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Article:

Alasmael, N, Mohan, R, Meira, LB et al. (2 more authors) (2016) Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential. Cancer Letters, 370 (2). pp. 250-259. ISSN 0304-3835

https://doi.org/10.1016/j.canlet.2015.10.031

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1

ActivationoftheFarnesoidX-receptorinbreastcancercelllinesresultsincytotoxicitybut

notincreasedmigrationpotential

NouraAlasmael*1,RatiMohan*

1,LisianeB.Meira

1,KarenE.Swales

2andNickJ.Plant

1

1.! SchoolofBiosciencesandMedicine,FacultyofHealthandMedicalSciences,University

ofSurrey,Guildford,Surrey,GU27XH,UK

2.! ClinicalPDBiomarkerGroup,TheInstituteofCancerResearch,Sutton,SM25NG,UK,

*Theseauthorscontributedequallytothemanuscript

AuthorforCorrespondence:DrNickPlant,SchoolofBiosciencesandMedicine,Facultyof

HealthandMedicalSciences,UniversityofSurrey,Guildford,Surrey,GU27XH,UK

Tel:+44(0)1483686412;Fax:+44(0)1483686401;email:n.plant@surrey.ac.uk

2

Abstract

Breastcanceristhecommonestformofcancerinwomen,butsuccessfultreatmentis

confoundedbytheheterogeneousnatureofbreasttumours:Effectivetreatmentsexistfor

hormone-sensitivetumours,buttriple-negativebreastcancerresultsinpoorsurvival.An

areaofincreasinginterestismetabolicreprogramming,wherebydrug-inducedalterationsin

themetaboliclandscapeofatumourslowtumourgrowthand/orincreasesensitivityto

existingtherapeutics.Nuclearreceptorsaretranscriptionfactorscentraltotheexpression

ofmetabolicandtransportproteins,andthusrepresentpotentialtargetsformetabolic

reprogramming.WeshowthatactivationofthenuclearreceptorFXR,eitherbyits

endogenousligandCDCAorthesyntheticGW4064,leadstocelldeathinfourbreastcancer

celllineswithdistinctphenotypes:MCF-10A(normal),MCF-7(receptorpositive),MDA-MB-

231andMDA-MB-468(triplenegative).Furthermore,weshowthatthemechanismofcell

deathispredominantlythroughtheintrinsicapoptoticpathway.Finally,wedemonstrate

thatFXRagonistsdonotstimulatemigrationinbreastcancercelllines,animportant

potentialadverseeffect.Together,ourdatasupportthecontinuedexaminationofFXR

agonistsasanovelclassoftherapeuticsforthetreatmentofbreastcancer.

Keywords:Apoptosis,Autophagy,NuclearReceptor,Triplenegativebreastcancer,Bile

acids,

Abbreviations:CDCA=chenodexoycholicacid;ER=estrogenreceptor;FXR=FarnesoidX-

receptor;Her2=Humanepidermalgrowthfactorreceptor2;PR=progesteronereceptor;

TNBC=Triplenegativebreastcancer

3

1.! Introduction

BreastcancerrepresentsoneofthelargestkillersofwomenintheWesternworld,witha

lifetimeriskofoneineight[1].Inthepastdecadesanumberofsuccessfultherapeutics

targetingbreastcancerhavebeendeveloped,usedinbothsingleandcombination

therapies.However,improvementsinthefiveyearsurvivalratehavenotbeenashighas

hoped.Thisistoalargedegreeduetotheheterogeneousnatureofbreasttumours,with

multiplemolecularlandscapesbeingclassifiedasasingledisease.Thesediversetumour

phenotypesresultinvariedresponsestotherapeuticintervention,oftenleadingtosub-

optimalpatientresponse[2].Toaidoptimisationoftreatmentregimens,breasttumoursare

commonlyclassifiedintothreeclinicallysignificantgroups:hormonesensitive,Her2positive

andtriplenegativebreastcancers(TNBC)[3].BothhormonesensitiveandHer2-positive

tumourshavearangeofgoodtherapeuticoptions,butTNBCtumoursarecharacterizedby

alackofmoleculartargetsandtendencytodevelopdrugresistance.Notsurprisingly,TNBC

representstheleadingcauseofdeathinbreastcancer[4].Itisthusimperativetodevelop

noveltherapeuticoptionsthatwouldexploittumourvulnerabilities,mitigatedrug

resistanceandleadtoimprovedpatientresponse,andespeciallyinthecaseofTNBC

tumours.

Twoareasoftherapeuticinterventionhavereceivedincreasingattentioninthepastfew

years:Syntheticlethalityandmetabolicvulnerability.Theserelatedconceptsexploitthe

greaterunderstandingofnetworkbiologytopredictsynergisticcombinationtherapies[5].

Insyntheticlethality,theconceptof‘rescuepathways’isexploited:Inessence,targetingof

asinglespeciesinabiologicalpathwayisoftennegatedbyre-routingofthebiological

networktoexploitasecondarypathway.Insyntheticlethality,drugcombinationsareused

4

totargetboththeprimaryandsecondarypathways,producingasynergisticcytotoxicity[6].

Inmetabolicvulnerability,thistheoryofnetworktargetingisfurtherexpanded,lookingfor

novelagentsthatwillworkwithexistingtherapeutics.Tumourcellshaveahighmetabolic

load,duetotheirrequirementtomakealltheprecursorsrequiredforconstantcell

proliferation[7].Assuch,thedevelopmentofdrugsthattargetmetabolismmayreducecell

proliferation,andsynergisewithexistingdrugstoproducemoreeffectivecombination

therapies[8].

Membersofthenuclearreceptorfamilyofligand-activatedtranscriptionfactorsgenerally

regulateexpressionofgenesthatencodeproteinsinvolvedinmetabolicandtransport

processes[9].Assuch,theymayrepresentimportanttargetstoinstigatemetabolic

reprogrammingoftumourcells,leadingtoareductioninsupplyofthecomponents

requiredforcellgrowth[10].Nuclearreceptorexpressioniswidespreadthroughoutthe

body[11],withanumberbeing(over)expressedinbreasttumours[12].Indeed,twoofthe

threemajorclassifiersforbreastcancer,theestrogenreceptor(ER;NR3A1)andthe

progesteronereceptor(PR;NR3C3),arenuclearreceptors,anddisruptionoftheirregulatory

actionisasuccessfultreatmentinreceptorpositivetumours[13,14].ThefarnesoidX-

receptor(FXR;NR1H4)isanadoptednuclearreceptorwithoxysterolssuchasprimaryand

secondarybileacidsasitsendogenousligands[15,16].Bileacidsarethemetabolicproducts

ofcholesterol,andcanbetoxictothebodyathighconcentrations.FXRactstoprevent

accumulationofbileacidsbyinducingexpressionofasecondnuclearreceptorsmall

heterodimerpartner(SHP,NR0B2),whichinhibitsexpressionofCYP7A1,therate-limiting

enzymeinbileacidsynthesis[17,18].Themainsiteofbileacidproductionandsecretion

aretheliverandintestine,respectively,andthisismirroredbyhighlevelsofFXRexpression

intheseorgans[19].However,asapproximately70%ofbileacidsarere-absorbedbythe

5

body,theycanreachmicromolarconcentrationsintheplasma,whichmayunderliethe

expressionofFXRinanumberofothertissues[20].FXRhasbeenshowntobeexpressedin

bothnormalbreasttissueandbreasttumours,withitsactivationinvitrostimulates

apoptosisandinhibitsaromatase[21].ThesefeaturessuggestthatFXRagonistsmay

representanovelclassofbreastcancertherapeuticsduetotheirabilitytoaltertheFXR-

regulatedmetabolicnetwork.

Inthepresentwork,wefirstfullydelineatethemolecularmechanismsbywhichFXR

agonistsactivateapoptosisinbreastcancercelllines,demonstratingthistobethroughthe

intrinsicpathway.Next,weexaminetheabilityofFXRagoniststostimulatemigrationin

thesecelllines,concludingthattheydonotpossessthisability.Together,thesedata

supportthefurtherexaminationofFXRagonistsascancerchemotherapeutics.

2.! MaterialsandMethods

2.1.! Materials

ThebreasttumourcelllineMDA-MB-468(ATCC-HTB-132),andthenormalbreastcellline

MCF10A(ATCC-CRL-10317)werepurchasedfromtheATCC(Teddington,UK),whilethe

breasttumourcelllinesMCF-7(ECACC86012803)andMDA-MB-231(ECACC92020424)

werepurchasedfromtheECACC(PortonDown,UK).CDCAandGW4064werepurchased

fromSigmaAldrich(Poole,UK)andTocrisBiosciences(Abingdon,UK),respectively.

2.2.! CellCulture

MCF7,MDA-MB-468andMDA-MB-231cellsweregrowninDulbecco’smodifiedeagle

medium(DMEM)withphenolredandL-glutamine,supplementedwith10%foetalbovine

serum,100units/mlpenicillin,0.1mg/mlstreptomycinsulphate,and0.25µg/ml

amphotericinB.MCF10AcellswereculturedinDulbecco’smodifiedeaglemedium/F12(1:1

6

v/vDMEM/F12)withphenolred,supplementedwith5%horseserum,100ng/mlcholera

toxin,0.5µg/mlhydrocortisone,10µg/mlinsulin,20ng/mlepidermalgrowthfactor,100

µg/mlstreptomycin,and100units/mlpenicillin.

2.3.! ViabilityAssay

Cellviabilitywasassessedbythe3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium

bromide(MTT)assay,aspreviouslydescribed[22].Briefly,breastcancercelllineswere

platedin96-welltissuecultureflasksatadensityof5x104cellsperwell,andallowedto

attachovernight.Forexperimentsusingserum-starvedconditions,cellsweregrownfora

further24hoursinserum-freemedium.Next,cellswereexposedtotestchemicalsforthe

requiredtime;duringthefinal60minofincubation,0.5mg/mLMTTwasadded.Attheend

oftheincubationperiod,theresultantformazansaltwasdissolvedinDMSOandits

absorbancemeasuredat540nm.Resultsareexpressedasapercentageofvehiclecontrol;

eachdatapointrepresentsthemeanofaminimumofthreeindependentexperimentsof6

wellsperexperiment,witherrorbarsrepresentingthestandarderrorofthemean(SEM).

2.4.! CaspaseAnalysis

CaspaseactivitywasmeasuredusingtheCaspase-Gloassaysystem(Promega,UK)as

previouslyreported[23].Briefly,breastcancercelllineswereseededin96-wellplatesata

densityof1x104cellsperwell,allowedtoattachovernight,serum-starvedfor24hours,

andthenexposedtotestchemicals(±specificcaspaseinhibitor)asrequired.Next,Caspase-

Gloreagentwasadded,mixed,incubatedatroomtemperatureforonehour,andthen

luminescencemeasured.Resultsareexpressedasapercentageofvehiclecontrol;eachdata

pointrepresentsthemeanofaminimumofthreeindependentexperimentsof6wellsper

experiment,witherrorbarsrepresentingthestandarderrorofthemean(SEM).

2.5.! ProteinAnalysis

7

Westernblotanalysiswasundertakenaspreviouslydescribed[22].Briefly,totalproteinwas

extractedusingRIPAbuffer,andproteinquantifiedbythemethodofSmith[24].Thirty

microgramsoftotalproteinwasseparatedonprecast6-18%polyacrylamidegels,andthen

transferredtoPVDFmembrane.Proteinweredetectedovernightat4˚Cusingthefollowing

primaryantibodiesanddilutions:FXR1:500(sc13063,SantaCruzBiotechnology);MMP2

1:200(sc-10736,SantaCruzBiotechnology);MMP91:200(sc-10737,SantaCruz

Biotechnology);PARP1:1000(#9542,CellSignalling);cytochromec1:500(sc-7159,Santa

CruzBiotechnology);Bax1:250(sc-493,SantaCruzBiotechnology);Bcl21:250(sc-7382,

SantaCruzBiotechnology);SHP1:250(sc-15283,SantaCruzBiotechnology);LC31:1000

(#2775,CellSignalling);and,B-actin1:2,000(A2228,Sigma-Aldrich).Next,membraneswere

incubatedwithanIRDye800CWgoat,anti-rabbitsecondaryantibody(1:10,000)forone

houratroomtemperature,andimagedusinganOdysseyCLxinfraredimagingsystem(LI-

CORBiosciences).

2.6.! AutophagyAnalysis

InadditiontoexaminationofautophagythroughWesternblottingofLC3cleavage(see

above),theaccumulationofwasp62/SQSTM1inautophagosomeswasquantifiedusingthe

PremoAutophagySensorGFP-p62assay(MolecularProbes,OR,USA)[25].Breastcancer

celllineswereseededin24-wellplatesatadensityof2x105cellsperwell,allowedto

attachovernight,exposedtopGFP-62inductionmixinserum-freemediumfor24hours,

andthenexposedtotestchemicalsasrequired.p62/SQSTM1fluorescencewasquantified

andexpressedasapercentageofvehiclecontrol;eachdatapointrepresentsthemeanofa

minimumofthreeindependentexperiments,witherrorbarsrepresentingthestandard

errorofthemean(SEM).3-methyladenineandrapamycin(Sigma-Aldrich)wereusedas

inhibitorsandactivatorsofautophagy,respectively[26-28].

8

2.7.! CellMigrationAnalysis

Forthetranswellassay,24-welltranswellinsertswithan8μmporesize(CorningInc,USA)

wereused.Breastcancercelllineswereseededintotheupperchamberatadensityof

1x105cellsperwellinserum-freemedium.Plateswereincubated24-72hours,andthen

cellsinthelowerchambercounted;toensurecountingofallmigratedcells,thelower

surfaceofthetranswellwasexposedto1xtrypsin:EDTAfor30minutes.

Forthewound-healingassay,35mmwoundhealing-inserts(Ibidi,Martinsried,Germany)

wereused.Breastcancercelllineswereseededintothechamberatadensityof1x105cells

perwellinserum-freemediumandallowedtoattachovernight.Insertswereremoved,and

gapclosuremeasuredattime0and12hoursafterexposuretotestchemicalusingtScratch

program[29].

2.8.! InhibitionofFXRactivitybyFXRdominant-negativeover-expression

TodemonstratethattheobservedeffectswereFXR-dependent,experimentswere

undertakenusingover-expressionofadominantnegativeFXRmutant[30].MCF-7and

MDA-MB-231cellsweretransfectedwiththeFXR-DNexpressingplasmidusingFugene6

(Promega,UK)accordingtothemanufacturer’sinstruction,andincubatedovernightto

allowproteinexpression.CellswerethenexposedtoGW4064andanalysedaspreviously

described.

2.9.! StatisticalAnalysis

StatisticalanalysiswasundertakenusingGraphPadPrismv6.01(GraphPadSoftwareInc.,La

Jolla,USA).Datasetswerecomparedthrougheitheraone-wayANOVAwithTukey’s

multiplecomparisontestoratwo-wayANOVAwithSidak’smultiplecomparisontest,as

appropriate.Thelevelofstatisticalsignificancewassetaprioriatp<0.05.

9

3.! ResultsandDiscussion

3.1.! ActivationofFXRinducescelldeathinbreastcancercelllines

ThenuclearreceptorFXRisclassicallyassociatedwithbileacidhomeostasisinthebodyand

itstargetgenesimpactonthemetabolicandtransporter-mediatedclearanceofbothbile

acidsandtheirprecursors[15].However,FXRactivationhasalsobeenreportedtoelicita

numberofotherphenotypes,includingcelldeath.Toconfirmthisphenotypeinbreast

cancercelllines,weexaminedtheeffectoftheendogenousligandCDCAandthemore

potentandselectiveartificialligandGW4064[31].Fourcelllineswerechosentorepresent

differentbreastcancerphenotypes:normal(i.e.MCF-10A),receptorpositivetumour(i.e.

MCF-7)andtriplenegativetumours(i.e.MDA-MB-231andMDA-MB-468).Weconfirmed

thatFXRisexpressedinthesecelllines,andforMCF-7andMDA-MB-231cellsthattheFXR-

dependentsignallingcascadeisintact,withactivationofSHPexpressioninresponsetoFXR

activation(supplementalfigureS1).

BothCDCAandGW4064causedaconcentration-dependenttoxicityinallcelllines(Figure

1),withtheeffectgenerallybeingmoremarkedinserum-freeconditions.Thisisconsistent

withtheobservationthatbothCDCAandGW4064arelipophilic,withLogPvaluesof4.15

and8.3,respectively[20].Inserum-containingmedium,thefreeconcentrationofboth

compoundsareexpectedtobereducedduetoproteinbinding[32].IC50valueswere

determinedforallconditionsandarepresentedinTable1.Wenotethat,asexpected,IC50

valuesforGW4064areapproximatelythreeordersofmagnitudelowerthanforCDCA,

consistentwiththehigheraffinityofthisligandforFXR[GW4064Kd=0.01μMversusCDCA

Kd=50μM[15,31]].DataconsistentwiththesevalueswerealsoobtainedwithMTTanalysis

following72hoursofexposureofcelllinestoeachcompound(datanotshown).

10

Despitethediversephenotypesofthefourcelllinesstudied,allrespondedinthesame

mannertoCDCAandGW4064,bothqualitativelyandquantitatively,withnostatistically

significantdifferencesinresponseforeitheragonistbetweencelllines.Thiswouldsuggest

thatFXR-dependentcytotoxicityisageneralcellresponse,andnotdependentofany

emergentphenotypicpropertyofthedifferentgenotypesofeachcellline.

3.2.! FXR-mediatedcytotoxicityismediatedthroughtheintrinsicapoptoticcascade

HavingdeterminedthatFXRagonistsstimulatecytotoxicityinallfourbreastcancercell

lines,wenextexaminedthemolecularmechanismsunderlyingthisresponseinthereceptor

positiveMCF-7andtriplenegativeMDA-MB-231celllines.

ActivationofFXRhasbeenpreviouslyreportedtocauseapoptoticcelldeathinbreast

cancerMCF-7andMDA-MB-468celllines[21].AsdepictedinFigure2,exposureofMCF-7

andMDA-MB-231cellstoGW4064orCDCAfor24hoursresultedinastatisticallysignificant

activationoftheexecutionercaspases3/7,andcleavageofitssubstratepoly(ADP-

ribose)polymerase-1(PARP)[33].ThisconfirmsthedataofSwalesetal,andextendsitto

coveranothertriplenegativecellline.Inaddition,wenoteSwalesetalusedhigh

concentrationsofGW4064(30µM;3,000xKd);hereweusealowerconcentrationthatwill

cause99.9%receptoroccupancywhilereducingtheriskofoff-targeteffects[31].FXR

agonist-mediatedactivationofcaspases3/7,andsubsequentPARPcleavage,ismorepotent

intheMDA-MB-231celllinecomparedtotheMCF-7cellline.Thisisconsistentwithreports

thattheMCF-7celllinelacksexpressionofcaspase3[34,35],meaningonlycaspase7

activityispresent.

HavingconfirmedthatFXRactivationbyGW4064orCDCAcausesapoptosisinbreastcancer

celllines,wenextexaminedthemolecularmechanism(s)underlyingthiseffect.The

apoptoticcascadecanoccurthroughtheintrinsicandextrinsicpathways,witheachbeing

11

composedofdistinctmoleculareventsthatconvergeattheactivationofexecutioner

caspasessuchascaspase3and7[36].Caspase8activationisclassicallyassociatedwiththe

extrinsicapoptoticpathway,whilecaspase9activationisindicativeofapoptosisviathe

intrinsicpathway.ExposureofMCF-7andMDA-MB-231cellstoeither3µMGW4064or

30µMCDCAfor9hourscausedastatisticallysignificantincreaseincaspase9activity,which

couldbepreventedbyco-incubationwiththecaspase-9inhibitorZ-LEHD-FMK(Figure3a).

Foldincreasesincaspase9activitywerethesameorderofmagnitudeasthatobservedwith

thepositivecontrol,1µMstaurosporine.Incontrast,neithercompoundwasabletoactivate

caspase8activityineitherMCF-7orMDA-MB-231celllines(Figure3b);presenceofa

functionalextrinsicpathwaywasdemonstratedthroughtheuseofthepositivecontrol

chemicalFasligand[37].Together,thesedataareconsistentwithactivationofFXRresulting

incelldeaththroughactivationoftheintrinsicpathwayofapoptosis.

ToconfirmtheabilityofGW4064andCDCAtoinduceintrinsicpathway-mediatedapoptosis,

weexaminedseveralothermolecularmarkersforthispathway.Akeystageintheintrinsic

pathway,andonethatdistinguishesitfromtheextrinsicpathway,ismitochondrial

dysfunction.MembersoftheBCL2familyarecentralregulatorsofthisprocess,withthe

relativeexpressionofpro-(e.g.Bcl2)andanti-(e.g.BAX)apoptoticfamilymembers

determiningcellfate[36].Figure4ademonstratesthatFXRactivationresultsinasignificant

increaseinBAX:Bcl2ratio,withGW4064increasingtheratioby2.9±0.3-foldand4.8±1.0-

fold,andCDCAby2.8±0.4-foldand2.5±0.2-foldinMCF-7andMDA-MB-231cells,

respectively.Alterationinthisratioisassociatedwithchangesinmitochondrialmembrane

integrity,leadingtocytochromecreleaseintothecytoplasm,whichcomplexeswithApaf-1

toformtheapoptosome[38].Weobserveatime-dependentreleaseofcytochromec

followingexposureofMCF-7andMDA-MB-231cellstobothGW4064andCDCA(Figure4b).

12

Asnotedabove,theMCF-7celllinemayhavelimitedapoptoticcapacityduetoalackof

expressionofcaspase3[34,35].Undersuchconditions,alternativepathwaysmaybe

initiated.Autophagyisacellrescuepathwayinvolvingdegradationandrecyclingof

damagedcellcomponents[39],andtherelativerolesofautophagyandapoptosisin

determiningcellfateappearstobehighlycontextdependent,withfactorsincludingcellular

background,typeofperturbation,anddegreeofdamage.ConjugationofcytosolicLC3-Ito

phosphatidylethanolamine,formingautophagosomalLC3-IIisanimportantmarkerof

autophagy[40],andinFigure5awepresenttheimpactofGW4064andCDCAontheLC3-

I:LC3-IIratioinbothcelllines.InMCF-7cells,GW4064isobservedtocausea4.4±0.3-fold

increaseinthisratio,consistentwithactivationoftheautophagypathway,butCDCAdoes

notelicitasimilarresponse.InMDA-MB-231cells,neithercompoundwasabletoelicita

significantchangeinLC3-I:LC3-II.Asecondexperimentalapproachwasusedtoconfirmthe

activationofautophagy,namelyaccumulationofp62/SQSTM1,aubiquitin-bindingprotein

thattargetsubiquitinylatedproteinsfordegradationviaautophagy[41].Both3µM

GW4064and30µMCDCAelicitedastatisticallysignificantincreaseinp62-GFPfluorescence

inMCF-7cells,butnotinMDA-MB-231cells.Thisincreasewasreducedbytheadditionof

5mM3-methyladenine,aninhibitorofautophagy.Assayspecificitywasdemonstratedby

thepositivecontrolcompound2.5µMrapamycin[26].

ThesedataareconsistentwithMCF-7cellsusingtheautophagicresponseasacomplement

toapoptosis,whichisnecessaryduetothecompromisedcapacitytoundergoapoptosis

causedbylackofexpressionoftheexecutionercaspase3.Insupportofthis,when

autophagyisinhibitedby3-methyladenine,nosignificantincreaseincaspase3/7activation

isseeninMCF-7cells,suggestingthatthecellsarealreadyutilisingthecaspase-dependent

apoptoticcelldeathpathwayatclosetoitscapacity(Figure5c).

13

3.3.! InductionofapoptosisandautophagyinbreastcancercelllinesisFXR-dependent

Aspreviouslynoted,whileGW4064isahighlyselectiveligandagainstFXR,theactionsof

CDCAaremorepromiscuous.Itisthusimportanttodemonstratethattheobservedeffects

aremediatedviaFXRandnotanalternatemechanism.Toexaminethis,wehaveuseda

dominantnegativeFXRmutant(FXR-DN)tosquelchFXR-mediatedsignalling[21,30,42].As

canbeseenfromfigure6a,transfectionoftheFXR-DNplasmidintoMCF-7andMDA-MB-

231cellseffectivelyblocksFXR-dependentsignalling,asevidencedbytheablationofSHP

induction,aclassicalFXRtargetgene,inresponsetoGW4064.

BlockadeofFXRsignallingbyFXR-DNalsoreducesGW4064andCDCA-dependentcelldeath

(Figure6b),consistentwithpreviousreportsthatthisisFXRdependent[21].Thereduction

incelldeathisduetodecreasedapoptosisinbothcelllines,asevidencebyreduced

activationoftheexecutioncaspases3/7(Figure6c).Aspreviouslydemonstrated,MCF-7

cells,whicharedeficientincaspase-3,arealsostimulatedtoundergoautophagyby

GW4064,andwedemonstratethatthisisalsoFXR-dependent,beingreducedinthe

presenceoftheFXR-DNmutant(figure6d).

Together,thesedatastronglysupportthattheobservedeffectsaremediatedthroughthe

actionofFXR,andnotviaanother,non-specific,mechanism.

3.4.! ActivationofFXRinbreastcancercelllinesdoesnotcauseanincreaseincell

migration

ThedatapresentedhereinsupportsthecytotoxicpotentialofFXRactivationinbreast

cancercelllines.Inaddition,wefurtherelucidatethemolecularmechanismsunderlying

this,whicharethroughtheprogrammedapoptoticandautophagicpathways.Assuch,these

datawouldsupportthepotentialofFXRagonistsascancerchemotherapeuticagents.

14

However,anumberofreportshavesuggestedthatactivationofFXRcanalterthe

migration/invasionphenotypeoftumourcells,althoughthedatainthisareais

controversial.Forexample,Fukaseetal.havereportedapositiveactiononmigrationinthe

Hep3Bhepatomacellline[43],whileStrauchetal.demonstratedasimilareffectin

intestinalepithelialcells[44].Conversely,ZhangetalreportedthatFXRsuppression

promotedproliferationandmigrationofHepG2andHep3Bhepatomacelllines[45],while

Yoyoetal.demonstratedarepressiveeffectonvascularsmoothmusclecellmigration[46].

Anypotentialforenhancedcellmigration/invasionisamajorliabilityinthedevelopmentof

atherapeuticagent,andwethereforeexaminedthepotentialofGW4064andCDCAto

effectmigrationinbreastcancercells.

Inthetranswellmigrationassaywewereabletodetectasignificantnumberofmigrated

cellsfollowing72hoursincubationforbothMCF-7andMDA-MB-231celllines,importantas

MCF-7areconsideredpoorlyinvasive[47].Exposureofcellsintheupperchambertoeither

3µMGW4064or30µMCDCAthroughouttheincubationperioddidnotresultinany

increaseinmigrationrates(Figure7a).Inaddition,wenotethatextensionoftheincubation

periodtoincludea1-hourpre-incubationwithFXRagonistalsodidnotimpactupon

migrationrates(datanotshown).Asecondexperimentalapproachwasalsousedto

examinemigration,thescratchassay.Datafromthisassaywasconsistentwiththetranswell

assay;whilesignificantmigrationcouldbeobservedoverthe24hourincubationperiod,

therewasnosignificantincreasewhenMCF-7orMDA-MB-231cellswereexposedtoFXR

agonists(Figure7b).

ThisdataappearsinconflictwiththatofSilvaetal,whodemonstratedthatthesecondary

bileaciddeoxycholatecouldinducemigrationofMDA-MB-231,butnotMCF-7cells[48].

However,themethodologyusedbySilvaetalwassignificantlydifferentfromthepresent

15

work,andonlythesecondarybileaciddeoxycholatewasexamined.Thisbileacidisa

productofbacterialmetabolismofCDCA,andattheconcentrationusedlikelytocauseboth

FXR-dependentandFXR-independenteffects[49].

Giventheimportantnegativeliabilityassociatedwithanypro-migratoryeffect,andthe

conflictingdatainthisarea,wealsoexaminedMCF10AandMDA-MB-468cellsfortheir

migratoryresponseuponFXRactivation.AspresentedinsupplementaryfigureS2,no

evidencewasfoundtosupportapro-migratoryphenotypeineitherofthesecelllines.As

such,thesedatasupportthenotionthatFXRactivationinbreastcancercelllinesdoesnot

enhancemigratorypotential.ThissuggeststhatanyinteractionbetweenFXRandcell

migration/invasioniscomplex,andalmostcertainlycontext-dependent.

Conflictofinterest

KESisanemployeeoftheInstituteofCancerResearch.NA,RM,LMandNJPdeclarethat

theyhavenoconflictsofinterest

Acknowledgements

NAwasfundedbytheKingdomofSaudiArabiaMinistryofEducation.

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22

Table1:IC50valuesforCDCA-andGW4064-elicitedcelldeathinbreastcancercelllines

CDCA GW4064

Complete

medium

Serum-free

medium

Complete

medium

Serum-free

medium

MCF-7 n.d. 244.7±9.5µM 10.6±2.6µM 12.6±2.8µM

MDA-MB-231 n.d. 273.3±32.2µM 9.5±3.9µM 3.7±3.3µM

MDA-MB-468 n.d. 188.5±124.4µM n.d. 4.8±1.4µM

MCF-10A n.d. 142.0±87.4µM 2.6±2.6µM 3.8±1.1µM

n.d.=notdeterminedasslopedidnotconverge

23

Figure1:TheFXRagonistsGW4064andCDCAcauseconcentration-dependenttoxicityin

breastcancercelllines.MCF-10A(A),MCF-7(B),MDA-MB-231(C)andMDA-MB-468(D)

cellswereexposedtovaryingconcentrationsofGW4064orCDCAincompleteorserum-free

medium.Following48or72hoursofexposure,cellviabilitywasassessedbyMTT.Eachdata

pointrepresentsthemeanofthreeindependentexperiments,whileerrorbarsrepresent

thestandarderrorofthemean(SEM).*=p<0.05,**=p<0.01,***=p<0.001

24

Figure2:TheFXRagonistsGW4064andCDCAcauseconcentration-dependentactivation

ofcaspase3andcaspase7intheMCF-7andMDA-MB-231celllines.(A)MCF-7andMDA-

MB-231cellswereexposedto3µMGW4064,30µMCDCA,or1µMstaurosporineinserum-

freemedium,inthepresenceorabsenceofspecificcaspaseinhibitorsfor24hours,and

thenCaspase3andcaspase7activitymeasured.(B)MCF-7andMDA-MB-231cellswere

exposedtoindicatedconcentrationsofGW4064,CDCAorstaurosporineinserum-free

mediumfor24hoursandthenpoly(ADP-ribose)polymerase-1(PARP)cleavageassessedby

Westernblot.Eachdatapointrepresentsthemeanofthreeindependentexperiments,

25

whileerrorbarsrepresentthestandarderrorofthemean(SEM).*=p<0.05,**=p<0.01,

***=p<0.001

Figure3:TheFXRagonistsGW4064andCDCAactivatecaspase9,butnotcaspase8,

activityintheMCF-7andMDA-MB-231celllines.MCF-7andMDA-MB-231cellswere

exposedto3µMGW4064,30µMCDCAor1µMstaurosporineinserum-freemediumfor24

hours,andthenCaspase8andcaspase9activitymeasured.Eachdatapointrepresentsthe

meanofthreeindependentexperiments,whileerrorbarsrepresentthestandarderrorof

themean(SEM).*=p<0.05,**=p<0.01,***=p<0.001

26

Figure4:TheFXRagonistsGW4064andCDCAstimulateapoptosisthroughtheintrinsic

pathwayinMCF-7andMDA-MB-231celllines.MCF-7andMDA-MB-231cellswere

exposedto3µMGW4064,30µMCDCAor1µMstaurosporineinserum-freemedium.(A)

Subcellularlocalisationofcytochromecwasexaminedfollowing0,4,6and8hoursof

exposure,withLDHasamarkerforthecytosolicfraction.(B)BaxandBcl2proteinlevels

weremeasuredintotalproteinfollowing24hoursofexposure.Eachdatapointrepresents

themeanofthreeindependentexperiments,whileerrorbarsrepresentthestandarderror

ofthemean(SEM).*=p<0.05,**=p<0.01,***=p<0.001

27

Figure5:TheFXRagonistGW4064stimulatesautophagyinMCF-7cells.MCF-7andMDA-

MB-231cellswereexposedto3µMGW4064or30µMCDCAinserum-freemediumfor24

hoursandautophagyassessedby(A)LC3cleavageand(B)p62/SQSTM1accumulation.5mM

3-methyladenineand2.5µMrapamycinwereincludedwhereindicatedasaninhibitorand

activatorofautophagy,respectively.Eachdatapointrepresentsthemeanofthree

independentexperiments,whileerrorbarsrepresentthestandarderrorofthemean(SEM).

*=p<0.05,***=p<0.001

28

29

Figure6:InductionofapoptosisandautophagyinbreastcancercelllinesbyGW6064and

CDCAisFXR-dependent.MCF-7andMDA-MB-231cellswereexposedtoGW4064orCDCA

inserum-freemediumfor24hoursinthepresenceorabsenceofadominantnegativeFXR

protein(FXR-DN).(A)Cellswereexposedto3µMGW4064or30µMCDCAandexpression

oftheFXRtargetgeneSHPmonitoredbyWesternblotting.(B)CellswereexposedtoIC50

concentrationofGW4064orCDCAandcellviabilityassessedbyMTTassay.(C)Cellswere

exposedto3µMGW4064or30µMCDCAandapoptosisassessedbycaspase3/7activity

assay.(D)Cellswereexposedto3µMGW4064or30µMCDCAandautophagyassessedby

p62/SQSTM1accumulation.Eachdatapointrepresentsthemeanofthreeindependent

experiments,whileerrorbarsrepresentthestandarderrorofthemean(SEM).*=p<0.05,

**=p<0.01,***=p<0.001

30

Figure7:TheFXRagonistsGW4064andCDCAdonotstimulatemigrationofMCF-7or

MDA-MB-231celllines.(A)MCF-7andMDA-MB-231cellswereseededintranswellplates

andexposedto3µMGW4064or30µMCDCAinserum-freemediumfor72hours.The

numberofcellsmigratingintothebottomchamberwasquantified.(B)MCF-7andMDA-

MB-231cellswereseededinawound-healingassayformatandexposedto3µMGW4064or

30µMCDCAinserum-freemediumfor24hours.Cellmigrationwasquantifiedbyclosureof

thescratchareausingthetScatchprogram.Eachdatapointrepresentsthemeanofthree

independentexperiments,whileerrorbarsrepresentthestandarderrorofthemean(SEM).

31

SupplementaryFigures

ActivationoftheFarnesoidX-receptorinbreastcancercelllinesresultsincytotoxicitybut

notincreasedmigrationpotential

NouraAlasmael,RatiMohan,LisianeB.Meira,KarenE.SwalesandNickJ.Plant

SupplementaryFigure1:BreastcancercelllinesexpressafunctionalFXRgeneregulatory

network

FigureS1:BreastcancercelllinesexpressafunctionalFXRgeneregulatorynetwork.(A)

MCF-10A,MCF-7,MDA-MB-231andMDA-MB-468cellswereexposedto3µMGW4064,

30µMCDCAorvehiclecontrolinserum-freemediumfor24hours,andthenFarnesoidX-

receptor(FXR)expressionassessedbyWesternblot.(B)MCF-7,MDA-MB-231orHepG2

(positivecontrol)cellswereexposedto3µMGW4064,30µMCDCAorvehiclecontrolin

serum-freemediumfor24hours,andthensmallheterodimerpartner(SHP)expression

assessedbyWesternblot.Eachdatapointrepresentsthemeanofthreeindependent

experiments,whileerrorbarsrepresentthestandarderrorofthemean(SEM).**=p<0.01,

***=p<0.001

32

SupplementaryFigureS2:TheFXRagonistsGW4064andCDCAdonotstimulatemigration

ofMCF-10AorMDA-MB-468celllines

FigureS2:TheFXRagonistsGW4064andCDCAdonotstimulatemigrationofMCF-10Aor

MDA-MB-468celllines.(A)MCF-10AandMDA-MB-468cellswereseededintranswell

platesandexposedto3µMGW4064or30µMCDCAinserum-freemediumfor72hours.

Thenumberofcellsmigratingintothebottomchamberwasquantified.(B)MCF-7and

MDA-MB-231cellswereseededinawound-healingassayformatandexposedto3µM

GW4064or30µMCDCAinserum-freemediumfor24hours.Cellmigrationwasquantified

byclosureofthescratchareausingthetScatchprogram.Eachdatapointrepresentsthe

meanofthreeindependentexperiments,whileerrorbarsrepresentthestandarderrorof

themean(SEM).