indole-3-carbinol mediated anti-proliferative regulation ... · cancer stem cell population in...

Indole-3-CarbinolMediatedAnti-ProliferativeRegulationofBreastCancerStemCellsand

MalignantMelanoma-InitiatingCellsBy

AnnaHyoPark

AdissertationsubmittedinpartialsatisfactionoftheRequirementsforthedegreeof

DoctorofPhilosophy

In

Endocrinology

Inthe

GraduateDivision

Ofthe

UniversityofCalifornia,Berkeley

Committeeincharge:

ProfessorGaryL.Firestone,ChairProfessorJen-Chywan(Wally)Wang

ProfessorThomasJ.Carlson

Fall2016

1

ABSTRACT

Indole-3-CarbinolMediatedAnti-ProliferativeRegulationofBreastCancerStemCellsand

MalignantMelanoma-InitiatingCells

By

AnnaHyoPark

DoctorofPhilosophyinEndocrinology

UniversityofCalifornia,Berkeley

ProfessorGaryL.Firestone,ChairThecancerstemcelltheoryascertainsasubpopulationoftumorigeniccellsthatselectivelypossess the ability for tumor initiation and self-renewal capacity to differentiate intononmalignant cancer cells, known as bulk tumor cells. Cancer stem cells were firstidentified in hematopoietic cancers but subsequently have been implicated in ovarian,melanoma, liver, sarcoma, head and neck, pancreatic, glioblastoma, and breast cancerssince.Asa result,malignant tumors canbecomposedofgeneticallyandmorphologicallyheterogeneouscellpopulationswithvaryingdegreesofdifferentiation,self-renewalability,and metastatic capacity. Experimental pursuits have sought to isolate and analyze thecancer stem cell population in primary tumors and molecularly strategize to identifyphenotypictargets.Alackofstandardizedcancerstemcellinvivoandinvitromodelshavesloweddownthedevelopmentofproper therapiesaswellas theelucidationofscientificmechanismsthatdictatecancerstemcells.Onemethodofdetectionhasbeentheutilizationof various stem cellmarkers to categorizemolecular phenotypes. In breast cancer stemcells, biomarkers such as the nucleolar GTPase nucleostemin; the detoxifying enzyme,ALDH-1; and cell surfacemarker, CD44aid in identification and isolationof cancer stemcells forresearchstudies.Furthermore,exclusionof theHoeschtDyeassays for thecell’sabilitytoeffluxcurrenttherapeuticmoleculesextra-cellullarly,evadingapoptosisandcellcycle arrest. Furthermore, melanoma cancer stem cells, also known as malignantmelanoma-initiating cells (MMICs), contain subpopulations expressingCD133, CD20, andABCG5 have defined features of unlimited self-renewal and proliferative capacities formelanomacancers.Whilecancerincidenceoverallhasdecreased,melanomacontinuestorise, with patients with metastatic and malignant melanoma continue to relapse fromcurrent therapeutic strategies. This thesis details the characterization of human breastcancer and melanoma cell lines of varying tumorigenic potential and their subsequentresponse to the phytochemical, indole-3-carbinol, and its synthetic derivative, 1-benzyl-I3C. I3C selectively targets breast cancer stem cells through different molecularmechanismssuchasprotein-proteininteractionsofthestemcellmarkernucleosteminanditsbindingpartner,murinedoublemutant2(MDM2)inthenucleolus,therebyfreeingp53to activate apoptosis. Furthermore, I3C sensitizes cells with a wild type p53, wild typePTEN phenotype or evenmore tumorigenicmelanoma cells that possess the rare BRAF

2

V600DmutationandPTENdeletion.GivenI3C’sanditsmorepotentderivative,1-benzyl-I3C’s, anti-proliferative modulation of the p53 pathway in breast cancer cells and thePTEN/Wnt pathways inmalignantmelanoma cell lines, preclinical results can implicateI3Casanovelchemopreventativemoleculethatselectivelytargetscancerstemcells.

i

AcknowledgmentsI would like to thank Professor Gary Firestone for your mentorship professionally andpersonallyinmylife.OursmallchatsaboutBerkeleysportstoourmeaningfuldiscussionsaboutourfutureshasbroughtmuchlightintomycareerhereatBerkeley.Ican’tspeaktothevolumesof engagement and thoughtfulness you’ve sharedwithme in591LSA.EventhoughmyyearsatBerkeleyendhere,Iknowtheinfluenceyou’vehadovermylifewillbeinfinite.MyextendedParkandWoo family–Myauntsanduncles.Thankyou forgivingme faithand trustingmewith your good guidance. I know every small act and lesson taughtmeimmenseintegrityanddeterminationtosucceed.WithoutyouIwouldn’thavebeenabletothinkindependentlyinsearchofdifferentthoughtsandideasthatenrichedmyeducation.Iaminfinitelygrateful.Eric–Youaremynumberonerockinmyadultlife.You’vealwayskeptmyheadstraight,given me pep talks at the right time, and cooked me food during my trying times. I’menthusiastic for our ever-changing futures whether we stay in San Francisco or moveacrosstheglobe.Tony–Ican’tthankyouenoughforyourunparalleledsupportthroughalltheseyearsbothemotionally and mentally. Thank you for giving me guidance when needed. And Iacknowledgemyselfforbeingsoawesome.Kevin–YouwerethefirstpersonIevermet in labwhenIwaswronglytoldIcouldtakeoneofyourredbullswithoutgettingyourpermissionfirstbutthatwasthebeginningofabeautiful and adventurous friendship! Thanks for mentoring me professionally andpersonallythroughgraduateschoolandnowinSF.Jeanne– JEANNE!!!!Ourspecial coffeedates, though too frequent,havebroughtsomuchexcitementintheafternoons.Iknowourfutureswillbebrightandhopefulaftergraduateschool where we can revel in more nourishment in far more exciting locations. I’m soexcitedforourfutureandallourconversationtocome.Randilea – Even though fate brought us together for MCB 32, it was no doubt ourquirkiness, deepening real talk, and love for fashionablepiecesbrought somuch light tograduate school. I don’t know how we would have survived 5th floor/ 4th floorawesomenesswithouteachother.IhopeafteryourmovetoLAwecontinueourchatsandheartwarmingconversationatrestaurantsandBlueBottlesallovertheworld.THANKYOUsomuchforyourlove.Jess–Our thoughtful friendshipallbloomed fromour friendaversaryatPhilzand Idon’tknow howmany cups and cups of coffee we drank together since then but the storiesyou’vesharedwithmeandthethoughtsyou’velistenedfrommehaspushedmetobecomeabetterpersonpersonally.Thankyouforalwaysbeingpresentandengagedwheneverweshare a beautiful moment. I hope we order two large Jacob Wonderbar’s and SilkenSplendorsuntilwegrowoldtogetherandbecometeacher/PTAqueen.

ii

Thisworkisdedicatedtomyparentsandmygrandmother

YounSukParkandYeunSilWooandKimKeumOak

Thegreatestpillarsofsupportinmylife.Thankyouforyourunconditionalsupport,unwaveringtruth,and

unparalleleddedicationtomyintegrity.

iii

TableofContentsListofFigures…………………………………………………………………………………..………….………………………………………………….………………….ivGeneralIntroduction…………………………………………………………………………………..………….…………………………………….………………viChapterIProtein-proteininteractionsofthestemcellmarkernucleostemininbreastcancerstemcells………………………………………………………………………………………..………….……………………………………………………….……………………..1 Abstract………………………………………………………………………………………..………….……………………………………………….2 Introduction………………………………………………………………………………………..………….……………………………………..3 MaterialsandMethods………………………………………………………………………………………..………….…………..…..5 Results………………………………………………………..………….…………..…………………………………………………………..…………9 Discussion………………………………………………………..………….…………..…………………………………………………………..25 References………………………………………………………..………….…………..………………………………………………………….27ChapterIIFiguresIndole-3-CarbinolRegulationofthetumorsuppressorp14ARFinbreastcancerstemcells………………………………………………………..………….…………..……………………………………………….…………..………………………………………………29 Abstract………………………………………………………………………………………..………….……………………………………………30 Introduction……………………………………………………………………………...………..………….………………………………...…31 MaterialsandMethods……………………………………………………….………………………………..………….…………..33 Results………………………………………………………..………….…………..…………………………………………………………..………36 Discussion………………………………………………………..………….…………..…………………………………………………………..59 References………………………………………………………..………….…………….………………………………………………………..61ChapterIIIFiguresIdentificationofmalignantmelanoma-initiatingcells(MMICs)andvaryingtumorigenicpotentialinfivephenotypicallydistinctmelanomacelllinesandtheirresponsivenesstoindole-3-carbinol………………………………..………………………….…………..………………………………………64 Abstract………………………………………………………………………………………..………….……………………………………………65 Introduction……………………………………………………………………………...………..………….………………………………...…66 MaterialsandMethods……………………………………………………….………………………………..………….…………..68 Results………………………………………………………..………….…………..…………………………………………………………..………71 Discussion………………………………………………………..………….…………..…………………………………………………………..88 References………………………………………………………..………….…………….………………………………………………………..90

iv

ListofFiguresGeneralIntroductionFiguresFigure1:CancerPreventativePhytochemicalsFigure2:Indole-3-Carbinol(I3C)Figure3:CancerStemCellProgressionFigure4:LocalizednucleosteminregulatesMDM2bindingandp53stabilityFigure5:CellularLocalizationofthetumorsuppressor,p14ARFFigure6:NFkBSignalingisinvolvedincanonicalandnon-canonicaltranscriptionalregulationFigure7:MelanomaStemCellHypothesis.ChapterIFiguresProtein-proteininteractionsofthestemcellmarkernucleostemininbreastcancerstemcellsFigure8:StemCell-LikeCharacteristicsofVariousCellLinesFigure9:QuantificationofALDH-1activityFigure10:Comparing10AT-Her2tumorsphereformingefficiencyFigure11:I3CinducesNucleostemin-MDM2interactionsandreleasep53incancerstemcells.Figure12:I3Cregulationofnucleostemin-MDM2andp53-MDM2protein-proteininteractionsinwell-establishedhumanbreastcancercelllinesFigure13:IncreasedexpressionofCancerStemCell-LikePhenotypeiscorrelatedwithI3CResponsivenessFigure14:SchematicDiagramofnucleostemin-MDM2-p53interactionsChapterIIFiguresIndole-3-CarbinolRegulationofthetumorsuppressorp14ARFinbreastcancerstemcellsFigure15:I3CEffectson10AT-Her2cellsFigure16:I3Cpromotesp14ARFinteractionwithMDM2anddiminishesnucleostemin-MDM2bindingFigure17:I3CinducedapoptoticresponseismediatedbyAkt1Figure18:ConstitutivelyactiveAkt1altersI3Cregulationonthemolecularinteractionsbetweenp14ARF,Nucleostemin,andMDM2Figure19:I3Creleasesp14ARFfromnucleophosminincancerstemcellsFigure20:I3Cdownregulatesproteinandtranscriptionallevelsofnucleophosminandatranscriptionfactor,MYCFigure21:I3CablatesbindingofMYCtotheNucleophosminpromoterandNFkBbindingtotheMYCpromoterFigure22:SummaryofI3C-mediatedp14ARFregulation

v

ChapterIIIFiguresIdentificationofmalignantmelanoma-initiatingcells(MMICs)andvaryingtumorigenicpotentialinfivephenotypicallydistinctmelanomacelllinesandtheirresponsivenesstoindole-3-carbinolFigure23:I3CinhibitsmelanomacelllinesofvaryingtumorigenicpotentialFigure24:I3CinducesstrongG1cellcyclearrestinG361andWM115cellsFigure25:I3CinhibitsformationofmelanomaspheroidsinG361andWM115cellsFigure26:Quantificationofcancerstemcellmarkers,CD133,CD20,andALDH-1Figure27:I3Cand1-benzyl-I3CselectivelymodulateG361andWM115cellsFigure28:Summaryofvaryingtumorigenicpotentialofhumanmelanomacelllines

vi

Anti-CancerPropertiesofPhytochemicalsPhytochemicals are naturally occurring chemicals in plants, and have been identified aspromisinganti-cancer,health-promotingagents.Theyincludephenolics,suchastaninsandflavonoids; alkaloids, such as caffeine; nitro-containing compounds; organosulfurcompounds,suchasindoles;phytosterols;andcarotenoids[1].Phytochemicalshavebeenidentifiedtohavepotentialinvariousmedicinalpathwayssuchas hormonal regulation, oxidative damage reduction, DNA repair, genomic stability,inflammation, and cellular proliferation. Some molecular reactions phytochemicals areknown to promote both in vivo and in vitro are apoptosis mechanisms such as PARPcleavage,ATP-dependentchromatinremodeling,tumorangiogenesis,differentiationintheWnt pathway, and tyrosine kinase receptor upregulation. Epidemiological studies dosuggestthatconsistentuptakeoffruitsandvegetables,andotherplantfoods, is linkedtoreduce cardiovascular and chronic diseases [2]. As a result, it has become critical tounderstand the underlying molecular mechanisms of phytochemicals in promoting itshealthbenefits.One of the toughest challenges of phytochemical usage is proving the effect of cancerprevention. Preventative epidemiological studies on a human population are difficult toprovewithinareasonabletimelines,asmostrequireevidenceofupto30years.Molecularstudies in identifying progression of a tumor after dosage of phytochemicals, however,have been critical in quantifying reduction of oncogenic biomarkers and stabilization oftumorsuppressors[3,4].Oneofthepromisingphytochemicalsexploredinmylaboratoryis,indole-3-Carbinol(I3C),which is found in Brassica vegetables, such as brussel sprouts, cauliflower, and napacabbage. It’s digestive derivative, is known as diindolylmethane (DIM), forms viacondensation in theacidicenvironmentof thestomach.Bothhaveexhibitedcriticalanti-proliferativeeffects inhormone responsive cancers suchasprostate,ovarian, andbreastcancers.Importantindolederivatives,suchas1-benzyl-I3C,hasbeenproveninourlabtobe effective at amuch lower dose, highlighting the potential for synthetic derivatives inbattlingthesecancers[5,6].

vii

Figure1CancerPreventativePhytochemicals.Variouschemopreventativeagentsinnaturalfruits,vegetables,andplantproductsshowntohaveantitumorandanti-proliferativeeffectsincancer,suchasovarian,breast,prostate,andmelanoma.

ix

Indole-3-CarbinolandBreastCancerIndole-3-carbinol is aphytochemicalnaturally occurring inBrassica vegetables andhavebeen associated with anticancer activities of cruciferous vegetables. It is produced bymembers of the family Cruciferae and the genus Brassica, including such vegetables ascabbage, brussel sprouts, cauliflower, radishes, and broccoli. Early studies have I3C hasbeenapromisingagentinreducingtheincidenceofspontaneousandcarcinogen-inducedtumorsofcertaincancerssuchascolon,skin,liver,cervix,andlung[7,8].In vitro, I3C has shown the suppress the proliferation of various tumor cells includingprostate,breast,endometrial,leukemic,andcoloncellsbyinducingG1/Scellcyclearrestbydownregulationofcyclindependentkinases,cyclinD1,andcyclinEorupregulationofp15, p21, and p27; or activating apoptosis by downregulation of antiapoptotic geneproducts,suchasBcl-2,Bcl-xL,andsurvivingorupregulationofproapoptoticproteinslikeBax[9-11].In vivo, I3C was found to be a potent chemopreventive agent for hormonal-dependentcancers such as breast and cervical cancer by inducing apoptosis, stimulating 2-hydroxylation of estradiol, inhibiting DNA-carcinogen adduct formation, and inhibitingangiogenesis and invasion. Initial clinical trials in women show that I3c is an effectivetherapyagainstbreastandcervicalcancers[8].I3C has been shown to activate or inactivate multiple nuclear receptors, decreasemitochondrial membrane potential induce endoplasmic reticulum stress, and regulatemultiplecellularsignalingpathways.I3Chasawidespreadeffectonmultiplecancercells,suchasbreastcancer,melanoma,andprostatecancers[5],ThisdemonstratestheI3Cplaysasignificantroleineitherthecellcyclearrestorapoptosisofvariouscancers.

x

Figure2Indole-3-Carbinol(I3C).I3C is produced by members of the family Cruciferae, and particularly members of thegenusBrassica.

xi

Indole-3-Carbinol (I3C)

•  Increased cruciferous vegetable intake in human diets is associated with lower risk of reproductive cancers

•  I3C and DIM (bioactive I3C

dimer) in clinical trials for reproductive cancers

xii

TheheterogeneityofacancerstemcellinawholetumorCancerstemcellswere firstdocumented in leukemiaandmyelomacancers, identifyingasmall side population of cancer cells as responsible for tumorigenesis and proliferation.Basic cancer research has questioned the effects of particular mutations on theproliferation,differentiation,andsurvivalofcertaincancercells. Ithabeenshownonlyasmall population of cells in a tumor are clonal, while the majority of solid cancers arecomprised of cells that are phenotypically heterogeneous [1, 12, 13]. Both normal stemcellsandtumorigeniccancerstemcellsgiverisetophenotypicallyheterogeneouscellsthatconveydifferentdegreesofdifferentiation.Whilesomeoftheheterogeneityofatumorcanbe due to prolonged mutagenesis, it is more likely that heterogeneity arises from theirregulardifferentiationofnormalcancercells[14-18].Themolecular signature of amalignancy iswell-characterized and strategies to enrich acancerpopulationandre-sensitizethemtotreatmentarebeingexplored.However,cancerstemcellsfromsolidtumorsaremuchmoreelusive,withambiguousphenotypicidentifiesandsignificantchallengesfordrugdevelopmentinmusttumortypes[12,19,20].However,based on certain characteristic properties such as tumor-initiating potential, nichedependence, detoxification/ drug resistance, activation of stem cell pathways, andclonogenicity, cancer stem cells can be identified and defined. In breast cancer cells, thepresenceofALDH1,CD44,andnucleosteminaswellasthelackofCD24expressionarekeycharacteristicsofabreastcancerstemcellphenotype[21-25].Stemcellsarecharacterizedby theirability todifferentiateandself-renew,whichallowsthemtogenerateall cell lineageswithinagiven tissue. Bothstemcellsandcancerstemcellsshowactivationofself-renewalassociatedpathwayssuchassonicHedgehog,Notch,Wnt/Beta-catenin,andPTEN[26,27].Through thisproperty, tumormayoriginate fromthe transformation of normal stem cells, similar signaling pathways pay regulate self-renewalinstemcellsandcancercells,andcancercellsmayincludecancerstemcells.As a result, mortality from breast cancer remains high because current therapies arelimitedbytheemergenceoftherapy-resistantcancercells[1].Metastaticbreastcancerstillremainsanincurablediseasebycurrenttreatmentstrategiesthereforeitbecomescriticalto understand themolecularmutations that underlie the biology of these cells thatmaylead toneweffective therapies [28,29]. Ifa smallpopulationof cancerstemcells causescancergrowthandproliferation,noveltherapiesmustexistthatnoonlytargetandablatesolidtumorsize,butalsotargetcancerstemcellsspecifically.

xiii

Figure3CancerStemCellProgression.Theheterogeneityofatumorrisesfromthedifferentiationofcancercells.Thiscanleadtotherapyresistanceandcancerrelapse,promotingpoorprognosisforcancerpatients.Novelmoleculartherapiescanmoreeffectivelytargetthesidepopulationofcancerstemcellsinaprimarytumorandengageincancerregression.

xv

Nucleostemin,asanactorofself-renewalandcellcyclearrestNucleosteminisaGTP-bindingproteinconcentratedinthenucleolusofmoststemcellsandnowimplicatedincell-cycleprogressionduetoitsp53-dependentactivity[30-32].Itwasdiscoveredbecauseofitshighexpressioninneuroepithelialstemandprogenitalcellsandwas laterdiscovered inother typesofstemcells, tumor initiatingcells (TICs),andtumorcells. In differentiated cells and tissues, however, nucleostemin is expressed at a muchlowerlevel,identifyingnucleosteminasfunctionallycriticalinundifferentiatedphenotypes[33-35].Recently,downregulationaswellasoverexpressionofnucleosteminhasbeenidentifiedincausingG1cell-cyclearrest incancercellsandstemcells.Nucleosteminupregulationwillinduce compartmentalization of murine double minute 2 (MDM2) in the nucleolus,stabilizing p53. Furthermore a new finding has identified depletion of nucleosteminpromotestheinteractionofMDM2withribosomalproteinsL5andL11,whichalsohindersp53degradation[36].Mechanistically, the functionofnucleostemin isunclearbutcertainstudies recentlyhavesought to explicate the genome-protective role of nucleostemin in stem cells and cancerstem cells. Several findings have implicated nucleostemin in reducing DNA damage ontelomeresandnon-telomericchromosomes throughmodificationofTRF1,alsoknownastelomericrepeatbindingfactor.NucleosteminalsohastheabilitytorecruitRAD41,acoreplayer of the homologous recombinationmachinery, to protect the genomic integrity ofstemorprogenitorcellsfromreplication-inducedDNAdamage[36].Anothermechanismcritical tonucleostemin’s activity is its ability to sequesterMDM2, anegative regulatorofp53, in thenucleolus [32,36].Therearesomestudies that supportp53-depdenncyofnucleosteminactivity,inwhichcellcyclearrest(G1orS)orapoptosisistriggeredbyknockdownorknockoutofnucleostemincanbereversedbyp53knockdown.Furthermore,nucleosteminhasbeenfoundtodirectlyinteractwithp53andMDM2aswellas p14ARF, a tumor suppressor that co-localizes MDM2 and enhances p53’s apoptoticactivity[30,31,37].

xvi

Figure4LocalizednucleosteminregulatesMDM2bindingandp53stability.Cancerstemcellsexpresshighlevelsofnucleostemin,capableofp53andMDM2regulationthroughproteinbinding.

xviii

Tumorigenicprotein-proteininteractionsofARFP14ARF tumor suppressor is a critical player in cancer as a key sensor of signals thatinstructacelltoproliferateandgrow[38,39].It is localizedinthenucleolitolimittheseprocesses and have been implicated in releasing from the nucleolus for its most activefunctions.ItisencodedfromthehumanINK4A/ARF(CDKN2A)locuswhichtranslatesintoboth the cyclin-dependent kinase inhibitor p16INK4A as well as the tumor suppressor,p14ARF. ARF is translated in an alternative reading frame, and creates distinctARF andINK4Aproteinsaftertranslation[40-42].Inhumancancers,oneofthemostrecurrentcytogeniceventsisthehomozygouslossoftheInk4Alocusasmutationatthislocusisonlysecondtothep53locus.Arf-/-micecommonlyare susceptible to sarcomas, lymphoid malignancies, carcinomas, and tumors in thenervous system [43]. Furthermore, they are commonly more sensitive to carcinogen-related tumor formation, such as when exposed to 7,12-dimethylbenz-α-anthracene(DMBA).Arflossalsoenhancesaggressivephenotypesfromothermutations,suchasthoseobserved inBcr-Abl inducedacute lymphoblastic leukemia.Micedisrupted foronlyexon1β develop tumors as early as eight weeks. After one year, 80% of the mice die fromspontaneoustumordevelopment,withameansurvivallatencyof38weeks[38,44].P14ARF has many oncogenic and tumor suppressive binding proteins due to its basicchemicalnatureconferredfromitshighconcentrationofarginineresidues,Itisrelativelysmall and has a half-life of about 6 hours, ablated by ubiquitin-mediated proteosomaldegradationintheN-terminus.Asaresult,ARFhasbeenshowntointeractwith30otherproteins, such as transcriptional activators, transcriptional repressors, posttranslationalmodifying enzymes, DNA modifying enzymes, and nuclear/ nucleolar proteins. ARF istypicallylocalizedinthenucleolihighlyboundinhighmolecularweightcomplexes,suchaswithnucleophosmin,andunleashedduringtimesofinstantaneousoncogenicstress.DuetonucleophosmininteractionwithARFinthenucleolus,basalARFmightbecorrelatedwithmaintainingnucleolarstructureandlimitingproteinsynthesisaswell[45,46].P14ARFisclassicallyinvolvedwiththep53axis,bysequesteringMDM2,anE3ligaseandpotentiatorofubiquitination,awayfromp53.Whenactivatedbyoncogenicsignals,ARF’sN-terminus associateswith the central region ofMDM2, which localizesMDM2 into thenucleolus away fromMDM2 and nearwhere ARF typically resides. The sequestration ofMDM2byARFprevents thebindingofMDM2 top53and theabilityofMDM2 to shuttlebetween thenucleus and cytoplasm, thereby impeding its ability to transportp53 to thecytoplasmfordegradation[47-51].

xix

Figure5CellularLocalizationofthetumorsuppressor,p14ARF.Thetumorsuppressor,p14ARF,isknowntoassociatewithmultiplebindingpartnersinthenucleolusandthenucleoplasminordertopromotep53-dependentapoptosis.

xxi

TranscriptionalRegulationofNucleophosmin,Myc,andNFkBinCancerNucleophosmin, also known as B23, is an acidic nucleolar protein with many bindingpartnersthatexhibitcriticalrolesinproliferationandcellgrowth.Ithasbeenidentifiedinawiderangeofdevelopmentalpathwayssuchascellularproliferation,ribosomalgenesis,DNA repair response, genomic stability, andmost importantly, in cancer, apoptosis [52,53].Asanacidicprotein,multipleproteinsinteractwithnucleophosmininthenucleolus,suchasthetumorsuppressorresponsibleforp53activation,p14ARF.Therelationshipbetweenp14ARFandnucleophosminiscomplex.Forexample,p14ARFbindstonucleophosmininthe nucleolus to promote degradation of the protein and induce cell death. There isincreasing evidence of nucleophosmin’s ability to retain p14ARF in the nucleolus andhinder its ability to bind to MDM2 and activate p53 [54]. However, other studies havefound that nucleophosmin is also able to bind to MDM2 directly, and protect p53 fromMDM2’s role in ubiquitin degradation. Furthermore, nucleophosmin is enhanced by itsbindingtoAkt1,anoncogeneimportantincellularproliferationandcanonicallyknowntophosphorylate and activateMDM2.Akt1, as a result of growth factor activation, protectsnucleophosmin from proteolytic degradation from caspase-3 and stimulates cellularsurvival.Byevidenceofbinding top14ARF,MDM2,p53, andAkt1directly, these studiessuggestthatnucleophosminhasakeyregulatoryroleinmediatingapoptosisandthep53-axis[47,49-51].Oneknowntranscriptionfactorofnucleophosminismyc,atranscriptionfactorresponsibleforunregulatedexpressionofmanygenesincancer,suchascellularproliferation,growthcontrol,differentiationandapoptosis.Themostcommonmutationofmycresults ingeneamplificationandoveractivationofchromosomalreplication.Because it isoverexpressedina largemajorityofhumancancers, it isaprimeanti-cancer therapeutic targetandhasbeen implicated in colon, breast, stomach, lung, and cervical cancers. Functionally,myc’sbindingtothecanonicalenhancerboxsequences,alsoknownasE-boxes,andrecruitmentofhistoneacetyltrasnferases(HATs)ininducingtranscriptionofdownstreamtargetgenes,suchasNF-kB[55-58].Thetargetgenesofmycregulatecellularsurvival,proteinsynthesis,cellcycle,celladhesionandmetabolism.Thenuclearfactor-kBfamilyofproteinsactsasdimerictranscriptionfactorsthatregulatetheexpressionofgenesregardinginflammationstressresponse,cellularproliferation,andinnateandadaptiveimmunity.ActiveNF-κB/Relcomplexesarefurtheractivatedbypost-translationalmodifications(phosphorylation,acetylation,glycosylation)andtranslocatetothe nucleus where, either alone or in combination with other transcription factorsincludingAP-1,Ets,andStat,theyinducetargetgeneexpression[57,58].

xxii

Figure6NFkBSignalingisinvolvedincanonicalandnon-canonicaltranscriptionalregulation.TheNFkBtranscriptionfactorisinvolvedinmanycancer-promotingpathwayssuchassurvival,proliferation,invasion,angiogenesis,andmetastasis.

xxiv

Melanomastemcells,ormalignant-melanoma-initiatingcells(MMICs)Malignantmelanomaishighlymetastaticcancerandincreasinglyresistanttoconventionaltherapiesduetoincreasedmolecularheterogeneityandexpressionofmetastaticmarkers.Heterogeneityofamelanomastemcellcanbeattributedtothecancerstemcellmodel.Thisevidence is supported by isolating cancer cells that express various levels of stem andprogenitor cell markers or chemo-resistance markers, and comparing their tumorigeniccapability[59].Severalbiomarkerscanbeidentifiedasmelanomacancerstemcellssuchascellsurfacemarkers,CD133andCD271;theATPbindingcassettetransporters,ABCG2,andABCG5;and the intracellulardetoxifyingenzymeexpressed inmanystemcells,AldehydeDehydrogenase(ALDH-1)[60-65].Throughthesestudiesofcancerorprogenitorstemcellmarkers,adistinct lineageofmelanomastemcellswas isolated,positingmarker-positivecells have greater tumor initiating capability in melanoma cells versus marker-negativecells which do not have the potential to recapitulate the phenotypic heterogeneity of aprimaryparentaltumor[66-68].Ahallmarkofmelanomastemcellsisinitiationoftumorgrowthandmelanomainitiation.Theyareaminorsubpopulation,capableofself-renewalanddifferentiation,enriched forhuman malignant-melanoma-initiating cells (MMIC). A unique strategy in cancertherapeuticsistoutilizemoleculartargetstoablateproliferationofMMICs.Geneprofilingof a marker-positive population can result in the identification of more markers toelucidate the mechanism and signaling pathways relevant for potential diagnostics andprognosis. Therefore, melanoma stem cell-directed therapeutic approaches representpromisingnovelstrategiestoimprovetherapy.Four types of melanoma cancer cells have been established: CD20(+), CD133(+), label-retainingorslow-cyclingcells,andside-populationcellswithhigheffluxactivities[13,69,70]. These properties are most likely overlapping for these subpopulations within amalignantlesion.Ongoingstudieshavebeenseekingtopurifyandcharacterizecapabilitiesthat determine prolonged self-renewal capability, immune evasion, vasculogenicdifferentiation,andmicrotumornichedevelopment[12,19,20].

xxv

Figure7MelanomaStemCellHypothesis.Asubpopulationofcellswithinmelanomawithstemcellpropertiessuchasindefiniteself-renewalanddifferentiationcapacitycanarisefromavarietyofcellswithvariouslineages,suchasadifferentiatedmelanocyte,melanoblast,transamplifyingprogenitorcell,oranadultmelanocytestemcell.

xxvii

References1. Dandawate,P.R.,etal.,Targetingcancerstemcellsandsignalingpathwaysby

phytochemicals:Novelapproachforbreastcancertherapy.SeminCancerBiol,2016.40-41:p.192-208.

2. Braakhuis,A.J.,P.Campion,andK.S.Bishop,ReducingBreastCancerRecurrence:TheRoleofDietaryPolyphenolics.Nutrients,2016.8(9).

3. Singh,S.,etal.,LeadPhytochemicalsforAnticancerDrugDevelopment.FrontPlantSci,2016.7:p.1667.

4. Oh,J.,etal.,TherapeuticEffectivenessofAnticancerPhytochemicalsonCancerStemCells.Toxins(Basel),2016.8(7).

5. Megna,B.W.,etal.,Indole-3-carbinolinducestumorcelldeath:functionfollowsform.JSurgRes,2016.204(1):p.47-54.

6. Tin,A.S.,etal.,Essentialroleofthecancerstem/progenitorcellmarkernucleosteminforindole-3-carbinolanti-proliferativeresponsivenessinhumanbreastcancercells.BMCBiol,2014.12:p.72.

7. Firestone,G.L.andL.F.Bjeldanes,Indole-3-carbinoland3-3'-diindolylmethaneantiproliferativesignalingpathwayscontrolcell-cyclegenetranscriptioninhumanbreastcancercellsbyregulatingpromoter-Sp1transcriptionfactorinteractions.JNutr,2003.133(7Suppl):p.2448S-2455S.

8. Licznerska,B.andW.Baer-Dubowska,Indole-3-CarbinolandItsRoleinChronicDiseases.AdvExpMedBiol,2016.928:p.131-154.

9. Brew,C.T.,etal.,Indole-3-carbinolactivatestheATMsignalingpathwayindependentofDNAdamagetostabilizep53andinduceG1arrestofhumanmammaryepithelialcells.IntJCancer,2006.118(4):p.857-68.

10. Brew,C.T.,etal.,Indole-3-carbinolinhibitsMDA-MB-231breastcancercellmotilityandinducesstressfibersandfocaladhesionformationbyactivationofRhokinaseactivity.IntJCancer,2009.124(10):p.2294-302.

11. Aronchik,I.,etal.,Targetproteininteractionsofindole-3-carbinolandthehighlypotentderivative1-benzyl-I3CwiththeC-terminaldomainofhumanelastaseuncouplescellcyclearrestfromapoptoticsignaling.MolCarcinog,2012.51(11):p.881-94.

12. Kozovska,Z.,V.Gabrisova,andL.Kucerova,Malignantmelanoma:diagnosis,treatmentandcancerstemcells.Neoplasma,2016.63(4):p.510-7.

13. Skvortsov,S.,P.Debbage,andI.Skvortsova,Proteomicsofcancerstemcells.IntJRadiatBiol,2014.90(8):p.653-8.

14. Owens,T.W.andM.J.Naylor,Breastcancerstemcells.FrontPhysiol,2013.4:p.225.15. Pinto,C.A.,etal.,Breastcancerstemcellsandepithelialmesenchymalplasticity-

Implicationsforchemoresistance.CancerLett,2013.341(1):p.56-62.16. Calhoun,K.E.,Breastcancerstemcells--readyfortheirclose-up?JAMASurg,2013.

148(9):p.878.17. Nigam,A.,Breastcancerstemcells,pathwaysandtherapeuticperspectives2011.

IndianJSurg,2013.75(3):p.170-80.18. Iqbal,J.,P.Y.Chong,andP.H.Tan,Breastcancerstemcells:anupdate.JClinPathol,

2013.66(6):p.485-90.

xxviii

19. Heryanto,Y.D.,etal.,Invivomolecularimagingofcancerstemcells.AmJNuclMedMolImaging,2015.5(1):p.14-26.

20. Kaur,S.,G.Singh,andK.Kaur,Cancerstemcells:aninsightandfutureperspective.JCancerResTher,2014.10(4):p.846-52.

21. Charafe-Jauffret,E.,etal.,ALDH1-positivecancerstemcellspredictengraftmentofprimarybreasttumorsandaregovernedbyacommonstemcellprogram.CancerRes,2013.73(24):p.7290-300.

22. Leon,G.,etal.,Cancerstemcellsindrugresistantlungcancer:Targetingcellsurfacemarkersandsignalingpathways.PharmacolTher,2016.158:p.71-90.

23. deBeca,F.F.,etal.,CancerstemcellsmarkersCD44,CD24andALDH1inbreastcancerspecialhistologicaltypes.JClinPathol,2013.66(3):p.187-91.

24. Bensimon,J.,etal.,CD24(-/low)stem-likebreastcancermarkerdefinestheradiation-resistantcellsinvolvedinmemorizationandtransmissionofradiation-inducedgenomicinstability.Oncogene,2013.32(2):p.251-8.

25. Kim,R.J.,etal.,Highaldehydedehydrogenaseactivityenhancesstemcellfeaturesinbreastcancercellsbyactivatinghypoxia-induciblefactor-2alpha.CancerLett,2013.333(1):p.18-31.

26. Oren,O.andB.D.Smith,EliminatingCancerStemCellsbyTargetingEmbryonicSignalingPathways.StemCellRev,2016.

27. Pires,B.R.,etal.,TargetingCellularSignalingPathwaysinBreastCancerStemCellsanditsImplicationforCancerTreatment.AnticancerRes,2016.36(11):p.5681-5691.

28. Korkaya,H.andM.S.Wicha,HER2andbreastcancerstemcells:morethanmeetstheeye.CancerRes,2013.73(12):p.3489-93.

29. Yenigun,V.B.,B.Ozpolat,andG.T.Kose,ResponseofCD44+/CD24-/lowbreastcancerstem/progenitorcellstotamoxifenanddoxorubicininducedautophagy.IntJMolMed,2013.31(6):p.1477-83.

30. Zhang,C.,etal.,Nucleosteminexertsanti-apoptoticfunctionviap53signalingpathwayincardiomyocytes.InVitroCellDevBiolAnim,2015.51(10):p.1064-71.

31. Yuan,F.,etal.,NucleosteminKnockdownSensitizesHepatocellularCarcinomaCellstoUltravioletandSerumStarvation-InducedApoptosis.PLoSOne,2015.10(10):p.e0141678.

32. Bao,Z.,etal.,NucleosteminpromotestheproliferationofhumangliomaviaWnt/beta-Cateninpathway.Neuropathology,2016.36(3):p.237-49.

33. Huang,G.,L.Meng,andR.Y.Tsai,p53ConfigurestheG2/MArrestResponseofNucleostemin-DeficientCells.CellDeathDiscov,2015.1.

34. Tsai,R.Y.,p53-guidedresponsetonucleosteminlossinnormalversuscancercells.CellDeathDis,2015.6:p.e2030.

35. Wei,B.,Q.Huang,andX.Zhong,Upregulationofnucleosteminincolorectalcanceranditseffectsoncellmalignancy.OncoTargetsTher,2015.8:p.1805-14.

36. Meng,L.,T.Lin,andR.Y.Tsai,NucleoplasmicmobilizationofnucleosteminstabilizesMDM2andpromotesG2-Mprogressionandcellsurvival.JCellSci,2008.121(Pt24):p.4037-46.

37. Wu,H.,etal.,Nucleosteminregulatesproliferationandmigrationofgastriccancerandcorrelateswithitsmalignancy.IntJClinExpMed,2015.8(10):p.17634-43.

xxix

38. Dominguez-Brauer,C.,etal.,TumorsuppressionbyARF:gatekeeperandcaretaker.CellCycle,2010.9(1):p.86-9.

39. Gallagher,S.J.,R.F.Kefford,andH.Rizos,TheARFtumoursuppressor.IntJBiochemCellBiol,2006.38(10):p.1637-41.

40. Maggi,L.B.,Jr.,etal.,ARFtumorsuppressioninthenucleolus.BiochimBiophysActa,2014.1842(6):p.831-9.

41. Ozenne,P.,etal.,TheARFtumorsuppressor:structure,functionsandstatusincancer.IntJCancer,2010.127(10):p.2239-47.

42. Sherr,C.J.,TheINK4a/ARFnetworkintumoursuppression.NatRevMolCellBiol,2001.2(10):p.731-7.

43. Kamijo,T.,etal.,TumorspectruminARF-deficientmice.CancerRes,1999.59(9):p.2217-22.

44. Wazir,U.,etal.,P14ARFisdown-regulatedduringtumourprogressionandpredictstheclinicaloutcomeinhumanbreastcancer.AnticancerRes,2013.33(5):p.2185-9.

45. Gjerset,R.A.,DNAdamage,p14ARF,nucleophosmin(NPM/B23),andcancer.JMolHistol,2006.37(5-7):p.239-51.

46. Carr-Wilkinson,J.,etal.,HighFrequencyofp53/MDM2/p14ARFPathwayAbnormalitiesinRelapsedNeuroblastoma.ClinCancerRes,2010.16(4):p.1108-18.

47. Agrawal,A.,etal.,Regulationofthep14ARF-Mdm2-p53pathway:anoverviewinbreastcancer.ExpMolPathol,2006.81(2):p.115-22.

48. Pare,R.,J.S.Shin,andC.S.Lee,Increasedexpressionofsenescencemarkersp14(ARF)andp16(INK4a)inbreastcancerisassociatedwithanincreasedriskofdiseaserecurrenceandpoorsurvivaloutcome.Histopathology,2016.69(3):p.479-91.

49. Qi,X.W.,etal.,Studiesonexpressionofp14ARFandMDM2inhumanthyroidneoplasms.PanminervaMed,2015.57(1):p.43-7.

50. Wang,J.,etal.,Roleofp14ARF-HDM2-p53axisinSOX6-mediatedtumorsuppression.Oncogene,2016.35(13):p.1692-702.

51. Xia,L.,A.Paik,andJ.J.Li,p53activationinchronicradiation-treatedbreastcancercells:regulationofMDM2/p14ARF.CancerRes,2004.64(1):p.221-8.

52. Grisendi,S.,etal.,Nucleophosminandcancer.NatRevCancer,2006.6(7):p.493-505.

53. Lim,M.J.andX.W.Wang,Nucleophosminandhumancancer.CancerDetectPrev,2006.30(6):p.481-90.

54. Lindstrom,M.S.andY.Zhang,B23andARF:friendsorfoes?CellBiochemBiophys,2006.46(1):p.79-90.

55. Li,Z.andS.R.Hann,TheMyc-nucleophosmin-ARFnetwork:acomplexwebunveiled.CellCycle,2009.8(17):p.2703-7.

56. Li,Z.andS.R.Hann,Nucleophosminisessentialforc-Mycnucleolarlocalizationandc-Myc-mediatedrDNAtranscription.Oncogene,2013.32(15):p.1988-94.

57. Ling,J.andR.Kumar,CrosstalkbetweenNFkBandglucocorticoidsignaling:apotentialtargetofbreastcancertherapy.CancerLett,2012.322(2):p.119-26.

58. Yeh,C.W.,etal.,Ras-dependentrecruitmentofc-Mycfortranscriptionalactivationofnucleophosmin/B23inhighlymalignantU1bladdercancercells.MolPharmacol,2006.70(4):p.1443-53.

59. Alamodi,A.A.,etal.,Cancerstemcellastherapeutictargetformelanomatreatment.HistolHistopathol,2016.31(12):p.1291-301.

xxx

60. Nguyen,N.,etal.,Understandingmelanomastemcells.MelanomaManag,2015.2(2):p.179-188.

61. Parmiani,G.,MelanomaCancerStemCells:MarkersandFunctions.Cancers(Basel),2016.8(3).

62. Roesch,A.,Melanomastemcells.JDtschDermatolGes,2015.13(2):p.118-24.63. Schmohl,J.U.andD.A.Vallera,CD133,SelectivelyTargetingtheRootofCancer.Toxins

(Basel),2016.8(6).64. Yiming,L.,etal.,CD133overexpressioncorrelateswithclinicopathologicalfeaturesof

gastriccancerpatientsanditsimpactonsurvival:asystematicreviewandmeta-analysis.Oncotarget,2015.6(39):p.42019-27.

65. Zimmerer,R.M.,etal.,PutativeCD133+melanomacancerstemcellsinduceinitialangiogenesisinvivo.MicrovascRes,2016.104:p.46-54.

66. Chan,K.S.,MolecularPathways:TargetingCancerStemCellsAwakenedbyChemotherapytoAbrogateTumorRepopulation.ClinCancerRes,2016.22(4):p.802-6.

67. Fulawka,L.,P.Donizy,andA.Halon,Cancerstemcells--thecurrentstatusofanoldconcept:literaturereviewandclinicalapproaches.BiolRes,2014.47:p.66.

68. Hashim,P.W.,P.Friedlander,andG.Goldenberg,SystemicTherapiesforLate-stageMelanoma.JClinAesthetDermatol,2016.9(10):p.36-40.

69. Rosa,R.,etal.,Approachesfortargetingcancerstemcellsdrugresistance.ExpertOpinDrugDiscov,2016.11(12):p.1201-1212.

70. Zeuner,A.,Thesecretlifeofquiescentcancerstemcells.MolCellOncol,2015.2(1):p.e968067.

1

ChapterI

Protein-proteininteractionsofthestemcellmarkernucleosteminandMDM2inbreastcancerstemcells

2

AbstractThecancerstemcell theoryarises toexplicate theheterogeneityofhumanbreastcancercells that possess the ability formulti-lineage differentiation and creates the survival oftherapy-resistanttumorpopulations.Inordertoaddresstheefficacyoftherapiesincancerstem cells, it becomes critical to isolate and propagate cancer stem cells from primarybreast tumors, which only represent 1-5% of the cells. Therefore, in order to study theaffectofanti-canceragentsthatcaninhibitcancerstemcellstopreventapoptosisevadingcellularmechanisms,anexperimentalcapabilitytoanalyzecancerstemcellswasnecessaryforthisstudy.ByexpressingexogenousHER2inapreneoplasticmammaryepithelialcelllines, the newly created MCF-10AT-Her2 cell line expresses high levels of cancerstem/progenitormarkerssuchasnucleosteminandALDH1aswellaspossessesincreasedtumorsphere forming efficiency than other cancer cell lines. Her2, or human epidermalgrowth factor receptor-2), is a member of the orphan epidermal growth factor (EGF)receptor gene family and is associated with poor prognosis in cancer patients andimplicated with aggressive and malignant forms of breast cancer. This cell line wascompared against other breast cancer cell lines of distinct lineages and of varyingtumorigenicpotential.Importantly,I3Cinducesap53-dependentapoptosisbyreducingtheaffinityofp53toitsnegativeregulators,MDM2.I3CenhancesbindingofnucleosteminwithMDM2(murinedoublemutant2),acanonicalE3 ligaseof thetumorsuppressor,p53.Byinductionofp53-dependentapoptosis throughdecreased interactionsofMDM2andp53,I3C initiates a cellular anti-proliferative response, inhibiting cellular growth and self-renewal capabilities. Our studies indicate the effectiveness of I3C over other establishedcancercell linesofvaryingtumorigenecityandhighlighttheselectivemolecularresponsein cells with high self-renewal capacity, increased enrichment of cell population subsetenriched in cancer stem cell marker profile expression, and marked notoriety to othercurrenttherapies.Thisnewparadigmhighlightstheineffectivenessofcurrenttherapiesattargetingcancerstemcellsovernon-cancerstemcellsanddefinesthenecessityfornoveltreatmentstrategiesthatchangetoday’scurrenttherapeuticstandards.

3

IntroductionAcriticalissueincancertreatmentistheaccurateidentification,isolationandtreatmentofmalignantandcancerstemcells,whichexhibitvariouspropertiesofbothacancercellandastemcell.Onedistinctionistheirabilitytoselfrenewinwhichtheircellfateissegregatedsymmetricallyorasymmetricallytodaughtercells,orotherwiseothertumorstemcellsorbulk, non-tumorigenic cells [1, 2]. Cancer stemcells also exhibit uniquemarkers suchashigh levels of the progenitor marker, nucleostemin [3, 4], and functional aldehydedehydrogenase isoform 1 (ALDH-1)—both predictors of poor prognosis. Very little isknown about the signaling pathways involved with these proteins and their abilities toinfluencephenotypesof cancer stemcellpopulations,but certainexpressionof stemcellmarkers, such as CD44 [5], nucleostemin, ALDH1, and lack of CD24 has allowed forisolation, but not propagation, of cancer stem cells [6-8]. These biomarkers have beenattributed tobreast cancer stemcells aswell as stemcells forproteins, suchasALDH-1,which prevent mutational agents from entering the cellular membrane. For example,patientswithCD44+/CD24-phenotypehavebeenassociatedwithpoorprognosisthroughthe Hedgehog signaling pathway [9]. Cancer stem cells are notoriously resistant toconventionaltherapies,suchaschemotherapyorradiotherapyandnoeffectivetherapeuticstrategyhasyetbeenidentified[10,11].Recentstudieshaveshownthatchemo-andradio-resistance of cancer stem cells in solid tumors explicates patient relapse and increasedincidenceofmetastasisinmanycancers[7].Currenttherapieswillattempttoablatetumorsizebytargetingcancercellsinawidefashion,ratherthanseekingtokillcancerstemcells,the causeof tumorburden.Asa result, efficacious therapiesareneeded in targeting thissmallelusivepopulationofcellsthatpreventtheablationoftheentirebreasttumoraftervariousroundsoftherapeutictreatments[12,13].However,inordertofirststudycancerstemcellsaeffectivemodelsystemmustbeestablishedasamodelforcancerstemcells.Tocultureandpropagatecancerstemcellsinvitro,astableandconsistentpopulationofcellsenrichedinacancerstemcellphenotypewascreatedbyourlaboratory.ByexpressingHER2,amemberof theorphanepidermalgrowth factor(EGF)receptor inthe parental preneoplastic human mammary epithelial cell line, MCF-10AT [14], weengineeredabreastcancercelllineinwhichthepopulationisenrichedwithastablestemcell-likecharacter.Asacontrolthe10ATcelllinewasalsotransfectedwiththeneomycincontrolvector,andlabeledasthe10AT-Neocontrolcelllinefromhereon.HER2expressioninbreastcancerhasbeenassociatedwithpoorprognosisinapproximately30%ofbreastcancersand implicated inmetastasis [15-17]. Analysisof thegeneratedMCF-10AT-Her2celllineshowspotentexpressionofcancerstemcellmarkerssuchascellsurfacemarker,CD44,thenuclearGTPasenucleostemin,andALDH-1,adetoxifyingenzymeassociatedwithpoor prognosis.We utilized thismodel system to elucidate themolecularmechanism ofsuchstemcellmarkersthroughinvitromammosphereassaysandinvivotumorxenograftcapabilities in nude athymic mice. One such promising anti-cancer therapy is thechemopreventative phytochemical indole-3-carbinol [18-22], which previous researchstudies identifieddirectly targets theneutrophil elastase.Binding of indole-3-carbinol toelastasestimulatesinteractionofnucleostemintoMurineDoubleMutant2(MDM2),anE3ligase for the tumor suppressor, p53 [23-25]. As a result, I3C-mediated interaction ofMDM2-Nucleosteminbindinginthenucleolusfreesp53toactivateapoptosisinbothHER2

4

expressing breast cancer stem cells, while the luminal cell line, MCF-7, and the triplenegative cell line, both with null p14ARF phenotype, are I3C insensitive and p53suppressive [26]. Protein levels of p14ARF, MDM2, p53, and the stem cell/ progenitormarker, nucleostemin, however, do not change, signifying the enhancement of I3C-mediatedprotein-protein interactionsof thep53-depedentapoptoticpathway.Ourstudyhas uncovered new mechanistic insights into how the cancer stem/progenitor cell-associated component nucleostemin is directly involved in an anti-proliferative cellularsignalingpathwaytriggeredbyI3C,anaturalanti-cancermolecule.

5

MaterialsandMethodsGeneratingtheMammaryEpithelialCancerStemCellLinePreneoplasticMCF-10AThumanmammaryepithelialcells(obtainedfromtheBarbaraAnnKarmanos Cancer Institute, Detroit, MI) were stably transfected with either the humanpCMV-HER2expressionvector,whichalsocontainstheneomycinresistancegene,orwiththe pCMV-Neo control vector forming 10AT-Her2 cells and 10AT-Neo cells, respectively.CellswerestablyselectedwithG418sulfate(Cellgro,Manassas,VA)for2months.CellCultureTheMCF-10ATparentcelllineandthe10AT-Her2and10AT-NeocelllineswereculturedinDMEM/F-12, 10% fetal bovine serum, 50U/mL penicillin, 50U/mL streptomycin (allmediacomponentspurchasedfromLonza,Allendale,NJ,andcellcultureplatespurchasedfromNUNC-Fischer,Pittsburgh,PA),0.02µg/mLepidermalgrowthfactor(purchasedfromPromega,Madison,Wisconsin,USA),0.05µg/mLhydrocortisone,10µg/mLinsulin,and0.1µg/mLcholeratoxin(obtainedfromSigma-Aldrich).BreastadenocarcinomacelllineMDA-MB-231was obtained from theAmericanType Culture Collection (Manassas, VA).MDA-MB-231cellsweregrowninIscove’sModifiedDulbecco’sMedium(IMDM)supplementedwith10%fetalbovineserum,50units/mlpenicillin/streptomycin,and2mML-glutamine(Cambrex Bio Science, Walkersville, MD). MCF-7 human breast cancer cell lines weregrowninDulbeccosmodifiedEaglesmediumsupplementedwith10%fetalbovineserum,1.25 ml 20,000 U/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 µg/ml insulin.SKBR3cell linewasobtained fromtheAmericanTypeCultureCollection (Rockville,MD)andwasculturedinMcCoy’s5Amediumsupplementedwith10%(v/v)FSC,100mg/lofstreptomycin and 100,000 U/l of penicillin G at 37 ◦C in 5% CO2 incubator. Cells weregrown to subconfluency in a humidified chamber at 37°C containing 5% CO2. For drugtreatments,a200mmol/lstocksolutionofI3C,10mmol/lstocksolutionof1-benzylI3C,50 mmol/l stock solution of Diindolymethane (DIM), 200 mmo/l stock solution ofTryptophol,and300mmol/lstocksolutionofArtemisinin(purchasedfromSigma-Aldrich,St. Louis,MO)was dissolved in dimethyl sulfoxide (DMSO) and then diluted in the ratio1:1000 in media before culture plate application. Before each drug treatment, cells arewashedinicecoldphosphate-bufferedsaline(PBS)(obtainedfromLonza).The10AT-Her2cellline,theparentalMCF-10ATcellsandtheSKBR3breastcancercelllinewerevalidatedbyshort tandemrepeat (STR)DNA fingerprintingusingDDCMedicalCellLineAuthenticationLabServices(Fairfield,OH,USA).TheSTRprofileswerecomparedtoknown ATCC fingerprints [83] and to the Cell Line Integrated Molecular Authenticationdatabase (CLIMA) version 0.1.200808 [84]. The STR profile of the 10AT-Her2 cell linematched that of its parental MCF-10AT cell line, whereas the SKBR3 cells matched theknownDNAfingerprints to thatof itself.Therefore, thecellpopulationof the10AT-Her2cell line,whichdisplayssignificantstemness-likecharacter, isnotcontaminatedwithanyother cell line, suchasSKBR3cells thatalsodisplay somestemness-like character in thecorrespondingcellpopulation.

6

TreatmentWithIndole-3-Carbinol

I3Cwas purchased from Sigma–Aldrich (St. Louis, MO), To study the effect of I3C, cellsweretreatedwithorwithout200 μMI3Cevery24 hfor72 handharvestedat48hoursforwesternblotandflowcytometricanalysis.I3Cwasdissolvedin99.9%HPLCgradeDMSO(Sigma–Aldrich,Milwaukee,WI)andthefinaldilutionwasperformedinthemediaaliquotsusedfortreatment.ALDEFLUORAssayALDEFLUORassaywasperformed followingmanufacture’s recommendedguidelinesandprotocol(StemCellTechnologies).LivesinglecellsweregatedforanalysisusingEpicsXL-MCL flow cytometer (Beckman Coulter) with single 488 nm blue laser filtered at 525BP/slot1.TumorsphereEfficiencyAssaySingle-cell suspensions of 10AT-Her2, 10AT-Neo, MCF-7, or SKBR3 cells were plated onultra-low attachment plates (Corning, Costar) inMammoCultHumanMedium (StemCellTechnologies, Vancouver) and cultured at 37oC, 5% CO2. Tumorsphere formation wasassessedvisuallybyphasemicroscopyandquantifiedbycountingthenumberofspheresformedinculture.WesternBlotsAfter the indicated treatments, cellswere harvested in radioimmune precipitation assaybuffer (150 mM NaCl, 0.5% deoxycholate, 0.1% NoNidet-p40 (Nonidet P-40, FlultaBiochemitra, Switzerland), 0.1% SDS, 50mMTris) containing protease and phosphataseinhibitors (50g/mlphenylmethylsulfonyl fluoride,10g/mLaprotinin,5 g/mL leupeptin,0.1 g/mLNaF, 1mMdithiothreitol, 0.1mM sodiumorthovanadate, and 0.1mMglycerolphosphate).Equalamountsoftotalcellularproteinweremixedwithloadingbuffer(25%glycerol,0.075%SDS,1.25mlof14.4Mβmercaptoethanol,10%bromphenolblue,3.13%0.5MTris-HCl,and0.4%SDS(pH6.8))andfractionatedon10%polyacrylamide/0.1%SDSresolvinggelsby electrophoresis.Rainbowmarker (AmershamBiosciences)wasusedasthe molecular weight standard. Proteins were electrically transferred to nitrocellulosemembranes(MicronSeparations,Inc.,Westboro,MA)andblockedfor1hourwithWesternwashbuffer5%NFDM(10mMTris-HCl(pH8.0),150mMNaCl,and0.05%Tween20,5%nonfat dry milk). Protein blots were subsequently incubated for overnight at 4oC inprimaryantibodies. Theantibodiesusedwereas follows,Rabbit anti-Nucleostemin, (sc-67012),Goatanti-ALDH-1(sc-22588),werepurchasedfromSantaCruzBiotechnologyanddiluted in theratio1:1000 inTBST. Rabbitanti-actin (AANO1;Cytoskeleton,DenverCO)wasdiluted1:1000 inTBST andused as a gel-loading control. Rabbit anti-HER2 (2165)was obtained from Cell Signaling. The working concentration for all antibodies was 1µg/mL inWesternwashbuffer. Immunoreactiveproteinsweredetectedafter incubationwithhorseradishperoxidaseconjugatedsecondaryantibodydilutedto3x104inWesternwash buffer (goat anti-rabbit IgG, rabbit anti-goat IgG, and rabbit anti-mouse IgG (Bio-

7

Rad)). Blotswere treatedwith enhanced chemiluminescence reagents (PerkinElmer LifeSciences), andall proteinsweredetectedby autoradiography.Equalprotein loadingwasconfirmedbyPonceauSstainingofblottedmembranes.

8

Results10AT-Her2andSKBR3cells,bothHER2+breastcancercelllines,revealthehighestcancerstemcellexpressionToassessthevaryingtumorigeniccapabilityofestablishedhumanbreastcancercelllines,proteinlevelsofestablishedandcriticalbreastcancerstemcellmarkerswereanalyzedin10AT-Neo,10AT-Her2,MCF-7,MDA-MB-231cells,andSKBR3cells.MCF-7cellsrepresentaluminalsubtypewithlowexpressionofHER2,whileSKBR3cellsexpresscomparablelevelsofHER2tothe10AT-Her2cellline.MDA-MB-231cellsaretriplenegativebasalsubtypecellwith slight expression of HER2. Western blot analysis shows that 10AT-Her2 cells andSKBR3 cells possess equivalent levels of HER2,Nucleostemin, CD44, and the detoxifyingenzyme,ALDH-1protein.Furthermore,bothMCF-7andMDA-MB-231cellsexpressmuchlowerlevelsofnucleostemincomparedtothemoretumorigenic10AT-Her2andSKBR3cellline.Through thequantificationofestablishedstemcellmarkerssuchasnucleostemin,astem/progenitorexpressionbiomarker,andALDH-1,adetoxifyingenzyme implicated intherapyresistance,thecharacteristicofenhancedandenriched“stemness”canbeanalyzedperbreastcancercellline.Toquantify thepopulationof cellswithin thevariousbreast cancercellpopulations thatexpress functional levelsofALDH-1, anALDEFLUORassaydetermined thepercentageofALDH-1+ cells within each cancer cell line. The assay revealed greater than 88% of theALDH-1 in10AT-Her2cellpopulation,44%inSKBR3,and less than1%inMCF-7,10AT-Neo, andMDA-MB-231 cells are enzymatically active.Upon200µM I3C treatment, therewas no change in functional ALDH-1 subpopulations within normal and cancer humanmammaryepithelialcells indicatingtotalproteinlevelsoffunctionalALDH1mightnotbeassociatedwithmalignancy.

9

Figure8StemCell-LikeCharacteristicsofVariousCellLines.10AT-Neo, 10AT-Her2, MCF-7, MDA-MB-231 and SKBR3 cells were harvested, total cellextracts electrophoretically fractionated and the levels of expressed HER2, CD44, CD24,ALDH-1,nucleostemin(NS)andactinproteindeterminedbyWesternblots.

10

Actin

Nucleostemin

CD44

HER2

CD24

ALDH1

10AT

-Neo

10AT

-Her2

MCF

-7

MDA

-MB-231

SkBR3

11

Figure9QuantificationofALDH-1activity.10AT-Her2,SKBR3,MCF-7,MDA-MB-231,and10AT-Neocellsweretreatedwithorwithout200µMI3Cfor48housrandALDH-1activitywasquantifiedin10AT-Her2and10AT-NeocellsbyALDEFLUORassay,10AT-Her2cells and10AT-Neocellswerequantifiedby flowcytometryof500,000cellsintriplicateindependentcellcultures.

13

InVitroFormationofTumorsphereofHumanBreastCancerCellLinesNext,varioushumanbreastcancercelllinesweresubjectedtolimitingdilutionstudiesinordertounderstandthe invitro tumor-initiatingcapability.Byassessingtheformationoftumorspheresincellsuspensionculturesof10AT-Neo,10AT-Her2,MCF-7,andMDA-MBA-231 cells, breast cancer cell linesbecomeenriched for cancer stemcells, capable of self-renewal.10AT-Her2cellswerethemostefficientinformingtumorsphere-likeformationswithin 2 days of culture and by 6 days the cells were identifiable as full, completedtumorspheres.However,otherlessmetastaticcelllinessuchasthepreneoplasticcellline,10AT-Neo,andMCF-7wereincapableofformingtumorspheresevenwhen50,000cellshadbeen cultured. 10AT-Her2 cells displayed almost 10-fold efficiency in which cultures of2000 cells performed similar to assays of 25,000 SKBR3 and 50,000 MCF-7 cells. SinceSKBR3 and 10AT-Her2 cells express similar levels of the HER2 protein, the capacity for10AT-Her2 cells to efficiently produce tumorspheres cannot be only ascribed to highexpressionlevelsofHER2.Therefore,tumorsphereformation,whichrepresentsahallmarkofstemcellself-renwalproperties,isonlyattributedtothe10AT-Her2cellline,despiteitssimilaritiesinproteinlevelsofcancerstemcellmarkerstoSKBR3cells.

14

Figure10Comparing10AT-Her2tumorsphereformingefficiency.10AT-Her2, 10AT-Neo, SKBR3 and MCF-7 breast cancer cells were incubated at theindicatedcelldensitiesandtumorsphereformationefficiencywasquantifiedafter6daysinculture under nonadherent conditions. The presented values are an average of threeindependent experiments. The gel inserts are western blots showing relative levels ofHER2proteinexpressionandactincontrolsfromelectrophoreticallyfractionatedtotalcellextractsofMCF-7,SKBR3and10AT-Her2cells.

16

I3CenhancesNucleostemin-MDM2bindinganddisruptsMDM2-p53interactions

Co-immunoprecipitationexperimentsrevealtheI3C-mediatedprotein-proteininteractionsof breast cancer cell lines. We examined whether cellular biomarkers that contributestowards the cancer stem/ progenitor-like phenotype might be correlated with I3CregulationoftheMDM2-p53pathway.Therefore,acriticalcomponentthatisintriguinglyexpressed in self-renewing cancer stem/ progenitor cells is nucleostemin, a nucleolarGTPaseshowntodirectlybindtoMDM2.BindingofMDM2andnucleosteminrequiresthecentraldomainofMDM2andthecoiled-coilandacidicdomainsofnucleostemin(xx).WhileI3ChadnoeffectonthetotallevelsofnucleosteminandMDM2proteininboth10AT-Her2andSKBR3cells,levelsofserine166-phosphorylatedMDM2decreasedincellstreatedwithI3C.I3CtreatmentpromotedtheinteractionsofnucleosteminwithphophrylatedMDM2aswellas totalMDM2protein.Thissuggests that theI3C-mediatedbindingofnucleosteminwith Ser166 phosphorylated MMD2 ablates p53 interactions with MDM2 and can beattributed for I3C’sability toactivateap53-dependentapoptotic response in10AT-Her2cells.

Secondly, to analyzewhether the I3C regulation ofMDM2 protein interactionswith p53and/or nucleostemin occurs in other indole-carbinol-sensitive breast cancer cells, threewell-establishedcelllines,SKBR3,MCF-7andMDA-MB-231,weretreatedwithorwithoutI3Cfor48hoursandMDM2–p53andnucleostemin–MDM2co-immunoprecipitationswereperformed.. As shown in ----, I3C disrupted MDM2–p53 interactions and stimulatednucleostemin–MDM2 interactions in SKBR3 cells, a cell line that expresses nucleosteminandother stem/progenitor cell-likemarkerproteinsapproximately to the same levelsasthe10AT-Her2cellpopulation(Figure2C).Therefore,theeffectsofI3Connucleostemin–MDM2andMDM2–p53interactionsthatweobservedwith10AT-Her2cellsisnotlimitedonly to thisnewlydevelopedbreast cancer cell line. In contrast, even thoughMCF-7andMDA-MB-231 cells are sensitive to the anti-proliferative effects of I3C, there were nodetectable changes in MDM2–p53 or nucleostemin–p53 protein interactions after I3Ctreatment (Figure5B,C). Based on expression of marker proteins, the relative stemnesscharacteroftheMCF-7andMDA-MB-231cellpopulationscanbeconsideredlessthanthatofeitherSKBR3or10AT-Her2cells,whichmaybeassociatedwiththelackofanyeffectsofI3Ctreatmentonnucleosteminprotein–proteininteractions.

17

Figure11I3CinducesNucleostemin-MDM2interactionsandreleasep53incancerstemcells.10AT-Her2cellsweretreatedwithorwithout200μMI3Cfor48hours.TotalcellextractswereimmunoprecipitatedwitheitherMDM2ornucleosteminantibodies.Asacontrol,non-immune antibodies (of immunoglobulin G or IgG) and samples not immunoprecipitated(No IP) were used. All extracts were electrophoretically fractionated and probed byWesternblotanalysisusingantibodiesspecific top53,serine-166phosphorylatedMDM2or totalMDM2orwith antibodies specific to either serine-166phosphorylatedMDM2ortotalMDM2

19

Figure12

I3Cregulationofnucleostemin–MDM2andp53-MDM2protein-protein interactionsinwell-establishedhumanbreastcancercelllines.

SKBR3(A),MCF-7(B)andMDA-MB-231(C)humanbreast cancer cellswere treatedwithor without 200 μM I3C for 48 hours. Total cell extracts were immunoprecipitated witheitherMDM2(toppanels for each cell line)ornucleostemin (lowerpanels for each line)antibodies. As a control, non-immune antibodies (IgG) and samples notimmunoprecipitated(NoIP)wereused.Allextractswereelectrophoretically fractionatedandprobedbyWesternblotanalysisusingantibodiesspecifictop53(toppanels)orwithantibodiesspecifictoserine-166phosphorylatedMDM2ortotalMDM2(lowerpanels).Thelevelsofactinproteinremaining inthecellextractsafter the immunoprecipitationswereused as gel-loading controls in each experiment. I3C, indole-3-carbinol; IgG,immunoglobulin G; IP, immunoprecipitated; MDM2, murine double mutant 2; NS,nucleostemin.

21

Figure13IncreasedexpressionofCancerStemCell-LikePhenotypeiscorrelatedwithI3CResponsiveness.

22

10AT-Neo

MCF-7

MDA-MB-231

SkBR3

10AT-Her2 High Expression of Her2

Increasing Stem Cell Character

23

Figure14Schematicdiagramofnucleostemin-MDM2-p53interactions.I3Cenhancesp53stabilizationthroughprotein-proteininteractionsafterbindingtothedirecttargetproteinelastaseextracelullarly.

25

DiscussionIndole-3-Carbinol, anantiproliferativephytochemical,hasbeenshown tohaveantitumoreffects in a rangeof cellular and animalmodels of cancer, and in the severalwell-testedsystems, indole-3-carbinol and its syntheticderivatives, like1-benzyl-I3Cwere shown toregulate oncogenic transcriptional and/ or cell signaling pathways [18, 20]. The MCF-10AT-Her2modelsystemrepresentsanenrichedpopulationofcellswithacancerstemcellphenotypecapableoftumorsphereformationandinvivotumorxenograftcapitulation[19].However, tumorigenic cell lines such asMDA-MB-231 andMCF-7 cells, display cell cyclearrest in response to indole-3-carbinol treatment, but do not promote protein-proteininteractionsbetweenthestemcellprogenitormarkernucleosteminandMDM2[3,25,27-29]. Nucleostemin is necessary for the I3C antiproliferative response inMCF-10AT-Her2cancer stem cell line and SKBR3 cell line, which represents a HER2 positive cell linewithoutself-renewalcapability.I3Ctargetsco-localizationofMDM2tonucleostemininthenucleolus inwhichMDM2 iscompartmentalizedaway fromp53onlyafter I3C treatmentdespitepresenceofnucleostemininthepresenceorabsenceofI3Ctreatment.Asaresult,the propensity for nucleostemin’s activation after indole-3-carbinol treatment remainsunclear.Althoughnucleosteminhasbeenimplicatedtoplayaroleinstemcelldevelopmentand the limitless replicative potential of cancer cells, its role in cancer stem cells withMDM2 and p53 has not been fully elucidated. No protein levels of p53, MDM2, andnucleosteminwereaffectedinanyofthefivebreastcancerstemcells.Previousstudiesinourlabhaveidentifiedelastaseasadirecttargetproteinin10AT-Her2cells,anditwouldbe critical for future experimental studies to elucidate the direct binding of indole-3-carbinol inothertumorigeniccancercell lines.Furthermore,sincedownstreamtargetsofindole-3-carbinolhavebeenelucidatedhere,itwillbecomecriticaltoprobetheupstreamHer2 regulatory signaling pathway that produces a cancer stem cell phenotype afterectopicHer2expression.Indole-3-Carbinolwasmore effective in inducing apoptosis in tumorigenic breast cancercell lines with a cancer stem cell like phenotype, such as 10AT-Her2 and SKBR3 cells,comparedtothep14ARFnegativecells,MDA-MB-231andMCF-7cells,ornontumorigeniccell lines,MCF-10A, thatwas resistant to indole-3-carbinol. The observation of indole-3-carbinol’s sensitivitydidnotdependonHER2expressionasSKBR3cells and10AT-Her2cellsdisplayedsimilarproteinlevelsofHER2.However,tumorigeniccelllines,MCF-7andMDA-MB-231 cells, which possess a p14ARF deletion, did not activate a p53 dependentapoptosis.ThiscouldpotentiallybeexplainedbyARF,whichisatumorsuppressorknownto associatewith p53 andMDM2, and their inability to form tumorspheres at the sameefficiency of SKBR3 and 10AT-Her2 cells. Even though all four tumorigenic cell linesrespond to I3C, only 10AT-Her2 and SKBR3 cells potentiated increased interactionsbetweennucleosteminandMDM2.Theselectivityof indole-3-carbinolhasbeenobservedinothercancertypesaswell.Forexample,indole-3-carbinolanditsmorepotentderivativehavebeenobservedtoablateovarian,prostate,andotherreproductivecancers.Thisstudypresentsindole-3-carbinol’smolecularmechanismforavarietyofcancerswitharangeoftumorigenicpotential. Indole-3-carbinoland1-benzyl-I3Crepresentpromisingantitumorpreventativeagentsforbreastcancertherapeuticmoleculesforavarietyofhumanbreast

26

cancer cell types due to their selective modulation of protein-protein interactions thataffectcellcyclearrestorapoptosis.This study presents I3C as an efficacious and novel antiproliferative phytochemical thatinduces apoptosis in cancer stemcell like cells bymodulating thenucleosteminandp53pathways in HER2 overexpressing cancers. These cancer cells are an opportunity toelucidate the molecular mechanisms of I3C in vitro in adherent and spheroid types ofbreast cancer cells, highlighting I3C as a potential therapeutic strategy for patientswithmalignantcancertumorsburdenedbyacancerstemcellpopulation.

27

References1. Al-Hajj,M.,Cancerstemcellsandoncologytherapeutics.CurrOpinOncol,2007.

19(1):p.61-4.2. Kapoor,A.andS.Kumar,Cancerstemcell:Arogueresponsiblefortumordevelopment

andmetastasis.IndianJCancer,2014.51(3):p.282-289.3. Meng,L.,T.Lin,andR.Y.Tsai,Nucleoplasmicmobilizationofnucleosteminstabilizes

MDM2andpromotesG2-Mprogressionandcellsurvival.JCellSci,2008.121(Pt24):p.4037-46.

4. Kobayashi,T.,etal.,Nucleosteminexpressionininvasivebreastcancer.BMCCancer,2014.14:p.215.

5. Leth-Larsen,R.,etal.,FunctionalheterogeneitywithintheCD44highhumanbreastcancerstemcell-likecompartmentrevealsagenesignaturepredictiveofdistantmetastasis.MolMed,2012.18:p.1109-21.

6. Orecchioni,S.andF.Bertolini,CharacterizationofCancerStemCells.MethodsMolBiol,2016.1464:p.49-62.

7. Charafe-Jauffret,E.,C.Ginestier,andD.Birnbaum,Breastcancerstemcells:toolsandmodelstorelyon.BMCCancer,2009.9:p.202.

8. Li,X.,etal.,Clinicalsignificanceofnucleosteminandproliferatingcellnuclearantigenproteinexpressioninnon-smallcelllungcancer.JBUON,2015.20(4):p.1088-93.

9. Cabuk,D.,etal.,ThedistributionofCD44+/CD24-cancerstemcellsinbreastcanceranditsrelationshipwithprognosticfactors.JBUON,2016.21(5):p.1121-1128.

10. Calcagno,A.M.,etal.,Prolongeddrugselectionofbreastcancercellsandenrichmentofcancerstemcellcharacteristics.JNatlCancerInst,2010.102(21):p.1637-52.

11. Hayashida,T.,etal.,Cooperationofcancerstemcellpropertiesandepithelial-mesenchymaltransitionintheestablishmentofbreastcancermetastasis.JOncol,2011.2011:p.591427.

12. Carnero,A.,etal.,Thecancerstem-cellsignalingnetworkandresistancetotherapy.CancerTreatRev,2016.49:p.25-36.

13. Chabner,B.A.andM.J.Murphy,Jr.,Breastcancer:ataleoftwocenturies:withimplicationsforunderstandingcancermetastasisandcancerstemcellbiology.Oncologist,2005.10(6):p.369.

14. Dua,R.,etal.,EGFRover-expressionandactivationinhighHER2,ERnegativebreastcancercelllineinducestrastuzumabresistance.BreastCancerResTreat,2010.122(3):p.685-97.

15. Korkaya,H.,etal.,HER2regulatesthemammarystem/progenitorcellpopulationdrivingtumorigenesisandinvasion.Oncogene,2008.27(47):p.6120-30.

16. Tiwari,R.K.,etal.,HER-2/neuamplificationandoverexpressioninprimaryhumanbreastcancerisassociatedwithearlymetastasis.AnticancerRes,1992.12(2):p.419-25.

17. Kallioniemi,O.P.,etal.,ERBB2amplificationinbreastcanceranalyzedbyfluorescenceinsituhybridization.ProcNatlAcadSciUSA,1992.89(12):p.5321-5.

18. Licznerska,B.andW.Baer-Dubowska,Indole-3-CarbinolandItsRoleinChronicDiseases.AdvExpMedBiol,2016.928:p.131-154.

28

19. Tin,A.S.,etal.,Essentialroleofthecancerstem/progenitorcellmarkernucleosteminforindole-3-carbinolanti-proliferativeresponsivenessinhumanbreastcancercells.BMCBiol,2014.12:p.72.

20. Firestone,G.L.andL.F.Bjeldanes,Indole-3-carbinoland3-3'-diindolylmethaneantiproliferativesignalingpathwayscontrolcell-cyclegenetranscriptioninhumanbreastcancercellsbyregulatingpromoter-Sp1transcriptionfactorinteractions.JNutr,2003.133(7Suppl):p.2448S-2455S.

21. Megna,B.W.,etal.,Indole-3-carbinolinducestumorcelldeath:functionfollowsform.JSurgRes,2016.204(1):p.47-54.

22. Poindexter,K.M.,etal.,Cooperativeantiproliferativesignalingbyaspirinandindole-3-carbinoltargetsmicrophthalmia-associatedtranscriptionfactorgeneexpressionandpromoteractivityinhumanmelanomacells.CellBiolToxicol,2016.32(2):p.103-19.

23. Huang,G.,L.Meng,andR.Y.Tsai,p53ConfigurestheG2/MArrestResponseofNucleostemin-DeficientCells.CellDeathDiscov,2015.1.

24. Tsai,R.Y.,p53-guidedresponsetonucleosteminlossinnormalversuscancercells.CellDeathDis,2015.6:p.e2030.

25. Zhang,C.,etal.,Nucleosteminexertsanti-apoptoticfunctionviap53signalingpathwayincardiomyocytes.InVitroCellDevBiolAnim,2015.51(10):p.1064-71.

26. Salcido,C.D.,etal.,Molecularcharacterisationofsidepopulationcellswithcancerstemcell-likecharacteristicsinsmall-celllungcancer.BrJCancer,2010.102(11):p.1636-44.

27. Wei,B.,Q.Huang,andX.Zhong,Upregulationofnucleosteminincolorectalcanceranditseffectsoncellmalignancy.OncoTargetsTher,2015.8:p.1805-14.

28. Wu,H.,etal.,Nucleosteminregulatesproliferationandmigrationofgastriccancerandcorrelateswithitsmalignancy.IntJClinExpMed,2015.8(10):p.17634-43.

29. Yuan,F.,etal.,NucleosteminKnockdownSensitizesHepatocellularCarcinomaCellstoUltravioletandSerumStarvation-InducedApoptosis.PLoSOne,2015.10(10):p.e0141678.

29

ChapterII

Indole-3-Carbinolregulationofthetumorsuppressorp14ARFinbreastcancerstemcells

30

AbstractIndole-3-Carbinol is a naturally occurring compound in Brassica vegetables such asbroccoli and brussel sprouts that can induce apoptosis in breast cancer stem cells,accompaniedby selective inhibitionofAkt1, anpotentkinase that activatesdownstreamoncogenes and prohibits activation of p53, the tumor suppressor. A series ofcoimmunoprecipitation experiments in a previously established breast cancer stem cellline, 10AT-Her2 cells, elucidate the indole-regulated changes in protein-proteininteractions of the p53 axis, beginning with the direct target protein of I3C, a humanneutrophilelastase.Analysisoftheseprotein-proteincomplexeshighlightedI3C’sabilitytopromoteanti-proliferativerelationshipsinwhichtumorsuppressorssuchasp14ARFwereable tosequester thep53E3 ligase,MDM2. Isolationofp14ARFfromastem/progenitorstemcellmarker,nucleostemin,whichnormallyresidesinthenucleolus,furtherrevealedp14ARF’s stabilization of MDM2. Furthermore, chromatin immunoprecipitationexperimentsrevealeddecreasedbindingofoncogenictranscriptionfactors,nuclearfactorκB andMYCDNA binding to its respective promoter sequences. In indole-treated cells,MYCandnuclearfactorκBdecreaseinatimedependentmannertranscriptionallyandattheproteinlevel,therebyreducingthepresenceofnucleophosmin,anucleolarinhibitorofp14ARF. Our results demonstrate that I3C’s transcriptional effects activate p14ARF, atumorsuppressor,toactivelyengagewithactivatedMDM2,therebyfreeingp53toregulateapoptosis.

31

IntroductionARFisabasicproteinproductoftheINK4Alocus,comprisedofonly132aminoacids,andknown to localize into thenucleuswhere itpromotes its tumorsuppressive functionsaswellasisstoredinthenucleolusuntilactivation[1-4].Itsbasicnaturelendsitselftohavemanypotentialbindingpartnersandasaresult,isacriticalcomponentofmanysignalingpathways. For example, it has been implicated in sequesteringmouse doubleminute 2(MDM2) in the nucleus, resulting in decreased stabilization of p53 and inactivation ofapoptosisincanceroustumorcells[2].Asacriticalcomponentoftumorsurveillance,ARFaccumulates in response to oncogenic and genotoxic stresses and activatesDNAdamagepathwaystohaltcell-cycledivisionand/oreliminatecellsthathavesustainedirreparabledamage.MutationsinARFincludinghomozygousdeletion,promotermethylation,andlossof heterozygosity, show that it is a critical component of tumor surveillance in cancers,suchas lung,oral,prostate,gastric, colon,andbreast.ARF-nullmicehavebeenshowntospontaneousdevelop tumorsearly in lifewithhighproliferationrates [1,5,6].AlsoARFinactivity in the nucleolus is promoted by its sequestration by the nucleolar protein,nucleophosmin(NPM),whichpreventsARF’s interactionwithMDM2in thenucleoplasm.Little is known of the functions of p14ARF in breast cancer stem cells, but due to itsstructureandlocalizationcouldrepresentatherapeutictargetforcancertreatmentduetoitscriticalcontributiontotumorsuppressivefunctions[7,8].Previous research has identified a natural phytochemical, indole-3-carbinol, (I3C),whichdirectly targets breast cancer stem cells by binding and inhibiting a serine protease,elastase, and ablates its enzymatic activity in theMCF-10AT-Her2 cell line [9-11].Whenexpressing exogenous HER2 into human preneoplastic mammary epithelial cells, theparent MCF-10AT cell line, populations are enriched with stem/progenitor cell-likecharacter.The10AT-Her2cell linepopulationexpresseshigh levelsof stemcellmarkerssuch as ALDH1, nucleostemin [12], and CD44+/CD24-; possesses high tumorsphereforming efficiency; and produces tumor xenografts in vivo in mice at significantly lownumbers—representing an ideal model system for studying the proliferation of breastcancerviacancerstemcells[13].Thesestemcellmarkershavebeenhighlyimplicatedinidentifying and isolating cancer stem cells as they are associated with self-renewalproperties. Furthermore, use of in vitro suspension assays, which plate cells in limitingdilution assays to assess for tumorsphere formation have been critical in understandingtumor-initiating capabilities [10,14-16].Theyare alsohighly sensitive to I3C,which is anatural plant compound, derived by hydrolysis from glycobrassicin produced incruciferousvegetablesoftheBrassicagenussuchasbroccoli,cabbage,andbrusselsprouts.Ithaspotentproapoptoticpropertiesandhasexhibitedawiderangeofanti-tumoreffectsinliver,colon,lung,melanomas,cervical,endometrial,prostate,andbreastcancers.I3Chasdirect target proteins in these systems, such as elastase, which triggers distincttranscriptional and cellular signalingpathways, particularlyp53-dependent apoptosis [9,17-19].I3C’sinhibitionofelastaseinthebreastcancerstemcelllineiscriticalinidentifyingfuturenaturalandefficacioustherapeuticsthatablatebreastcancerstemcellsamongstatumor population [10]. Cancer stem cells are notoriously resistant to conventionaltherapies,suchaschemotherapyorradiotherapyandnoeffectivetherapeuticstrategyhasyetbeenidentified.Recentstudieshaveshownthatchemo-andradio-resistanceofcancer

32

stemcellsinsolidtumorsexplicatespatientrelapseandincreasedincidenceofmetastasisinmanycancers[14,15,20,21].Asaresult,efficacioustherapiesareneededintargetingthissmallelusivepopulationofcells thatprevent theablationof theentirebreast tumoraftervariousroundsoftherapeutictreatments.We demonstrate that I3C activates a p53-dependent apoptosis in the 10AT-Her2 breastcancer stem cell population by promoting interactions of the tumor suppressor proteinp14ARF and its oncogenic substrate, MDM2 [3, 22, 23]. As an alternate reading frameproteinproductoftheCDKN2Alocus,p14ARFisresponsibleforinhibitionofabnormalcellgrowthbyblockingtheMDM2oncogenepathway.Inactivationviahypermethylationofthepromoter region of p14ARF and other cell cycle modulators have been implicated inattenuation of p53 activity [4, 23-26]. I3C causes p14ARF’s sequestration of themurinedouble mutant 2 (MDM2) protein into the nucleus along with its binding partner,nucleostemin, thereby allowing the p53 tumor suppressor protein to escape from theMDM2 inhibition of apoptotic activity. Furthermore, I3C’s stabilization of p14ARF bydepleting p14ARF’s negative regulator nucleophosmin (NPM) further promotes p53apoptoticactivity.AlsoknownasB23,thisphosphoproteinisamolecularchaperonethataggregates and sequesters proteins like p14ARF into the nucleolus, preventing MDM2-p14ARF apoptotic complexes [7, 27-31]. I3C’s depletion of the nucleophosmin protein isattributed to inhibited transcriptional activity upstream transcription factors, myc, andproteincomplexes,NFkB[27,32-34].Therefore,I3C’spromotionofp14ARFdemonstratesitscriticalabilitytotargetandablateMCF-10AT-Her2cells,whichrepresentanopportunemodel system to study nucleostemin-p14ARF-MDM2-p53 cellular pathways in breastcancer stem cells. Our study has identified novel pathways into how the cancerstem/progenitor cell-associated marker, nucleostemin, and a p53-stabilizing tumorsuppressor, p14ARF, are directly involved in an anti-proliferative and pro-apoptosiscellularcascadesdisruptedbyI3C.

33

MaterialsandMethodsExpressionplasmidsandtransfectsHuman cytomegalovirus CMV-HER2 and CMV-Akt1 expression plasmids were kind giftsfromDrLeonardBjeldanes(DepartmentofNutritionalSciencesandToxicology,Universityof California at Berkeley). Transfection of expression vectors was performed usingSuperfect transfection reagent from QIAGEN (Germantown, MD, USA) per themanufacturer’srecommendedprotocol.Transfectionof10AT-Her2celllinePreneoplasticMCF-10AThumanmammaryepithelialcells(obtainedfromtheBarbaraAnnKarmanos Cancer Institute, Detroit, MI, USA) were stably transfected with either thehumanpCMV-HER2expressionvector,forming10AT-Her2cells.Cellswerestablyselectedfor2monthswithG418sulfate,purchasedfromCellgro(Manassas,VA,USA).10AT-Her2and10AT-Neocell lineswerecultured inDMEM/F-12,10%fetalbovineserum,50U/mlpenicillin/streptomycin (Lonza, Allendale, NJ, USA), 0.02 μg/ml epidermal growth factor(Promega,Madison,WI,USA),0.05μg/mlhydrocortisone,10μg/mlinsulinand0.1μg/mlcholeratoxin(Sigma-Aldrich,StLouis,MO,USA).The10AT-Her2cellline,theparentalMCF-10ATcellsandtheSKBR3breastcancercelllinewerevalidatedbyshort tandemrepeat (STR)DNA fingerprintingusingDDCMedicalCellLineAuthenticationLabServices(Fairfield,OH,USA).TheSTRprofileswerecomparedtoknown ATCC fingerprints and to the Cell Line Integrated Molecular Authenticationdatabase(CLIMA)version0.1.200808.TheSTRprofileofthe10AT-Her2celllinematchedthatofitsparentalMCF-10ATcellline,whereastheSKBR3cellsmatchedtheknownDNAfingerprintstothatofitself.Therefore,thecellpopulationofthe10AT-Her2cellline,whichdisplayssignificantstemness-likecharacter,isnotcontaminatedwithanyothercellline.CellProliferationAssayThe cellular response to I3C was examined using the Cell-Counting Kit-8 (DojindoMolecular Technologies, Inc, Santa Clara, CA, USA) based on the manufacturer’srecommendedprotocol[53).Cellswereplatedatadensityof5,000to7,000cellsperwellin24-wellplatescontaining500μlof culturemedium.After the indicated treatmentandincubation times at 37°C, 40μl CCK-8 reagentwas then added to eachwell,whichwerethenincubatedfor2hoursbeforereadingatawavelengthof450nm.FlowCytometryCells were plated onto six-well tissue culture plates at 70% confluency and treated asindicatedintriplicatewiththemediumchangedevery24hours.Followingtreatment,thecellswerewashedoncewithphosphatebuffered saline (Lonza), alsoknownasPBS, andsubsequently harvested with 1 mL of the reagent. Cells were lysed in 300µL of DNAstainingsolution(0.5mg/mLpropidiumiodide,0.1%%sodiumcitrate,and0.05%TritonX-

34

100).Nuclearfluorescenceofwavelengthmorethan585nmwasmeasuredonaBeckman-CoulterEPICSXLinstrumentwithlaseroutputadjustedtodeliver15megawattsat488nm.Foreach sample10,000nucleiwereanalyzedand thepercentageof cells inG1, S,G2/Mphase of the cell cyclewas determined by analysiswithMulticycle provided by PhoenixFlow Systems in the Cancer Research Laboratory, Flow Cycle Cytometry Facility of theUniversityofCalifornia,Berkeley.WesternBlotAfter indicated treatments, all cells were harvested in ice-cold PBS and lysed using aradioimmunoprecipitationassaybuffer(150mMNaCl,0.5%deoxycholate,0.1%NoNidet-p40, 0.1% SDS, 50 mM Tris) containing protease and phosphatase inhibitors (50 g/mlphenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 5 g/ml leupeptin, 0.1 g/mlNaF, 1mMdithiothreitol, 0.1 mM sodium orthovanadate and 0.1 mM β-glycerol phosphate). Aftercentrifugation,totalproteininthelysatewasestimatedusingtheproteinquantificationkit(Bio-Rad,Hercules,CA,USA).CelllysateswereelectrophoreticallyfractionatedusingSDS-PAGEandtransferredtonitrocellulosemembranes.Theblotswereblockedwith5%nonfatdry milk for an hour at room temperature and incubated with primary antibodiesovernight at 4°C. Immunoreactiveproteinsweredetected after a1-hour incubationwithhorseradish peroxidase conjugated secondary antibodies. Blots were then treated withenhanced chemiluminescence reagents (Estman Kodak, Rochester, NY, USA) forvisualizationonfilm. Primaryantibodies,nucleostemin,(sc-67012),CD44(sc-65412),CD-24(sc-70598),ALDH1(sc-22588), MDM2 (sc-5304), p53 (sc-6243), and lamin (sc-7293), MYC (sc-764),Nucleophosmin (sc-47725), and Akt1 (sc-5298) were purchased from Santa CruzBiotechnology.Actin (AANO1)wasobtained fromCytoskeleton (Denver, CO,USA).HER2(2165), was obtained from Cell Signaling Technology, Inc (Danvers, MA, USA). Allantibodieswerediluted1:1000inTBST(0.1MTris,150mMNaCl,0.05%Tween20).ReverseTranscriptase-PolymeraseChainReactions(RT-PCR)Cellsweretreatedwithmentionedcompoundsandharvestedin1mlofTri-reagent(Sigma,St.Louis,MO).RNAwasextractedand1mgwassubjectedtoRTusingrandomhexamers,GAPDHForward5’-TGAAGGTCGGAGTCAACGGATTTG-3’,GAPDHReverse:5’-CATGTGGGCCATGAGGTCCACCAC-3’.PCRproductswereanalyzedon1.2%agarosegelalongwith1-kb Plus DNA ladder from Invitrogen (Carlsbad, CA) and the products were visualizedwithGelRedfromBiotium(Hayward,CA).qPCRreactionsweresubjectedtoaninitialstepof10minat95°CtoactivatetheAmpliTaqGold,followedby40cyclesconsistingof15sat95°C,45sat57°C,15sat70°C.Fluorescencewasmeasuredat theendofeachelongationstep.DatawasanalyzedusingtheStepOnePlusRealTimePCRsystem(AppliedBiosystems,CarlsbadCalifornia),andathresholdcyclevalueCtwascalculatedfromthelinearphaseofeachPCRsample.

35

Co-immunoprecipitationAfter the indicated treatments, pre-cleared samples were then incubated with 50 μg ofspecificantibodiesas indicatedovernightat4°C. Immunoprecipitatedproteinwaselutedfrom beads by addition of gel-loading buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10%glycerol, 1% β-mercaptoethanol, 12.5 mM ethylenediaminetetraacetic acid (EDTA), 0.02mMbromophenolblue)andheatingthesampleat100°Cfor5min.SampleswereanalyzedbyWesternblot.The immunoprecipitationprocedurewasnearly100%efficientbecausethe resulting supernates did not contain any of the proteins of interest. If any of theintended immunoprecipitated proteinwas detected in a given supernatant fraction, thatexperiment was not used for the study. Also, the “No IP” control in each experimentrepresents samples that were subjected to the usual immunoprecipitation procedureexceptthatthebeadswereaddedwithouttheantibody.ChromatinImmunoprecipitation10AT-Her2cellsweregrownto~60%confluencyandtreatedfor48hrwith200uMI3CorDMSO vehicle control. DNA was cross-linked to proteins through the addition of 1%formaldehydeandincubatedatroomtemperaturefor5minutes.Thefixationreactionwasquenchedwithglycineforafinalconcentrationof125mMandincubatedfor5minutesatroom temperature. Cells were then harvested and lysted with chromatinimmunoprecipitations lysis buffer (50 mM NaCl, 1% Triton X-100, and 0.1% sodiumdeoxycholate)withprotease inhibitors(50g/mLphenylmethylsulfonyl fluoride,10g/mLaprotonin, 5 g/mL leupeptin, 0.1 g/mL NaF, 1mM dithiothreitol. 0.1 mM sodiumorthovanadate, and 0.1mM glycerol phosphate). Cells were sonicated and supernatantswere standardizedusingBio-RadProteinAssay (Bio-Rad,Hercules, CA). 1mgof proteinwas used for each IP. Protein-DNA complexses were incubated overnight with 15 µLantibody recognizingEts-1 fromSantaCruzBiotechnology. ComplexeswereprecipitatedbyaddingSepharose-Gbeads fromGEHealthcare (Piscataway,NJ)and incubatingat4°Cfor1hour.Beadswere thenwashed2xwithChIPLysisBuffer, followedby2xwithChIPwashbuffer(10mMTris,pH8.0,250mMLiCl,0.5%NP-40,0.5%sodiumdeoxycholateand1mM ethylendiaminetetraacetic acid). Sampleswere elutedwith ChIP elution buffer (50mMTrispH8.0,1%sodiumdodecylsulfate,and10mMethylenediaminetetraaceticacid)at65°Cfor18hourse.PCRwascarriedoutinatotalof50µL.PrimersforChIPexperimentswere as follows: MYC-forward 5’ GTAGAAGAGACGTGGAAG 3’, MYC-reverse5’TCGCCGCACGTATATATG3’NF-κB-forward:5'-TCTTTATCTCCCAGCCCTTTGG-3;reverse:5'-AGTGGAAGGAGTGGCAAATAGGGT-3. Productswere then visualizedon a 1.6%agarosegelbufferedwithTrisborate-EDTA,andtheproductswerethenvisualizedwithEthidiumBromide.

36

ResultsI3C stimulates interactions of the tumor suppressor p14ARF with the apoptoticregulatorMDM2anddisruptsp14ARFinteractionswithnucleosteminTodefine theproliferativecontextassociatedwith the I3Cregulationofp14ARFprotein-protein interactions, the anti-proliferative effects of this indolecarbinol compound wereinitially analyzed in 10AT-Her2 breast cancer cells treated for 48 hours with aconcentrationrangeofI3C.Cellswereinitiallyculturedinadherentmonolayersandtotalcell number was determined using cell-counting kit-8 assays. As shown in Figure 1A,similartootherhumancancercelltypes,200µMI3Cinducedanearmaximaldecreaseintotalcellnumberwithahalfmaximalresponsecalculatedtobeapproximately125µMI3C.Flowcytometry(Fig.1B)ofnuclearDNAstainedwithpropidiumiodiderevealedthat48hourstreatmentwith200µMI3C inducedasignificant increase in10AT-Hercellswithasub-G1DNA content compared to vehicle-controlled cells, which is indicative of theactivationofapoptosis.Therefore,200µMI3Cwasusedthroughoutthisstudy.Wepreviouslydemonstrated that I3C treatmentdisrupted the interactionofMDM2withthep53 tumor suppressor that freesp53 to trigger its apoptotic response in10AT-Her2breastcancercellsaswellasstimulateMDM2 interactionswith thestemcell/progenitormarker protein nucleostemin ([10]). In several other cell systems, p14ARF tumorsuppressorwasshowntosequesteractivatedandphosphorylatedMDM2awayfromp53,althoughlittleisknownaboutp14ARFproteininteractionsnucleosteminintheapoptoticresponse.Co-immunoprecipitaitonswerecarriedouttoassessthepotentialI3Cregulationof p14ARF protein-protein interactions in the context of the apoptotic response. 10AT-Her2breastcancercellsweretreatedwithorwithout200µMI3Cfor48hoursandthenwesternblotsof immunoadsorbedp14ARFprobedforeitherMDM2ornucleostemin. Asshown inFigure1C,under conditions inwhich there areno changes in the totalproteinlevelsofMDM2andnucleostemin,I3Ctreatmentsignificantlyincreasedtheinteractionofp14ARF with MDM2 and concomitantly decreased the interaction of p14ARF withnucleostemin. Complementary co-immunoprecipitations from cell extracts isolated fromI3C treated and untreated cells showed that immunoprecipitated MDM2 pulls downsignificantly more p14ARF protein after I3C treatment (Fig 1D), whereas,immunoprecipitated nucleostemin pulls down significantly less p14ARF after I3Ctreatment(Fig1E).I3Chadnoeffectonthetotallevelofp14ARFprotein.Takentogether,theseresultsdemonstratethatinthecontextofap53-dependentapoptoticresponsethatresultsfromreleaseofp53fromap53-MDM2proteincomplex[3],I3Ctreatmentinducedp14ARF-MDM2protein-proteininteractionsanddisruptedp14ARF-nucleosteminprotein-proteininteractions.

37

Figure15I3CEffectson10AT-Her2cells.I3C inhibits the proliferation of cultured MCF-10AT Her2 human breast cancer cells.Culturedcellsweretreatedfor48hoursatindicateddosesandquantifiedbycellcountingkit. 10AT-Her2 cells were treated with 200 µM I3C and the cell population contentsquantifiedbyflowcytometry.

38

Vehicle Control

200 µM I3C

DNA Content

Cel

l Cou

nt

0%

20%

40%

60%

80%

100%

0 50 100 200 300

Cel

l Via

bilit

y (%

)

I3C [µM]

39

Wepreviouslydemonstrated that I3C treatmentdisrupted the interactionofMDM2withthep53 tumor suppressor that freesp53 to trigger its apoptotic response in10AT-Her2breastcancercellsaswellasstimulateMDM2 interactionswith thestemcell/progenitormarker protein nucleostemin [10]. In several other cell systems, p14ARF tumorsuppressorwasshowntosequesteractivatedandphosphorylatedMDM2awayfromp53,althoughlittleisknownaboutp14ARFproteininteractionsnucleosteminintheapoptoticresponse.Co-immunoprecipitaitonswerecarriedouttoassessthepotentialI3Cregulationof p14ARF protein-protein interactions in the context of the apoptotic response. 10AT-Her2breastcancercellsweretreatedwithorwithout200µMI3Cfor48hoursandthenwesternblotsof immunoadsorbedp14ARFprobedforeitherMDM2ornucleostemin. Asshown inFigure1C,under conditions inwhich there areno changes in the totalproteinlevelsofMDM2andnucleostemin,I3Ctreatmentsignificantlyincreasedtheinteractionofp14ARF with MDM2 and concomitantly decreased the interaction of p14ARF withnucleostemin. Complementary co-immunoprecipitations from cell extracts isolated fromI3C treated and untreated cells showed that immunoprecipitated MDM2 pulls downsignificantly more p14ARF protein after I3C treatment (Fig 1D), whereas,immunoprecipitated nucleostemin pulls down significantly less p14ARF after I3Ctreatment(Fig1E).I3Chadnoeffectonthetotallevelofp14ARFprotein.Takentogether,theseresultsdemonstratethatinthecontextofap53-dependentapoptoticresponsethatresultsfromreleaseofp53fromap53-MDM2proteincomplex[4],I3Ctreatmentinducedp14ARF-MDM2protein-proteininteractionsanddisruptedp14ARF-nucleosteminprotein-proteininteractions.

40

Figure16I3Cpromotesp14ARF interactionwithMDM2anddiminishesnucleostemin-MDM2binding.10AT-Her2cellsweretreatedwithorwithout200µMI3Cfor48hours.Totalcellextractswereimmunoprecipitatedwithp14ARF,Nucleostemin,MDM2antibodiesrespectively.Asacontrol,non-immuneantibodies(IgG)andsamplesnot immunoprecipitated(NoIP)wereused. All extracts were electrophoretically fractionated and probed by western blotanalysis.AntibodiesspecifictoNucleostemin,MDM2,andp14ARFwereused.

42

EffectsofexpressingaconstitutivelyactiveAkt1ontheI3Capoptoticresponseandp14ARFandnucleosteminassociatedprotein-proteininteractionsIt iswellestablished thatMDM2functionandbindingwithp53 is facilitatedby itsAkt1-mediated phosphorylation, which is mechanistically connected to the control of cellularapoptosis. Therefore, the role of Akt1 in the I3C apoptotic response and regulation ofprotein-protein interactionswas functionally evaluatedby expression stable transfectionof10AT-Her2cellswitheitheraconstitutiveactiveAkt1expressionvector(CA-Akt1)oranemptyvectorcontrol(EV).Cellsweretreatedfor48hourswithorwithout200µMI3CandwesternblotswereusedtoevaluatetheeffectivenessofthetransfectionsbyprobingtotalcellextractsforAkt1proteinusingAkt-1specificprimaryantibodies.AsshowninFigure2A,innon-transfected(Non-Trans)andemptyvectortransfectedcells,treatmentwithI3CdownregulatedAkt1proteinlevels,whereasincellstransfectedwithCA-Akt1,I3ChadnoeffectonthelevelsofAkt1immunoreactiveproteininthecells(Fig2A). Flowcytometryanalysisofsub-G1DNAcontent,whichisindicatedofapoptosis,revealedthatexpressionofconstitutivelyactiveAkt1in10AT-Her2cellspreventedI3Cinducedapoptosis,whereas,inemptyvectortransfectedcellsI3Cefficientlyinducedanapoptoticresponse(Fig2B).

43

Figure17I3CinducedapoptoticresponseismediatedbyAkt1.10AT-Her2cellstransfectedwiththecontrolvector,“EmptyVector,”orwithconstitutivelyactive, “CA-Akt1”were treatedwith orwithout 200µM I3C for 48hours. Expression ofAkt1 was confirmed by western blot. DNA content was assessed by flow cytometry.

45

BecauseoftheefficientrescuefromtheI3C-mediatedapoptoticresponse,weassessedtheability of expressed constitutively active Akt1 to reverse or attenuate I3C regulatedprotein-protein interactions involving p14ARF, nucleostemin, MDM2 and p53. The co-immunoprecipitationswerecarriedoutfromcellstransfectedwitheithertheconstitutivelyactiveAkt1expressionvector(CA-Akt1)ortheemptyvectorcontrol(EV)aftertreatmentfor 48 hours with or without 200 µM I3C. Expression of constitutively active Akt-1completely reversed the I3C stimulated protein-protein interactions involving p14ARF-MDM2 and nucleostemin-MDM2 when either p14ARF or nucleostemin wereimmunopreciptated(Fig2Cand2D)orwhenMDM2wasinitiallyimmunoprecipitated(Fig2E). Furthermore,expressionofconstitutivelyactiveAkt-1preventedtheI3CdisruptionofMDM2-p53 interactionsandofp14ARF-nucleostemin interactions(Fig2C,2Dand2E),andalsoreversedtheI3Cinducedlossoftotalphospho-MDM2(Fig2D).Expressionoftheempty vector (EV) had no effect on the I3C regulation of protein-protein interactions.Takentogether,theI3ClossofAkt-1proteinlevels,anditsphosphorylationofitscellulartargets, including MDM2, plays an important role in the control of protein-proteininteractionsassociatedwiththep53apoptoticresponse.

46

Figure18Constitutively active Akt1 alters I3C regulation on the molecular interactionsbetweenp14ARF,Nucleostemin,andMDM2.10AT-Her2cellsweretransfectedwitheitherconstitutivelyactiveAkt1oranemptyvectorcontrolandthentreatedwithorwithout200µMI3Cfor48hours.Totalcellextractswereimmunoprecipitatedwithp14ARF,nucleostemin,orMDM2antibodies,electrophoreticallyfractionatedandwesternblotsprobedwithMDM2,nucleostemin,p14ARForactinspecificantibodies as indicated. Negative controls were immunoprecipitations carried out withnon-immuneantibodies(IgG)orsamplesthatwerenotimmunoprecipitated(NoIP).

47

E

I3C

I3C

I3C

48

I3Cdownregulationofnucleophosmingeneexpressionaccountsforthelossofthep14ARF-nucleophosminproteincomplexTo further explore the mechanism underlying the I3C control of protein-proteininteractionsandp14ARFfunction,10AT-Her2breastcancercellstreatedwithorwithoutI3Candcoimmunoprecipitatedp14ARFproteinwasprobedforp14ARFbindingpartners.One important regulator of p14ARF is nucleophosmin, a nucleolar phosphoproteinexpressed at high levels in certain highly mutated tumor types [6] that can inhibitp14ARF’stumorsuppressorpropertiesandcanpreventapoptosisbysequesteringp14ARFinthenucleolus[2].Co-immunoprecipitationanalysisfromI3CtreatedanduntreatedcellsshowedthatI3Cstronglytriggeredasignificantlossofp14ARFboundwithnucleophosminthatwasaccountedforthedownregulationoftotalnucleophosminproteinlevels(Fig3A).Weproposethatthelossoftotalnucleophosminproteinallowsunboundp14ARFproteinto bind to and sequester MDM2 in the nucleolus and thereby allow the p53 apoptoticresponse.

49

Figure19

I3Creleasesp14ARFfromnucleophosminincancerstemcells.10AT-Her2cellsweretreatedwithorwithout200µMI3Cfor48hours.Totalcellextractswere immunoprecipitatedwithp14ARFantibodies.As a control, non-immuneantibodies(IgG) and samples not immunoprecipitated (No IP) were used. All extracts wereelectrophoreticallyfractionatedandprobedbywesternblotanalysis.Antibodiesspecifictonucleophosminwereused.

51

To assess the kinetics of I3C down regulation of nucleophosmin gene expression, 10AT-Her2cellswere treatedwith I3Covera48hour timecourseandnucleophosminproteinlevels were determined by western blot analysis and reverse transcriptase polymerasechainreaction(RT-PCR)wasusedtomonitorthelevelsofnucleophosmintranscripts. AsshowninFigure20,I3Cstronglydownregulatednucleophosminproteinlevelsby48hoursand its corresponding transcript levels were down regulated within 24 hours of I3Ctreatmentandcompletelyablatedby48hours.Undertheseconditions,thelevelsofactinand GAPDH transcripts remained constant. These results suggest that the loss ofnucleophosmintranscriptsaccountsforthelossofnucleophosminprotein.Theexpressionofmycproteinandtranscript levels,aknowntranscriptionalregulatorofnucleophosminexpression, was examined over the same time course. As also shown in Figure 20, I3Cmore rapidly down regulated Myc gene expression compared to the observed loss ofnucleophosminexpression. MycProteinexpressionisalmostcompletelyabolishedby48hoursandtranscriptlevelsarediminishedcompletelyby36hours.Thesekineticssuggestthat the I3C down regulation of Myc may be causal for the down regulation ofnucleophosminexpression.

52

Figure20I3C downregulates protein and transcriptional levels of nucleophosmin and atranscriptionfactor,MYC.10AT-Her2cellsweretreatedwithorwithout200µMI3Cfortheindicateddurations.Totalcellextractswereelectrophoretically fractionatedandwesternbltosprobedforMYCandNucleophosmin.Actinwasusedasagelloadingcontrol.ToassessI3Cmediatedregulationatthetranscriptlevel,totalRNAwasisolated.ThetranscriptlevelsofNucleophosmin,MYC,and GAPDH were determined by reverse transcriptase-polymerase chain reaction usingspecific primers and the products fractionated by electrophoresis on a 1.2%agarose gelandvisualizedonanultraviolet transilluminator.GAPDHservedasa gel loading control.

54

I3CtreatmentdisruptsMycinteractionswiththenucleophosmingenepromoterandNFkBinteractionswiththeMycgenepromoter.Nucleophosmin contains a consensus Myc DNA binding site in its promoter at -730 bpupstream of the transcriptional start site, suggesting that the I3C-mediated loss of Mycprotein levels should result in the loss of Myc interactions with the nucleophosminpromoter. Chromatin immunoprecipitations (ChIP) assaywas used to directly test thispossibility. 10AT-Her2cellsweretreatedwithorwithoutI3Cfor48hours,andgenomicfragments that were cross-linked to protein were immunoprecipitated with anti-Mycantibodies or with an IgG control antibody. As shown in Figure 21, PCR analysis usingprimers specific to the Myc consensus binding site in the nucleophosmin promoterrevealed that I3C significantly down regulated endogenous Myc interactions with thispromoter.Onepercentinputwasusedasaloadingcontrol.

TheabilityofconstitutivelyactiveAkttopreventtheI3Cregulatedp14ARFprotein-proteininteractions(seeFigure17),suggeststhatadownstreamcomponentofAktsignalingmaybelinkedtotheI3CdownregulationofMycexpression.Onesuchtranscriptionalregulatoris NFkB because the Akt mediated phosphorylation of IkB and of NFkB functions tomaintain nuclear localized NFkB in an active form. A functional NFkB binding site islocated at -1096 bp upstream of the Myc gene transcriptional start site. Therefore, aparallel ChIP assay was completed 10AT-Her2 cclls treated with or without I3C for 48hourstoassessendogenousinteractionsofNFkBwiththeMycgenepromoter. Asshownin Figure 21, although I3Cdid not down regulate totalNFkBprotein levels, I3C stronglydownregulatedtheabilityofendogenousNFkBtointeractwiththeMycpromoter,whichweproposemaymediatetheI3CdownregulationofMycgeneexpression.

55

Figure21I3CablatesbindingofMYCtotheNucleophosminpromoterandNFkBbindingtotheMYCpromoter.10AT-Her2cellsweretreatedwithorwithout200µMI3Candsubjectedtowesternblotanalysisandchromatinimmunoprecipitationassay.ExpressionofMYCandNFkBwereconfirmedbyanti-MYCandanti-NFkBantibodiesthroughwesternblot.Genomicfragmentsthatwerecross-linkedtoproteinwereimmunoprecipiatedwithanti-MYCoranti-NFkBantibodies,orwithacontrolIgGantibody.

57

Figure22SummaryofI3C-Mediated14ARFregulation.

59

DiscussionInhumanbreastcancercells,protein-proteininteractionsofthetumorsuppressorp14ARFanditsoncogenicsubstrate,MDM2,areabiologicallysignificantrelationshipthatactivatesp53-dependentapoptosis. In thep53pathway,p14ARFmaintainsMDM2 localized in thenucleolussothatMDM2,itsE3ligase,cannotdegradep53,andthuspromotesapoptosisinbreastcancerstemcells.PreviousstudiesinourlabhaveshownI3Cmediatedlocalizationof MDM2 into the nucleolus in 10AT-Her2 cells to initiate a p53-dependent apoptosis.Furthermore, direct binding of indole-3-carbinol to its target protein, elastase, in breastcancer stemcellshas illustrated indole-3-carbinolsapoptotic regulation. I3Cdisrupts theinteraction of elastase and CD40, preventing it from associating with a tumor necrosisfactor familyprotein, calledTRAF6.TRAF6 isknownasanE3 ligase that stabilizesAkt1,andthustheimpairmentofAkt1’subiquitinationactivatesp53-dependentapoptosis[9,10,35]. Disruption of the p14ARF-MDM2-p53 pathways is commonly developed in manycancers, preventing the shuttling of p53 in an auto-regulatory feedback loop [22]. Ourstudies show that I3C inhibits MDM2-p53 interactions through increased p14ARFactivation, which is promoted by ablation of protein levels of its negative regulator,nucleophosmin[28].Thissuggestspromisingopportunitiesandmanyanti-cancer targetsinpatientswithhighlymetastaticandproliferativebreastcancertumors.OurstudyestablishesthatI3Cinhibitscellularproliferationofbreastcancerstemcellsbyregulating the protein-protein interactions of the tumor suppressor, p14ARF, anddownstreamtranscriptional targetssuchasMYCandNFkB. These findings implicate thecriticalinvolvementofp14ARFsignalingthroughitsbindingpartnersnucleophosminandnucleostemininattenuatingcellularproliferationandinducingapoptosisinhumanbreastcancer stem cells. The p14ARF tumor suppressor plays an important role in cancer bypromotingtheaccumulationofthep53tumorsuppressorfollowedbytheinductionofp53-mediated cell growth arrest or cell death [24, 36-38]. Loss of either p53 or p14ARFcontributestotheunrestrainedgrowthofmostcancers.Furthermore,p14ARF’sregulationcan be attributed specifically to its binding partner, nucleophosmin. Nucleophosminfunctionstosequesterp14ARF,arolethatisinhibitedbyI3C’stranscriptionalregulationofnucleophosmin, aswell as its transcriptional regulator,myc [39]. Based onwestern blotand PCR studies, I3C downregulation of nucleophosmin can be directly attributed todecreased protein and transcript levels of its transcription factor,myc aswell as one ofmyc’s own transcription factor, NFkB. NFkB is known to be a dimeric complex oftranscriptionfactorsthattransactivatethemycpromoterinhighlymetastaticcancercells.Reduced myc expression and growth arrest is accompanied by p53 activation.Furthermore,I3CwaseffectiveininhibitingAktactivationandablatingproteinexpressionofp14ARF’snegativeregulator,nucleophosmin.However,I3Cdidnotalterproteinlevelsofp53,nucleostemin,MDM2,andp14ARF,suggestingI3C’sregulationcanbeonproteinlevelaswellastheprotein-proteininteractions.StudyingtheDNAbindingregulatoryroleofI3Cis a powerful approach to understanding the therapeutic value of I3C as a naturallyoccurring,cost-accessibleplantmoleculewithmanyanti-cancerabilities.Togetherwithpreviousresultsfromourlaboratory,I3Cshowstobepromisingtherapeuticagent forablationofbreast cancerstemcellsbystabilizingp53 throughenhancementof

60

protein-protein interactions of p14ARF and MDM2, localization of MDM2 into thenucleolus, and downregulation of oncoproteins implicated in p14ARF deregulation andrampantcellularproliferation, suchasMYCandNFkB [7,27,32,33,40,41].Overall, theidentificationofmolecular targets in tumorigenicbreast cancer cellshasbeen importanttherapeuticidentificationsasmostcancerstemcellsarenotoriouslyresistanttotherapiesandshowpoorprognosisforpatientswithhighexpressionofstemcellprognosisfactors,such as CD44, ALDH1, and nucleostemin. For example, tumors associated with highexpressionofthecellsurfacemarker,CD44,andlackofCD24,presentcellswhichhaveself-renewalcapacityandabilitytoproliferateinsuspensionasmammospheresinnonadherentsuspensionmedia.Furthermoremammospheresfromthesebreastcancercellsretaintheirtumor-initiating ability in vivo when isolated and injected into nude, athymic mice.Therefore,understandthemolecularphenotypeof10AT-Her2cellsanditsresponsivenessto indole-3-carbinol is critical in developingmore effective therapeutic strategies beforethedeathofbreastcancerpatientsfrommetastasis.[14,21,42] Weobservedthat10AT-Her2 cells are highly sensitive to the antitumor effects of I3C and triggers a molecularresponsethatnormallycausesproliferationincancerstemcells.Because10AT-Her2cellsexhibit increasedexpressionofnucleosteminandconfersselectiveresponsivenesstoI3C,regulationof thep53’sprotein-protein interactionswithMDM2,p14ARF,andAkt1showconsistentablationoftumorigenicgrowth.BreastcancercontinuestobediagnosedinoneineightAmericanwomenandpersistsasthe second leading cause of cancer death in women [15, 21, 43, 44]. Further effectivetreatmentsforbreastcancermaybedevelopediffullknowledgeofmechanismsofactioninvarious proteins involved in tumor suppressor pathways, including the p53-p14ARFpathwayisrevealed.NucleosteminasanearlyprogenitorstemcellmarkermaybecameanimportantprognosticmarkerintreatmentofpatientswithhighlymetastaticandmalignantHer2positivetumors.Furthermore,previousresearchinourlaboratoryhasdemonstratedelastase as a direct target protein for indole-3-carbinol. I3C’s anti-proliferative signalingbegins by disrupting downstream targets of elastase, such as CD40. I3C disrupts thebindingofCD40toitsdownstreameffector,tumornecrosisfactorreceptoractivatorfactor-6 (TRAF6),which is also is an E3 ubiquitin ligase for Akt1.We observed that the directinteraction between elastase on the cellularmembranewith I3C triggers a downstreamsignalingcascadeinvolvingCD40andTRAF6potentiatesap53apoptoticmechanismthatablates the growth of cancer stem cells [9, 19, 35]. Through exploitation of the p53-dependentapoptoticpathwayanditsupstreamregulator,elastase,adirecttargetproteinofindole-3-carbinol,thephytochemicalcanprovideeaseintreatmentaswellaslong-termprevention.Theoverallsignificanceofourpre-clinicaleffortscanpotentiallybecriticalinfurthering development of cheaper, more effective low-cost indole based molecules fortargeted treatment of cancer stem/ progenitor cell populations in solid breast cancertumorsforwomenimpactedby latestageandmetastaticclasscancers.Cancerstemcellspossess the capacity to self-renewand to generateheterogenous lineages of cancer cellsthat comprise tumors, and as a result, are critical in understanding their role in tumorinitiationandmetastasis.

61

References1. Gallagher,S.J.,R.F.Kefford,andH.Rizos,TheARFtumoursuppressor. IntJBiochem

CellBiol,2006.38(10):p.1637-41.2. Maggi,L.B., Jr.,etal.,ARFtumorsuppressioninthenucleolus.BiochimBiophysActa,

2014.1842(6):p.831-9.3. Qi, X.W., et al., Studies on expression of p14ARF and MDM2 in human thyroid

neoplasms.PanminervaMed,2015.57(1):p.43-7.4. Wang, J.,etal.,Roleofp14ARF-HDM2-p53axisinSOX6-mediatedtumorsuppression.

Oncogene,2016.35(13):p.1692-702.5. Lindstrom,M.S. andY. Zhang,B23andARF: friendsor foes? CellBiochemBiophys,

2006.46(1):p.79-90.6. Kamijo,T.,etal.,TumorspectruminARF-deficientmice.CancerRes,1999.59(9):p.

2217-22.7. Li,Z.andS.R.Hann,Nucleophosminisessentialforc-Mycnucleolarlocalizationandc-

Myc-mediatedrDNAtranscription.Oncogene,2013.32(15):p.1988-94.8. Chin,L.,J.Pomerantz,andR.A.DePinho,TheINK4a/ARFtumorsuppressor:onegene-

-twoproducts--twopathways.TrendsBiochemSci,1998.23(8):p.291-6.9. Aronchik, I., et al., Target protein interactions of indole-3-carbinol and the highly

potent derivative 1-benzyl-I3C with the C-terminal domain of human elastaseuncouplescellcyclearrest fromapoptoticsignaling.Mol Carcinog, 2012.51(11): p.881-94.

10. Tin,A.S.,etal.,Essentialroleofthecancerstem/progenitorcellmarkernucleosteminfor indole-3-carbinol anti-proliferative responsiveness in human breast cancer cells.BMCBiol,2014.12:p.72.

11. Nguyen,H.H.,etal.,Thedietaryphytochemicalindole-3-carbinolisanaturalelastaseenzymaticinhibitorthatdisruptscyclinEproteinprocessing.ProcNatlAcadSciUSA,2008.105(50):p.19750-5.

12. Li,X.,etal.,Clinicalsignificanceofnucleosteminandproliferatingcellnuclearantigenproteinexpressioninnon-smallcelllungcancer.JBUON,2015.20(4):p.1088-93.

13. Shi,C.,etal.,CD44+CD133+populationexhibitscancerstemcell-likecharacteristicsinhumangallbladdercarcinoma.CancerBiolTher,2010.10(11):p.1182-90.

14. Al-Hajj, M., Cancer stem cells and oncology therapeutics. Curr Opin Oncol, 2007.19(1):p.61-4.

15. Perou, C.M., et al., Molecular portraits of human breast tumours. Nature, 2000.406(6797):p.747-52.

16. Pfeiffer,M.J. and J.A. Schalken,Stemcellcharacteristics inprostatecancercell lines.EurUrol,2010.57(2):p.246-54.

17. Aronchik, I., et al., The antiproliferative response of indole-3-carbinol in humanmelanomacells is triggeredbyan interactionwithNEDD4-1anddisruptionofwild-typePTENdegradation.MolCancerRes,2014.12(11):p.1621-34.


62

19. Brew, C.T., et al., Indole-3-carbinol inhibitsMDA-MB-231breast cancer cellmotilityand induces stress fibers and focal adhesion formation by activation of Rho kinaseactivity.IntJCancer,2009.124(10):p.2294-302.

20. Carnero,A., et al.,Thecancerstem-cellsignalingnetworkandresistancetotherapy.CancerTreatRev,2016.49:p.25-36.

21. Charafe-Jauffret,E.,C.Ginestier,andD.Birnbaum,Breastcancerstemcells:toolsandmodelstorelyon.BMCCancer,2009.9:p.202.

22. Agrawal, A., et al., Regulation of the p14ARF-Mdm2-p53 pathway: an overview inbreastcancer.ExpMolPathol,2006.81(2):p.115-22.

23. Carr-Wilkinson, J., et al., High Frequency of p53/MDM2/p14ARF PathwayAbnormalitiesinRelapsedNeuroblastoma.ClinCancerRes,2010.16(4):p.1108-18.

24. Pare,R., J.S.Shin,andC.S.Lee,Increasedexpressionofsenescencemarkersp14(ARF)and p16(INK4a) in breast cancer is associated with an increased risk of diseaserecurrenceandpoorsurvivaloutcome.Histopathology,2016.69(3):p.479-91.

25. Wazir, U., et al.,P14ARF isdown-regulatedduring tumourprogressionandpredictstheclinicaloutcomeinhumanbreastcancer.AnticancerRes,2013.33(5):p.2185-9.

26. Oliner, J.D., et al., Oncoprotein MDM2 conceals the activation domain of tumoursuppressorp53.Nature,1993.362(6423):p.857-60.

27. Li, Z. andS.R.Hann,TheMyc-nucleophosmin-ARFnetwork:acomplexwebunveiled.CellCycle,2009.8(17):p.2703-7.

28. Yung,B.Y.,Oncogenicroleofnucleophosmin/B23.ChangGungMedJ,2007.30(4):p.285-93.

29. Weber,J.D.,etal.,CooperativesignalsgoverningARF-mdm2interactionandnucleolarlocalizationofthecomplex.MolCellBiol,2000.20(7):p.2517-28.

30. Lim, M.J. and X.W. Wang, Nucleophosmin and human cancer. Cancer Detect Prev,2006.30(6):p.481-90.

31. Grisendi, S., et al.,Nucleophosminandcancer. Nat Rev Cancer, 2006.6(7): p. 493-505.

32. Ling, J. and R. Kumar, Crosstalk between NFkB and glucocorticoid signaling: apotentialtargetofbreastcancertherapy.CancerLett,2012.322(2):p.119-26.

33. Yung, B.Y., c-Myc-mediated expression of nucleophosmin/B23 decreases duringretinoicacid-induceddifferentiationofhumanleukemiaHL-60cells.FEBSLett,2004.578(3):p.211-6.

34. Yeh,C.W.,etal.,Ras-dependentrecruitmentofc-Mycfortranscriptionalactivationofnucleophosmin/B23 in highly malignant U1 bladder cancer cells. Mol Pharmacol,2006.70(4):p.1443-53.

35. Aronchik,I.,L.F.Bjeldanes,andG.L.Firestone,Directinhibitionofelastaseactivitybyindole-3-carbinol triggersaCD40-TRAFregulatorycascadethatdisruptsNF-kappaBtranscriptional activity in human breast cancer cells. Cancer Res, 2010. 70(12): p.4961-71.

36. Bennecke, M., et al., Ink4a/Arf and oncogene-induced senescence prevent tumorprogressionduringalternativecolorectaltumorigenesis.CancerCell,2010.18(2):p.135-46.

37. Ozenne,P.,etal.,TheARFtumorsuppressor:structure,functionsandstatusincancer.IntJCancer,2010.127(10):p.2239-47.

63

38. Dominguez-Brauer, C., et al.,Tumor suppressionbyARF:gatekeeperandcaretaker.CellCycle,2010.9(1):p.86-9.

39. Sherr, C.J., The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol,2001.2(10):p.731-7.

40. Rayet, B. and C. Gelinas, Aberrant rel/nfkb genes and activity in human cancer.Oncogene,1999.18(49):p.6938-47.

41. Watson, J.D., et al., Identifying genes regulated in a Myc-dependent manner. J BiolChem,2002.277(40):p.36921-30.

42. Orecchioni, S. and F. Bertolini,CharacterizationofCancerStemCells.MethodsMolBiol,2016.1464:p.49-62.

43. Calcagno,A.M.,etal.,Prolongeddrugselectionofbreastcancercellsandenrichmentofcancerstemcellcharacteristics.JNatlCancerInst,2010.102(21):p.1637-52.

44. Freudenberg, J.A., et al.,TheroleofHER2 inearlybreastcancermetastasisandtheoriginsofresistancetoHER2-targetedtherapies.ExpMolPathol,2009.87(1):p.1-11.

64

ChapterIII

Identificationofmalignantmelanoma-initiatingcells(MMICs)andvaryingtumorigenicpotentialinfivephenotypicallydistinctmelanomacelllinesandtheir

responsivenesstoindole-3-carbinol

65

AbstractThecancerstemcelltheoryindicatesthattumorigenesisispropelledbyasubpopulationoftumorcellsthatareabletoself-renew,giverisetoaheterogenousprogeny,andinitiatethegrowth of new tumors. Current therapies have been ineffective in targeting cancer stemcells to reduce tumor size, cancer incidence, and mitigating poor cancer prognosis. Achemopreventativeagentisindole-3-carbinol,hydrolyzedinthevegetablesoftheBrassicagenussuchasbrusselsproutsandradish.I3CpromotesaG1cellcyclearrestinmetastaticcancers with a wild type PTEN, wild type p53 mutation, and the most common BRAFmutation in melanomas, the V600E mutation. However, our results also indicate themolecular responseof a cancer cell line enriched inmalignantmelanoma initiating cells,indicatinga strong sensitizedproliferative response in cancerswithahighphenotype tocancer stem cells, a PTEN mutation deletion, and a rare BRAF mutation, the V600Dmutation.Since melanoma cancer stem cells, also known as malignant melanoma-initiating cells(MMICs)havethecapabilitytoself-renewandpotentiateintotumorswithpoorprognosisfor patients, it becomes evenmore critical to createmolecularly targeted therapies. Theorigin of a cancer stem cell is poorly understood as cancer cells have propertiesreminiscent of both stem cells aswell as cancer cells. Although not necessarily a clonalderivationof a stemorembryonic cell, cancer stemcellshavepresumablyarisen fromamalignant transformation event that deregulated the self-renewal ability of normal stemcells. Furthermore, perhaps even differentiated cells could experience a transformativemutationthatledtoacquiringpropertiesofcancerstemcells.Advancementsingeneticandmolecular therapies to identifysignalingcomponents thatpromoteself-renewalcapacity,apoptosisevasion,andinitiatecarcinogenesisgeneratefutureabilitiestoreverserelapseinpatientswithraremelanomamutations.Ourstudiesspecifythefirstevidencethatanaturalanti-tumoragentregulatesthecellularproliferativeresponseofmalignantmelanoma-initiatingcells,whichinducesaG1cellcyclearrestandreducestheformationofincreasingmulticlonaltumorigencity.

66

IntroductionEven though overall cancer incidence has decreased, the incidence of humanmalignantmelanoma continues to increase in theUnited States. As a result,melanoma is the sixthmostcommoncancerinmalesandtheseventhinfemalesintheUnitedStates.Metastaticmelanoma is far more dangerous than local melanoma, which can be surgically excisedbeforeithasspread.Metastaticmelanomahasbeenassociatedwithpoorprognosisduetoitsdrug-resistantandaggressivecancerswithamediansurvivaltimeof6monthsanda5-yearsurvivalrateoflessthan5%[1-3].Sincenocurrenttherapyselectivelytargetsthesemalignantmelanoma-initiating cells and there isno indicationof themolecular signalingpathwaysitutilizestoevadeapoptosis,thereexistsasignificantneedtodevelopefficaciousandtherapeuticdrugsformetastaticmelanoma.A promising dietary anti-proliferative phytochemical is indole-3-carbinol, which hasdemonstratedchemopreventativepropertiesaswellaspromisinganti-tumorcapabilitiesin ovarian, prostate, breast, and other reproductive cancers. Most recently, indole-3-carbinol has also been shown to sensitizemelanoma cells to cell cycle arrest. Indole-3-Carbinolisderivedfromthehydrolysisofglycobrassicinin,aphytochemicalproducedfromcruciferous vegetables such as broccoli, cabbage, and brussel sprouts. I3C, aswell as itspotent synthetic derivative, 1-benzyl-I3C, are natural agents capable of inducing G1 cellcycle arrest in a phenotypic range of cancers, such as melanoma, prostate, breast, andovarian [4-7].Ourresultsdemonstrate that I3Chasantiproliferativeeffects inaselectivemannerofhumanmelanomacelllineswithvaryingtumorigenicpotential.TheG1cellcyclearrestoccursinamutantBRAFdependentmanner,targetinghumanmelanomacellswitheither a wild type PTEN and mutant V600E phenotype, or a null-PTEN and V600Dphenotype, which constitutes the most cancer stem cell-like systems. While the BRAFV600E mutation is common but malignant in many melanoma patients, the V600Dmutation is equally as metastatic but demonstrates worse prognosis as less treatmentoptions exist [8]. Treatment with I3C downregulated oncogenes such as MITF-M andvariousstemcellmarkerssuchasCD20andALDH1inmelanomacell linesthatexhibitedthehighestcancerstemcell-likebehavior. Incontrast,cellswithmutantPTENormutantp53exhibitednoI3Csensitiveresponse.Toisolateandstudythecancerstemcell,biomarkersforidentificationofstemcellsfromnormal and cancerous tissues have been crucial [9]. Some biomarkers critical inacquisitionofcancerstemcellsforstudyofself-renewalcapabilitiesare:

• CD133, a cell surface differentiation antigen: associated with self-renewaldifferentiationinbraintumors,melanoma,andovariancancer[10]

• ABCG2/ABCG5:ATPBindingCassette:signalspathwaydrug-resistancerecurrenceandprognosisoftumors[11,12]

• ALDH1,aldehydedehydrogenase1:adetoxifyingenzymefoundinbothstemcellsandcancerstemcells,andlinkedwithdrugresistancecapabilities[13-15]

67

CD133isoriginallyidentifiedasatransmembraneproteinforhumanhematopoieticstemcells and subsequently, present in progenitor and stem cells, tumors, and regenerativetissues.ThesefunctionalrolesforCD133associateCD133asaprominentstemcellmarkerfor melanoma tissues where it is most likely involved in self-renewal, metabolism, anddifferentiation. Previously isolated CD133+ cells have given rise to tumorspheres andxenograftstumorsinNOD/SCIDmiceinmultiplecancers,fromcolorectalcancer,ovariancancer, glioblastoma, and gallbladder carcinomas [16, 17]. CD133 also has directinteractionswithtwostemcellfactors,OCT4andSOX2,twotranscriptionfactorsthatbindonP1promoterregionofCD133geneloci.Asaresult,theroleofCD133expressioninself-renewal and tumorigenesis is likely [18]. Therefore, our results demonstrate the use ofindole-3-carbinol in rare BRAF V600Dmutant to improve prognosis for a population ofmalignantmelanoma patients aswell as a potential regulator of transcriptional and cellcycleeffectsformelanomapatientswithmutantBRAFandwildtypep53.One of the reasons for malignant melanoma-initiating cells is the composition ofheterogeneous cancer cell subpopulations in which only the cancer stem cell will self-renew,differentiation,andinitiatetumorigenesis[19,20].Currenttherapies inducegreatside effects by also killing bystander healthy wild type cells or mass targeting non-malignantbulktumorcellswhichcauseforincreasedclonaldifferentiation,andasaresult,therapyresistance.Therefore,giventheimportantfunctionandpresenceofacancerstemcellinpropagatingaggressivetumorsubpopulations,itwillbequitecriticaltotreatMMICs.Todate,therearenoeffectivetherapiesthatcantargetmalignantmelanomainitiatingcellsto improve the prognosis of patientswith aggressive anddrug-resistant tumors [11, 20-22].Asaresult,thereisaprofoundneedfortheinclusionofnewtherapies,suchasindole-3-carbinol,torepresentapromisingnoveltargetformelanomatherapy.

68

MaterialsandMethodsCellCulture

MelanomacelllinesG-361,SK-MEL-2,SK-MEL-28,SK-MEL-30andWM115werepurchasedfrom American Type Culture Collection (ATCC) (Manasas, VA), and were authenticatedaccording to the ATCC guidelines. The G361 melanoma cells were cultured in ModifiedMcCoy's5Acellmediasupplementedwith10%fetalbovineserum(GeminiBioProducts,West Sacramento, CA), 2 mML-glutamine, and 2.5 ml of 10,000 U/mlpenicillin/streptomycinmixture (Gibco, LifeTechnologies, Carlsbad, CA). SK-MEL-28 andSK-MEL-30cellswereculturedinDMEMwith4.5g/Lglucosesupplementedwith10%fetalbovine serum, 2 mmol/L L-glutamine, 2.5 mL of 10,000 U/mL penicillin/streptomycinmixture in addition to 1 of MEM nonessential amino acid (Gibco, Life Technologies).WM!!%cellswereculturedin“WMMedia”:MCDB153mediumcontaining20%LeibovitzL-15 medium, 2% FBS, 0.2% sodium bicarbonate, 5μg/ml insulin, and 1%penicillin/streptomycin (P/S). SK-MEL-2 melanoma cells were cultured in DMEMcontaining 4.5 g/L glucose, 114 mg/L sodium pyruvate, and 2 mML-glutamine,supplemented as described above. The cells were incubated in tissue culture dishes(NalgeneNunc,Penfield,NY)at37°Cwithcontrolledhumidityand5%CO2aircontent.TreatmentWithIndole-3-Carbinol

I3Cwas purchased from Sigma–Aldrich (St. Louis, MO), To study the effect of I3C, cellsweretreatedwithorwithout200 μMI3Cevery24 hfor72 handharvestedat24,48,and72 hforwesternblotandflowcytometricanalysis.I3Cwasdissolvedin99.9%HPLCgradeDMSO(Sigma–Aldrich,Milwaukee,WI)andthefinaldilutionwasperformedinthemediaaliquotsusedfortreatment.MTTProliferationAssayMelanoma cell lines were seeded on a 48-well plate in triplicates and upon 80–90%confluencywereeither treatedwithdifferent concentrationsof I3CorDMSO, thevehiclecontrol, for 48 hours. Subsequently, inhibition of proliferation was assessed using theDojindoCell countingKit-8 asper theprotocol in theuser'smanual.Briefly, 50 μl of theCCK-8solutionwasadded toeachwell alongwith450 μlmediaand incubated for2–3 h.The absorbance was read at 450 nm and percent inhibition was calculated for eachconditionstandardizingDMSOtozero.FlowCytometryMelanomacellswereeithertreatedwiththeindicatedconcentrationsofI3Cintriplicatescell cultures for48 h, or treatedwith200 μM I3C in triplicate cultures for48hours. TheDNAcontentofpropidiumiodidestainednucleifromharvestedcellsweredeterminedbyflowcytometry.Briefly,cellswerehypotonically lysed in300 mlofDNAstainingsolution(0.5 mg/ml propidium iodide, 0.1% sodium citrate, and 0.05% Triton-X 100). Emittedfluorescence from the nuclear of wavelengths more than 585 nm was measured with a

69

Coulter Elite instrument with laser output adjusted to deliver 15 mW at 488 nm. Tenthousand nuclei were analyzed from each sample at a rate of 300–500 nuclei/s. ThepercentageofcellswithintheG1,S,andG2/MphasesofthecellcycleweredeterminedbyanalysiswiththemulticyclecomputerprogramprovidedbyPhoenixFlowSystemsintheCancer Research Laboratory Microchemical Facility of the University of California,Berkeley.Fluorescence-ActivatedCellSorting(FACS)

Fluorescence-activated cell sorting (FACS) was done on a Cytomation MoFlo cytometer(DakoCytomation,FortCollins,CO).FITC-conjugatedmAbagainsthumanCD133wasusedfor separation. Polyclonal antibodies were labeled by PE-conjugated goat antibodiesagainstrabbitIgG(Sigma)andmAbsbyFITC-conjugatedrabbitantibodiesagainstmouseIgG(Sigma). AldefluorAssayALDH1activitywasmeasuredusing theAldefluorkit (StemCellTechnologies) followingthe manufacturer's instructions. ALDEFLUOR assay was performed followingmanufacture’srecommendedguidelinesandprotocol(StemCellTechnologies).Livesinglecellsweregated foranalysisusingEpicsXL-MCLflowcytometer(BeckmanCoulter)withsingle488nmbluelaserfilteredat525BP/slot1.TumorsphereEfficiencyAssaySingle-cell suspensions of 10AT-Her2, 10AT-Neo, MCF-7, or SKBR3 cells were plated onultra-low attachment plates (Corning, Costar) inMammoCultHumanMedium (StemCellTechnologies, Vancouver) and cultured at 37oC, 5% CO2. Tumorsphere formation wasassessedvisuallybyphasemicroscopyandquantifiedbycountingthenumberofspheresformedinculture.WesternBlotAfter indicated treatments, all cells were harvested in ice-cold PBS and lysed using aradioimmunoprecipitationassaybuffer(150mMNaCl,0.5%deoxycholate,0.1%NoNidet-p40, 0.1% SDS, 50 mM Tris) containing protease and phosphatase inhibitors (50 g/mlphenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 5 g/ml leupeptin, 0.1 g/mlNaF, 1mMdithiothreitol, 0.1 mM sodium orthovanadate and 0.1 mM β-glycerol phosphate). Aftercentrifugation,totalproteininthelysatewasestimatedusingtheproteinquantificationkit(Bio-Rad,Hercules,CA,USA).CelllysateswereelectrophoreticallyfractionatedusingSDS-PAGEandtransferredtonitrocellulosemembranes.Theblotswereblockedwith5%nonfatdry milk for an hour at room temperature and incubated with primary antibodiesovernight at 4°C. Immunoreactiveproteinsweredetected after a1-hour incubationwithhorseradish peroxidase conjugated secondary antibodies. Blots were then treated withenhanced chemiluminescence reagents (Estman Kodak, Rochester, NY, USA) forvisualizationon film.Actin (AANO1)wasobtained fromCytoskeleton (Denver, CO,USA).

70

HER2 (2165),was obtained from Cell Signaling Technology, Inc (Danvers,MA, USA). Allantibodieswerediluted1:1000inTBST(0.1MTris,150mMNaCl,0.05%Tween20).

71

ResultsHumanmelanomacellsareselectivelyresponsivetoIndole-3-CarbinolThepotentialeffectsofI3Conmelanomacellularproliferationwastestedinfivedifferenthumanmelanomacelllineswithdistinctmutationprofiles.Eachofthecellswastreatedfor48 hours with or without 200 µM indole-3-carbinol, and cellular proliferation wasdeterminedusingaCCK-8proliferationassay.AsshowninFigure 23, the mostsensitive to the treatment were G631, SK-MEL-30 cells, andWM115 cells. The cellularproliferationofSk-Mel-28andSk-Mel-2cells,whichexpressmutantp53,wasnotaffectedbyI3Ctreatment.

72

Figure23I3Cinhibitshumanmelanomacelllinesofvaryingtumorigenicpotential.G361,Sk-Mel-30,WM115,Sk-Mel-24,andSk-Mel-2cellsweretreatedwithorwithout200µMI3Cfor48hoursandcellnumberwasquantifiedbytheCCK-8proliferationassay.

73

0

10

20

30

40

50

60

G361 SK-MEL-30 WM115 SK-MEL-24 SK-MEL-2

% In

hibi

tion

of P

rolif

erat

ion

Human Melanoma Cell LInes

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 200 300

Surv

ival

Fra

ctio

n

I3C Dosage (µM)

G361

SK-MEL-30

WM115

SK-MEL-24

SK-MEL-2

74

I3C-MediatedG1cellcyclearrestofG361andWM115cellsOur studies indicate that the antiproliferative effect of the antitumor agent indole-3-carbinol is cell type specific between cells from one tissue through regulation of cellsignalingpathwaysandinductionofapoptosis.Humanmelanomacellsweretreatedwithorwithout200µM/Lindole-3-carbinolfor48hours,andpotentialcell-cycleeffectswereanalyzedbyflowcytometryofpropidiumiodide-stainednuclearDNA.Thisconcentrationof I3C has been proven to be optimally effective in other human cancer cell lines as thefunctionalintracellularconcentrationofthisindolecarbinolenteringcellsislessthan0.3%ofthefinalconcentrationinthecellculturemedia.AsshowninFigure24,aG1cellcyclearrestwasobservedinG361andSK-MEL-30cells,whicharephenotypicallywildtypep53,wild typePTEN,andV600EB-rafmutation.However, incells thatdisplayA499G-mutantPTEN ormutant p53, such as the Sk-Mel-28 cells, and Sk-Mel-2 cells, respectively, therewasnoindicationofacellcyclearrest.Inadoseresponse,Sk-Mel-28cells,showedslightsensitivity--anarrestafter300µMoftreatmentorpast48hours(datanotshown).Sk-Mel-2cells,whichexpressawild typeBRAF,aremostly insensitive to theeffectsof indole-3-carbinol. Interestingly, WM115 cells, which are considered metastatic and malignantmelanomacells,werealso responsive to I3Cdespite theirnullPTEN,wild typep53, andV600D-mutantB-RAF geneticmolecular profile. This suggests that I3Cmight function intwodifferentsignalingmechanismsthatisnotonlyspecifictoawild-typePTEN,wild-typep53,andV600E-mutantB-RAFgenotype.

75

Figure24I3CinducesstrongG1cellcyclearrestinG361andWM115cells.Humanmelanomacellsweretreatedwithorwithout200µMI3Cfor48hours,andthecellpopulation DNA contents quantified by flow cytometry after staining with propidiumiodide. The results are an average of triplicate results. The average error wasapproximately-/+1%foreachoftheresults.

76

VehicleControl Indole-3-CarbinolCellLine Phenotype G1 S G2/M G1 S G2/M

G361WildTypePTENWildTypep53B-RafV600EMutation

36.3% 38.6% 25.2% 70.4% 16.8% 12.8%

Sk-Mel-28A499GPTENMutantp53B-RafV600EMutation

60% 28% 12 63% 24% 13%

Sk-Mel-30WildTypePTENWildTypep53B-RafV600EMutation

48% 36% 14% 77% 16% 6%

Sk-Mel-2WildTypePTENMutantp53WildTypeB-Raf

59% 28% 13% 60% 27% 13%

WM115PTENDeletionWildTypep53B-RafV600Dmutation

40% 41% 19% 73% 18% 9%

77

I3Cablatesspheroidformationincelllines,WM115cellsandG361cellsEarlier studies have demonstrated a correlation between tumor initiation and adherentfreesuspensions.Theabilityofcellstoinitiatemelanospheregrowthinsuspensionmediahasbeensuggestedtodefinethecancerstemcell.Therefore,thepresenceoftumorspheregrowthcanbeutilizedtoidentifyasidepopulationofcells inatumortoelucidatetumorinitiation capabilities. Cancer cells that lack stem cell properties have limited sphere-formingpotentialduetotelomereloss,cellularsenescence,andinactivationofself-renewalsignalingpathwaysasaresultofdifferentiation.G361andWM115cellswereplatedat adensityof10,000cellsperwell in tumorsphereculture conditions and incubated with or without I3C. Only one dose of I3C wasadministered in tumorsphere culture media at day 0 and after 6 days, tumorsphereformation was assessed visually by phase microscopy and quantified. Only G361 andWM115cellswerecapableofmelanosphereformation,suggestingthemelanomastemcellliketumorigenicpotentialofthetwocelllines.I3CablatedspheroidformationinG361cellstwofold; while WM115 cells were affected approximately more than three fold by thetreatmentofI3C.

78

Figure25I3CinhibitsformationofmelanomaspheroidsinG361andWM115cells.10,000G361cellsorWM115perwellwereplatedinserumfreenonadherenttumorspherecultureconditionsandtreatedwiththeindicatedindole-3-carbinolorthevehiclecontrol.Treatmentwasadministeredatday0andincubatedforsixdays.Tumorsphereformationwas quantified in three independent experiments by determining the total number oftumorspheresineachcellculturewell.

79

0

5

10

15

20

25

30

35

40

G361 WM115

TumorspherespermL

VehicleControl

I3CTreated

80

Molecular Profiling ofWM115 and G361 cells reveal presence of cancer stem cellmarkersWe profiled the responses ofmutationally distinctmelanoma cell lines and investigatedwhichcelllinesarecapableofformingspheroids,whethercommonlyusedcancerstemcellmarkers were enriched in these spheroids, and whether they were responsive to I3C.Cancerstemcellmarkersareupregulated incells culturedasspheres.To investigate theenrichmentofcancerstemcellmarkersinspheroidsversusadherentsuspensioncells,wesorted cells on the basis of cancer stem cell markers, CD133, CD20, and ALDH1. Weanalyzed the expression of cell surfacemarkers, CD133 and CD20, through fluorescenceactivatedcellsorting(FACS)andcomparedlevelsoffunctionalALDH1activitythroughtheALDEFLUOR assay, amongst five distinct cell lines under adherent and nonadherentconditions.Whencancerstemcellmarkers,suchasCD133andCD20wereanalyzed,inbothadherentcellsandspheroids,tumorsphereswereenrichedforALDH-1expression.However,CD133expressionwhichwasnormallyhigher inadherent cells forG361cells andWM115overI3Cirresponsivecelllines,suchasSk-Mel-28andSk-Mel-2cells,ortheI3CresponsiveSk-Mel-30cellline.

We analyzed the molecular and functional phenotype of adherent cells and melanomaspheroids by sorting for melanoma-associated markers. Gene expression profiling andspheroidmodeling allow for a greater grasp ofmelanosphere behavior and allowed forenrichment of cancer stem cells with biomarker expression and enhanced self-renewalcapabilities.OutofthewildtypePTENandwildtypep53phenotypiccelllines,onlyG361cells were able to form spheroids in nonadherentmedia. Even though Sk-Mel-30whichshow a reasonably strong response to I3C expressed small levels of CD133, CD20, andfunctionalALDH1at1%,2%,and8%respectively, itwasnotable to formmelanosphereenrichedwith stemcell factors.However, theWM115cell line,,whichwereable to formmelanospheresinnonadherentsuspensionandexpressedstemcellmarkersCD133,CD20,and ALDH1 in both adherent and spheroid conditions, interestingly did not changebetweentwoconditions.Asaresult,itcouldbehypothesizedthattheWM115celllines,asawell-recognizedmetastaticandmalignantmelanomacellline,hasreacheda“capacity”inexpression of melanosphere markers. However, when quantifying the number oftumorspherespermLinmedia,WM115cellswereabletoformalmostdoublethenumberofspheroidsofthatoftheG361cellsafter4days.

81

Figure26Quantificationofcancerstemcellmarkers,CD133,CD20,andALDH-1.Cell surfaceexpressionofCD133andCD20 inG361,Sk-Mel-28,Sk-Mel-30,Sk-Mel-2,andWM115cellswerequantifiedbyflowcytometryof500,000cellsintriplicateindependentcell cultures. ALDH1 activity was measured through the ALDEFLUOR kit by STEMCELLBIOLOGIES. G361, Sk-Mel-28, Sk-Mel-30, Sk-Mel-2, andWM115 cells were quantified byflowcytometryof500,000cellsintriplicateindependentcellcultures.

82

Adherent SpheroidCellLine Phenotype CD133 CD20 ALDH1 CD133 CD20 ALDH1

G361WildTypePTENWildTypep53B-RafV600E

7.6% 1% 9.2% 8% 7.5% 78.7%

Sk-Mel-28

A499GPTENMutantp53B-RafV600E 2% 3% 1% -- -- --

Sk-Mel-30

WildTypePTENWildTypep53B-RafV600E

1% 2% 8% -- -- --

Sk-Mel-2WildTypePTENMutantp53WildTypeB-Raf

0% 0% 0% -- -- --

WM115PTENDeletionWildTypep53B-RafV600D

5.2% 3.1% 22.2% 5.9% 3.5% 82.1%

83

WesternBlotanalysisidentifiesselectivemodulationoftheG361andWM115cellsToverifythemolecularmechanismofindole-3-carbinolregulation,G361andWM115cellswere treated with the vehicle control, DMSO, I3C, and the potent indole derivative, 1-benzyl-I3C. WM115andG361cellswereselectivelyresponsive to I3C.LevelsofMITF-Mdecreasedwith I3Cand1-benzyl-I3C treatment,but inWM115cells,onlyafter1-benzyl-I3C treatment was MITF-M signal lost. MITF-M is a “lineage survival” oncogene that isrequired for both tissue-specific tumorigenesis and progression and elevated MITF-Mlevelscorrelatewithdecreasedoverallpatientsurvival.InthenullPTENline,WM115,noPTENchangesweredetectedbut,inG361cells,proteinlevelsofPTEN,atumorsuppressor,increased upon I3C and 1-benzyl-I3C treatment. This effect was similar for the Axinprotein,whichremainunchangedinWM115cells,butincreasedinG361cellsafterI3Candthemore potent 1-benzyl-I3C derivative.Wnt levels decreased in G361 cells, but not inWM115cells, identifyingadifferentmolecularmechanismtoablate tumor initiatingcellsforWM115cells.Thecontrolprotein,actin,remainunchanged.

84

Figure27I3Cand1-benzyl-I3CselectivelymodulateG361andWM115cells.G361 and WM115 cells were treated with 15 µM 1-benzyl-I3C, 200 µM I3C, or DMSOvehiclecontrolfor48hours.TotalcelllysatesweresubjectedtoSDS-PAGEandthelevelsofMITF-M,B-catenin,Wnt,PTEN,AxinandActin(loadingcontrol)wasmonitoredbywesternblotanalysis.

85

PTEN

Wnt

B-catenin

Actin

MITF-M

Axin

G361 WM115

D I 1-B D I 1-B

86

Figure28Summaryofvaryingtumorigenicpotentialofhumanmelanomacelllines.

87

Increasing Stem Cell Character

RESPONSIVENESS TO I3C

SK-MEL-2

SK-MEL-28

SK-MEL-30

G361

WM115

88

DiscussionThe aim of this study is to investigate whether establishedmelanoma cell lines containsubpopulations of cells that aggregate as cancer stem cells and to understand theirresponsiveness to the phytochemical, indole-3-carbinol. Previous research in ourlaboratorydetectedaG1cellcyclearrestofG361melanomacells,whichretainawildtypep53 and wild type PTEN, molecular phenotype and no response from Sk-Mel-28 cells,which possess mutant PTEN, p53, and mutant B-RAF. Melanomas show phenotypicheterogeneity bothinvivoandinvitro, suggesting an origin froma cellwithmultilineagedifferentiation abilities [23, 24]. Melanoma cells retain their morphologic and biologicalplasticity despite repeated cloning and contribute to increased resistance to therapyselection.To assess whether the decrease in cell proliferation due to I3Cwas either cytostatic orcytotoxic,wedeterminedtheeffectof I3Concellcycleprogression.Cellcycleanalysisbyquantification of DNA content indicates the strongest G1 cell cycle arrest in G361 cells,followedbyacellcyclearrestinSk-Mel-30cells.Genotypically,bothcellsexhibitwildtypePTENandwildtypep53,highlightingsensitivitytoindole-3-carbinol.ThisiscontrastedbySk-Mel-28 cells and Sk-Mel-2 cells which exhibit a distinct mutant PTEN and a p53mutation,respectively.Theyshowednoresponsetoindole-3-carbinolincellcyclestudies.Inthedoseresponsestudy,Sk-Mel-28cellsminutelybegantorespondtoI3Ctreatment,at300µMtreatmentat48hours,indicatingaminutecellularsignalingresponse.WM115cellline isresistanttomanytypesof therapeuticmolecules includingVemurafenib[25] Asaresult,I3Ccanbeapromisingmalignantmelanoma-initiatingcelltherapyduetoitsabilitytoablatenotonly typicalB-rafmutationmelanomas,butalsoVemurafenib-resistant,butnotoriously malignant melanomas [26, 27]. Our findings indicate I3C as a potentialtherapeutic for melanomas that express low levels of wild type PTEN protein in abackground of either wild type BRAF or BRAF (V600E) by disrupting the NEDD4-1mediatedubiquitinationofPTENandenhancing the total levelsof this tumorsuppressorprotein.PreviousresearchinourlaboratoryhasshownI3Candthesyntheticderivative(1-benzyl-I3C) strongly upregulates PTEN protein levels without altering the level of PTENtranscripts inwild typePTENexpressing cell lines. InWM115cells,whichdisplayanullPTENgenotypeandamutantB-rafV600Dmutation,I3Cand1-benzyl-I3Cstillconferredastrong apoptotic response. This suggests the I3C response in melanoma stem cells isdifferentthanthosecelllinesthatdisplayawildtypePTEN,wildtypep53,andmutantB-rafV600Emutation[4,5,8].We profiled the responses ofmutationally distinctmelanoma cell lines and investigatedwhichcelllinesarecapableofformingspheroids,whethercommonlyusedcancerstemcellmarkers were enriched in these spheroids, and whether they were responsive to I3C.Cancerstemcellmarkersareupregulated incells culturedasspheres.To investigate theenrichmentofcancerstemcellmarkersinspheroidsversusadherentsuspensioncells,wesorted cells on the basis of cancer stem cell markers, CD133, CD20, and ALDH1.UpregulationofCSCmarkerssuchasALDH1andCD133isconsistentwiththehypothesis

89

that growth ofmelanospheres in nonsuspensionmedia enriches for CSCsmarkers. OnlyG361 cells andWM115 cells were capable for spheroid formation consistent with theirhigherexpressionofCD133,CD20,andALDH-1overI3Cnon-responsivecelllinessuchasSk-Mel-28 and Sk-Mel-2 cells, as well as the I3C responsive cell line, Sk-Mel-30. This isindicativeofa lessactivemelanomastemcellphenotype in theSk-Mel-28,Sk-Mel-2,andSk-Mel-30 cell lines. Melanoma spheres were significantly enriched for expression ofALDH-1,butnotCD133andCD20.Melanomasareasignificantcauseofmortalityduetotheirresistancetocurrenttherapies.Thiscanbeduetothepresenceofheterogeneouspopulationofcancercellsthatarepoorlycharacterized,withasidesubpopulationoftransformedstemcells.Thesecells,alsoknownas melanoma stem cells or melanoma initiating cells, possess the ability to self-renew,differentiateintodiverseprogenies,anddrivecontinuousgrowth.Giventhis,itiscriticaltomolecularly understand the responsiveness of melanoma to various nonconventionaltherapiesaswell[28,29].Thesetumor-initiatingcellsaredistinctfrommalignantcancercellsornon-malignantstemcells due to their self-renewing capability, propensity to differentiate into activelyproliferating tumor cells, elevated expression of stem cell biomarkers, resistance toconventional therapies, and low proliferative rates (similar to a stem cell capacity).Targetingandpersonalizingtherapiestothesecellscannotonlygreatlyablatetumorsizebutalsoattacktumormetastasisandtumorinitiationofamelanomatumor,reducingpoorprognosis and high rates of remission [1, 29-31]. This is especially pertinent due to themulticlonaloriginsofaheterogeneouspopulationofbulktumorcellsandrarecancerstemcells. Agentssuchasindole-3-carbinolthatenhancesensitivityofthesetherapy-resistanttumor stem cells to current or emerging anti-proliferative drugs could potentiate higherinhibitionabilitiesaswell.

90

References1. Nguyen,N.,etal.,Understandingmelanomastemcells.MelanomaManag,2015.2(2):

p.179-188.2. Parmiani, G.,MelanomaCancerStemCells:MarkersandFunctions. Cancers (Basel),

2016.8(3).3. Roesch,A.,Melanomastemcells.JDtschDermatolGes,2015.13(2):p.118-24.4. Aronchik,I.,L.F.Bjeldanes,andG.L.Firestone,Directinhibitionofelastaseactivityby

indole-3-carbinol triggersaCD40-TRAFregulatorycascadethatdisruptsNF-kappaBtranscriptional activity in human breast cancer cells. Cancer Res, 2010. 70(12): p.4961-71.

5. Aronchik, I., et al., Target protein interactions of indole-3-carbinol and the highlypotent derivative 1-benzyl-I3C with the C-terminal domain of human elastaseuncouplescellcyclearrest fromapoptoticsignaling.Mol Carcinog, 2012.51(11): p.881-94.


7. Brew, C.T., et al., Indole-3-carbinol inhibitsMDA-MB-231breast cancer cellmotilityand induces stress fibers and focal adhesion formation by activation of Rho kinaseactivity.IntJCancer,2009.124(10):p.2294-302.

8. Aronchik, I., et al., The antiproliferative response of indole-3-carbinol in humanmelanomacells is triggeredbyan interactionwithNEDD4-1anddisruptionofwild-typePTENdegradation.MolCancerRes,2014.12(11):p.1621-34.

9. Skvortsov, S., P. Debbage, and I. Skvortsova, Proteomics of cancer stem cells. Int JRadiatBiol,2014.90(8):p.653-8.

10. Shi,C.,etal.,CD44+CD133+populationexhibitscancerstemcell-likecharacteristicsinhumangallbladdercarcinoma.CancerBiolTher,2010.10(11):p.1182-90.

11. Pfeiffer,M.J. and J.A. Schalken,Stemcellcharacteristics inprostatecancercell lines.EurUrol,2010.57(2):p.246-54.

12. Hayashida, T., et al., Cooperation of cancer stem cell properties and epithelial-mesenchymal transition in the establishment of breast cancer metastasis. J Oncol,2011.2011:p.591427.

13. Kim,R.J., et al.,Highaldehydedehydrogenaseactivityenhancesstemcell features inbreastcancercellsbyactivatinghypoxia-inducible factor-2alpha. CancerLett, 2013.333(1):p.18-31.

14. Charafe-Jauffret, E., et al., ALDH1-positive cancer stem cells predict engraftment ofprimarybreasttumorsandaregovernedbyacommonstemcellprogram.CancerRes,2013.73(24):p.7290-300.

15. Leon,G.,etal.,Cancerstemcellsindrugresistantlungcancer:Targetingcellsurfacemarkersandsignalingpathways.PharmacolTher,2016.158:p.71-90.

16. Yiming,L.,etal.,CD133overexpressioncorrelateswithclinicopathologicalfeaturesofgastric cancer patients and its impact on survival: a systematic review and meta-analysis.Oncotarget,2015.6(39):p.42019-27.

91

17. Zimmerer, R.M., et al., Putative CD133+melanoma cancer stem cells induce initialangiogenesisinvivo.MicrovascRes,2016.104:p.46-54.

18. Schmohl,J.U.andD.A.Vallera,CD133,SelectivelyTargetingtheRootofCancer.Toxins(Basel),2016.8(6).

19. Al-Hajj, M., Cancer stem cells and oncology therapeutics. Curr Opin Oncol, 2007.19(1):p.61-4.

20. Carnero,A., et al.,Thecancerstem-cellsignalingnetworkandresistancetotherapy.CancerTreatRev,2016.49:p.25-36.

21. Orecchioni, S. and F. Bertolini,CharacterizationofCancerStemCells.MethodsMolBiol,2016.1464:p.49-62.

22. Salcido, C.D., et al.,Molecular characterisation of side population cells with cancerstemcell-likecharacteristicsinsmall-celllungcancer.BrJCancer,2010.102(11):p.1636-44.

23. Henson, A., et al., Outcomes for Metastatic Melanoma Treated With StereotacticRadiosurgeryIntheEraofTargetedSystemicTherapies.IntJRadiatOncolBiolPhys,2016.96(2S):p.E135-E136.

24. Zeng, K., et al., BRAF V600E mutation correlates with suppressive tumor immunemicroenvironmentandreduceddisease-free survival inLangerhanscellhistiocytosis.Oncoimmunology,2016.5(7):p.e1185582.

25. Ravnan,M.C.andM.S.Matalka,VemurafenibinpatientswithBRAFV600Emutation-positiveadvancedmelanoma.ClinTher,2012.34(7):p.1474-86.

26. Kawaguchi, K., et al., Vemurafenib-resistant BRAF-V600E-mutated melanoma isregressedbyMEK-targetingdrugtrametinib,butnotcobimetinibinapatient-derivedorthotopicxenograft(PDOX)mousemodel.Oncotarget,2016.

27. Lott,J.P.,VemurafenibinmelanomawithBRAFV600Emutation.NEnglJMed,2011.365(15):p.1449-50;authorreply1450.

28. Byeon, H.K., et al., Acquired resistance to BRAF inhibition induces epithelial-to-mesenchymal transition inBRAF (V600E)mutant thyroid cancer by c-Met-mediatedAKTactivation.Oncotarget,2016.

29. Hashim, P.W., P. Friedlander, andG. Goldenberg,SystemicTherapies forLate-stageMelanoma.JClinAesthetDermatol,2016.9(10):p.36-40.

30. Kozovska, Z., V. Gabrisova, and L. Kucerova, Malignant melanoma: diagnosis,treatmentandcancerstemcells.Neoplasma,2016.63(4):p.510-7.

31. Lopez-Bertoni,H.,Y.Li,andJ.Laterra,CancerStemCells:DynamicEntitiesinanEver-EvolvingParadigm.BiolMed(Aligarh),2015.7(Suppl2).

indole-3-carbinol mediated anti-proliferative regulation ... · cancer stem cell population in...

Documents