorioCraveri,BuenosAires)inthecontralateral flank, daily injections in the4th mammary glandof5μgFGF2orsaline.Allgroupsweresimultane-ouslytreatedsubcutaneously(s.c.)withvehicleorRU486(12mg/kgbodyweight;Sigma-Aldrich,StLouis,MO)intheback.FGF2wassynthesizedasdescribedpreviously9.Animalswereeuthanizedand the4thmammaryglandswereexcised,fixedinbufferedformalinandembedded

inparaffin.Vehicle-treatedglandsshowedductstructuressurroundedbyadipocytes(Fig.1A).MPAinducedbranch-ingorparaductalhyperplasiaaspreviouslypublished10 (Fig. 1C).RU486 inhibitedmost of theMPA-inducedeffects(Fig.1D),althoughithadapartialgrowthagonisteffectwhenadministeredalone(Fig.1B).FGF2-treatedmammaryglands showed solid intraductal hyperplasiawithout branching, associatedwith a dense inflamma-toryinfiltratecomposedofpolyandmononuclearcellsinthesurroundingadiposetissue(Fig.1E).ThecombinedtreatmentofFGF2togetherwithRU486didnotinhibittheintraductalhyperplasiasordiminishedtheinflammatoryresponse (Fig.1F).The fact thatRU486 inhibitedonlytheeffectsinducedbyMPAsuggeststhattheintraductalhyperplasiainducedbyFGF2isPRindependent.

Next,weevaluatedtheeffectofRU486onMPA-orFGF2-induced tumorgrowth.Althoughwehadalreadydemonstrated thatRU486 induces tumor regression inhormone-independenttumorsofourmodel8,wehadnot

Fig.1.–Mammaryglands(4th)fromvirginBALB/cmicetreatedforoneweekwithsaline (A),RU486 (B),MPA(C),MPA+RU486(D),FGF2(E)orFGF2+RU486.MPAinducedbranching,RU486inhibitedMPA-inducedeffectsbutwhenadministeredaloneinducedaslightprogestinagonisticef-fect.FGF2 inducedan intraductalhyperplasia.Thewhitearrow shows the multilayered ducts and the black arrow shows the inflammatory cells intermingledwith the adi-pocytes.InFGF2-treatedglands,RU486inducedglandulardifferentiation (whitearrow)within themultilayeredductsanddid not reduce the inflammatory response; bar 20X:100μm,bar40X:10μm.

RU486 and FGF2 in mammaRy caRcinoGenesis 531

yettestedwhetherRU486revertedMPA-inducedtumorgrowthin vivo.AsshowninFig.2A8,C4-HDonlygrowsinMPA-treatedmiceandRU486inducedcompletetumorregressioninspiteofthepresenceofMPA.ToevaluatetheeffectofFGF2theexperimentswerecarriedoutinBALB/candinNOD/SCIDmicetoruleoutpossibleeffectsofFGF2actingasanimmunogen.Animalsweretransplanteds.c.withC4-HDtumors,andtreatedwithMPA(20mgpellets)as described previously8.When tumors reachedasizeofabout25mm2,thepelletswereremovedandthemicewere administeredwith intratumor injections of FGF2,saline, or theywere reinoculatedwithMPA.After oneweek,halfoftheFGF2-treatedmiceweresimultaneouslytreatedwithdailydosesofRU486asdescribedaboveforanotherweek.Tumors inuntreatedmice regressedalmostcompletely.Asreportedpreviously9,tumorstreatedonlywith FGF2 continued growing,while a significantdecrease in growthwas observedwith the combinedtreatmentwithRU486(Fig.2B;p<0.001Studentt test comparingthetumorsizesattheendoftreatment).MPA-and fGf2-treated tumors showed a similar mitotic [MpA: 3.5±0.27;FGF2:2.96±0.48mitoticfigures/highpowerfield (x±SE)]andapoptotic index [MPA:1.26±0.21;FGF2:1.231±0.26apoptoticfigures/highpowerfield(x±SE)].Asignificantdecreaseinmitosis(p<0.01)andanincreaseinapoptosis(p<0.01)wasobservedin(FGF2+RU486)-treatedmicecomparedtoFGF2-treatedtumors.the outcome was similar in either BAlB/c or NoD/sCID mice.TheseresultsconfirmtheexistenceofacrosstalkbetweentheFGF2andPRsignalingpathways.

TumorsgrowinginthepresenceofMPAarecomposedofnestsofepithelialcellswithlowdegreeofglanddifferentiationsurroundedbystromalcells(Fig.2C,left).FGF2-treated,aswellas(FGF2+RU486)-treatedtumorsshowedglandulardifferentiation(Fig.2C,middleandrightrespectively).Ahighreactivestromawasobservedinthelatter.

The postnatal development of themammary glandinvolvesaseriesofsequentialchangesunderthecontrolofseveralsteroidhormonesandsignaltransductionpath-ways,amongthemtheEGFandIGFfamilymembers11.LessinformationisavailableregardingtheroleofFGFs,althoughithasbeensuggestedthattheymayactactivat-ingFGFRoftheepithelialcells12.InthisstudyweshowthatFGF2inducesductalcellproliferation,inthenormalmammarygland,whereasMPAactionsareassociatedwith branching and paraductal hyperplasia, a type ofabortivebranchingof theductal structures, suggestingthatbotheffectsareregulatedbydifferentmechanisms.The hormone effects associatedwith changes in theorganhistoarchitecture,suchasthoseinducedbyMPA,areprobablycomplexphenomenainvolvingtheorches-trationofmanygrowthfactorpathways.TheexogenousadministrationofFGF2inducedaproinflammatorymilieuthatmaybeparticipatingintheinductionoftheintraductalhyperplasia.Theantiglucocorticoideffectsdescribedfor

RU48613mightberesponsibleformaintainingtheinflam-matoryenvironmentinthemammaryglandstreatedwithFGF2combinedwithRU486.AninterestingfindingwasthatRU-486-treatmenthadaslightagonisticeffectonthemammaryglands.Asitwassuggestedthatantiprogestinsprevent BrCA1 induced hyperplasia14, these findingswereunexpected.

Intumors,bothFGF2andMPAinducedtumorgrowth.As shown in our recent array study15,FGF2isupregulatedinMPA-treatedtumors,suggestingthatFGF2maybeamediatorofthehormoneeffect.Inthiscase,noinhibitionofFGF2-inducedtumorgrowthwouldhavebeenexpectedwiththeantiprogestintreatment.Thedatashowedherein,pointtoeitheraninteractionofFGF2withthePRpath-way16,oraninvolvementofPRincellsurvival.Inaddi-tion,theysupportthehypothesisthatstromalFGF2maybeparticipating inhormone independent tumorgrowth.fGf2 activates stAt5 in our model (unpublished data) andactivatedSTAT5hasbeenobservedindifferentiated

Fig.2.–EffectsofRU486onMPA-orFGF2-inducedC4-HDtumorgrowth.A:AnimalsweretreatedwithMPAdepotscinthecontralateralflankoftumorinoculumthesamedayof tumor transplantation.RU486 treatment startedwhentumorsreachedasizeof25mm2.Tumorsregressedcom-pletelyafteroneweekoftreatment.B:Miceweretreatedwith20mgpelletsofMPA toallow tumorgrowth.Whentumors reached a size of 25-50mm2,MPA pelletswereremoved and tumors were treated with intratumor daily injectionsofFGF2,salineorreinoculatedwithMPA.AfterconfirmingthatFGF2stimulatedtumorgrowth,halfoftheanimals receivedRU-486 treatment foroneweek.Asig-nificantdecreaseintumorsizewasobservedwithRU-486treatment(p<0.001,ttest)C.H&Eimagesofrepresenta-tiveC4-HDtumorsgrowinginMPA-treated,FGF2-treatedor(FGF2+RU486)-treatedmice.Differentiatedstructureswere observed in fGf2-treated tumors as well as vessels ofdifferentcalipers.Differentiatedglandsandsmallnestsof epithelial cells intermingled in a reactive stromawereobservedinRU-486plusFGF2-treatedtumors.Insetsshowdetailsofmitoticfigures(left),differentiatedstructures(mid-dleandright:FGF2orFGF2+RU486-treatedtumors).Notumorgrowthwasobservedinuntreatedanimals;Bar:70μm;Barinset:10μm.

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tumors,suggestingthattheactivationofSTAT5mayhaveapivotalroleinhormoneindependenttumorgrowth.

Insummary,inthisstudyweprovideevidencethattheeffectsinducedbyFGF2orMPAinthemammaryglandsaredifferent,suggestingthatthecrosstalkobservedbe-tweenMPAandFGF2inmammarycarcinomasmighthavebeenacquiredduringtheprocessofcarcinogenesis.Ourresults support the idea that fGfr inhibitors and antipro-gestinsmaybeusedinselectedbreastcancerpatients.

Acknowledgements:WeareverygratefultoCraveriLabo-ratorios,BuenosAiresforMPA.WealsothankP.DoCampoandJBoladoforexcellenttechnicalassistance.

funding: this work was supported by sECyt (pICts 2005and2007932 toC.Lanari);andFundaciónSales.Dr.MolinoloissupportedbytheIntramuralResearchProgram ofthe Institute of Dental andCraniofacial Research, National InstitutesofHealth,Bethesda,MD,USA.

Conflicts of interest: the author(s) declare that they have nocompetinginterests.

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- - - -Notuverdad:laverdad.Yvenconmigoabuscarla.Latuya,guárdatela.

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