Detecting gene-gene interactions in prostate disease in African American men

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Detecting gene-gene interactions in prostate disease in African American men
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Infectious Agents and Cancer 2011, 6(Suppl 2):S1
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Reams, R. Renee
Kalari, Krishna Rani
Wang, Honghe
Odedina, Folakemi T.
Soliman, Karam F. A.
Yates, Clayton
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Background: The most common male malignancy in the United States is prostate cancer; however its rate of occurrence varies significantly among ethnic groups. In a previous cDNA microarray study on CaP tumors from African American (AA) and Caucasian (CA) patients, we identified 97 candidate genes that exhibited opposite gene expression polarity with respect to race groups; genes up-regulated in AA were simultaneously down-regulated in CA. Purpose: The purpose of this study was to narrow the 97 member gene list, to a smaller number of genes in order to focus studies on a limited number of genes/SNPs that might explain prostate cancer disparity in African Americans. Methods: We performed genotype-phenotype, SNP and expression transcript levels correlations using HapMap Yoruba population with 85 of our 97 prostate candidate genes using SCAN database. Results: Findings revealed an association of SNPs surrounding ABCD3 gene with basal gene expression of RanGAP1 is important in prostate tumors in AA. Hence, to confirm our results in clinical biospecimen, we monitored expression of ABCD3 in a novel panel of African American and Caucasian prostate cancer paired cell lines. The LNCaP, C4-2B showed 2-fold increase; MDA-2PC-2B cell line, derived from AA, showed highest foldchange, 10-fold. The EGFR over expressing DU-145 WT cell line exhibited a 4-fold increase in expression relative to non transfected DU-145 prostate cell lines. Furthermore, Ingenuity Network analysis implicated our AA prostate candidate genes are involved in three network hubs, ERK, MapK and NFkB pathways. Conclusions: Taken together, these findings are intriguing because other members of the ABC gene family, namely, ABCC3, ABCD1, and ABCD2 have been shown to confer chemoresistance in certain cancer types. Equally important, is the fact that activation of the MapK/ERK pathway via EGFR stimulation is vital for increased transcription of numerous cancer related genes. It is especially noteworthy that overexpression of EGFR has been widely observed in AA prostate tumors. Collectively our findings lead us to think that a novel signaling cascade, through which increased aggressiveness and chemoresistance is achieved, may explain prostate cancer health disparity in AA males and the nature of aggressive CaP tumors in general.
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Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.

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PROCEEDINGS OpenAccessDetectinggene-geneinteractionsinprostate diseaseinAfricanAmericanmenRReneeReams1*,KrishnaRaniKalari2,HongheWang3,FolakemiTOdedina4,5,KaramFASoliman1,ClaytonYates3From TheScienceofGlobalProstateCancerDisparitiesinBlackMen Jacksonville,FL,USA.27-29August2010AbstractBackground: ThemostcommonmalemalignancyintheUnitedStatesisprostatecancer;howeveritsrateof occurrencevariessignificantlyamongethnicgroups.InapreviouscDNAmicroarraystudyonCaPtumorsfromAfrican American(AA)andCaucasian(CA)patients,weidentified97candidategenesthatexhibited opposite geneexpression polaritywithrespecttoracegroups;genesup-regulatedinAAweresimultaneouslydown-regulatedinCA. Purpose: Thepurposeofthisstudywastonarrowthe97membergenelist,toasmallernumberofgenesinorderto focusstudiesonalimitednumberofgenes/SNPsthatmightexplainprostatecancerdisparityinAfricanAmericans. Methods: Weperformedgenotype-phenotype,SNPandexpressiontranscriptlevelscorrelationsusingHapMap Yorubapopulationwith85ofour97prostatecandidategenesusingSCANdatabase. Results: FindingsrevealedanassociationofSNPssurroundingABCD3genewithbasalgeneexpressionof RanGAP1isimportantinprostatetumorsinAA.Hence,toconfirmourresultsinclinicalbiospecimen,we monitoredexpressionofABCD3inanovelpanelofAfricanAmericanandCaucasianprostatecancerpairedcell lines.TheLNCaP,C4-2Bshowed2-foldincrease;MDA-2PC-2Bcellline derivedfromAA,showedhighestfoldchange,10-fold.TheEGFRoverexpressing DU-145WT celllineexhibiteda4-foldincreaseinexpressionrelativeto nontransfected DU-145 prostatecelllines.Furthermore,IngenuityNetworkanalysisimplicatedourAAprostate candidategenesareinvolvedinthreenetworkhubs,ERK,MapKandNFkBpathways. Conclusions: Takentogether,thesefindingsareintriguingbecauseothermembersoftheABCgenefamily,namely, ABCC3,ABCD1,andABCD2havebeenshowntoconferchemoresistanceincertaincancertypes.Equallyimportant,is thefactthatactivationoftheMapK/ERKpathwayviaEGFRstimulationisvitalforincreasedtranscriptionofnumerous cancerrelatedgenes.ItisespeciallynoteworthythatoverexpressionofEGFRhasbeenwidelyobservedinAAprostate tumors.Collectivelyourfindingsleadustothinkthatanovelsignalingcascade,throughwhichincreased aggressivenessandchemoresistanceisachieved,mayexplainprostatecancerhealthdisparityinAAmalesandthe natureofaggressiveCaPtumorsingeneral.IntroductionProstatecancer(CaP)isthesecondleadingcauseof cancer-relateddeathamongallmenintheUnitedStates. However,incidenceandmortalityratesforthisdisease varysubstantiallyamonggeographicareasandethnic groups.MostnotablyAfricanAmericanmen(AA)inthe UnitedStateshavethehighestrisk(19%)ofdeveloping prostatecancer,andduetothedevelopmentofmore aggressivedisease,theyhavemorethantwicethemortality rateobservedforotherracialandethnicgroups[1].The explanationforthesedifferencesisstillunknown;however proposedexplanationsincludegeneticfactors,dietaryfactors,behavioralfactors,bio logicaltumoraggressiveness, socio-economicfactorsandgene-environmentinteraction [2-35].WhileAArace/ethnicityisoneofthethreeprimarynon-modifiableriskfactorsconfirmedforCaP,there areonlyafewpublishedcDNAmicroarraystudies[36-38] thathavefocusedongeneexpressiondifferencesinAA *Correspondence:renee.reams@famu.edu1CollegeofPharmacyandPharmaceuticalSciences,FloridaA&MUniversity, Tallahassee,Florida,USA FulllistofauthorinformationisavailableattheendofthearticleReams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 2011Reamsetal;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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tumorscomparedtoCAinanattempttounderstand prostatecancerhealthdisparity.Previouslyweidentified 97genesdifferentiallyexpressedinAAprostatetumors. Tonarrowdownthisnumberofgenes,weutilized advancebioinformaticsmethods.Inthepresentstudywe performedgenotype-phenotypeorSNPandexpression transcriptlevelcorrelation sofHapMaplymphoblastoid celllinesfromYorubapopulationtothe97prostatecandidategenesinAA,inanattempttoferretoutgenetic variantsassociatedwithAApopulation.Inaddition,we usedIngenuitypathwayanalysistocalculatetheprobabilityoffindingoursetofcandidategeneswithinagiven pathway(s)toestablishpro bablesignaltransduction mechanisms.MethodsMicroarrayprostatecandidategenelistforAAtumorsThegenelistusedinthisstudywasobtainedfromour previouslypublishedcDNAmicroarraystudy[36].SCANdatabaseanalysistolookforgene-gene interactionsSCANisalarge-scaledatabaseofgeneticsandgenomics dataassociatedtoaweb-interfaceandasetofmethods andalgorithmsthatcanbeusedforminingthedatainit (http://www.scandb.org/newinterface/about.html).InformationontherelationshipbetweenSNPsandexpression transcriptlevels(eQTLs)thatisservedbySCANcomes fromaseriesofpublicationsdescribingstudiescharacterizingeQTLsinlymphoblastoidcelllinesfromHapMaP Caucasian(CEU)andYoruba(YRI)samplesforwhich transcriptlevelshavebeenassayedusingtheAffymetrix HumanExon1.0STArray[39-44]. TheSCANdatabasecontainstwocategoriesofSNP annotations:(1)Physical-basedannotationorSNPscategorizedaccordingtotheirpositionrelativetogenes(intronic,antigenic,etc.)andaccordingtolinkagedisequilibrium (LD)patterns(anintergenicSNPcanbeannotatedtoa geneifitisinLDwithvariationinthegene).(2)Functional annotationwhereSNPsarecl assifiedaccordingtotheir effectsonexpressionlevels,i.e.whethertheyareeQTLsfor thatgene.Informationonphysical,functionalandLD annotationservedontheSCANdatabasecomesdirectly frompublicresources,includingHapMap(release23a), NCBI(dbSNP129),orisinformationcreatedbyusingdata downloadedfromthesepublicresources.InSCANdatabase,genotypedatafortheYRIsampleswasobtainedfrom HapMapproject(http://www.hapmap.org).Genotypeand geneannotationswereobtainedfromNCBI,dbSNP129. WeuploadedappropriategeneidentifiersforourprostatecandidategenesandqueriedforSNPsthataresignificantlyassociatedwithexpressionofprostatecandidate genesinYoruba(YRI)populationinlymphoblastoidcell lines.SCANgeneticandgenomicdatafortheYoruba PopulationinIbaden,Nigeria,Africawasusedbecauseof closeancestraltiesofNigerianstoAfricanAmericans. TheSCANanalysisoutputreportsalistofSNPsingene (s)thatpredictexpressionquantitativetraitlocifoundin mRNAprofilesfromYRIwithp-valueslessthan0.0001.HugogenesymbolsToenteralistofgenesintoSCAN,itisfirstnecessaryto usetheHUGO( Human Genome Organization)genesymbol-theuniquegenenameandsymbolgiventoeach humangenebyTheHUGOGeneNomenclatureCommittee(HGNC).Fromour97genelist,wewereabletoobtain HUGOgenesymbolsfor85ofthe97genes(hereafter referredtoas85/97).IngenuitypathwayanalysisIngenuitysoftware(http://www.ingenuity.com/)wasused forpathwayanalysis.Ingenuitysoftwarecalculatesp-value fortheprobabilityoffindingasetofgeneswithinagiven pathway.Fisher sexacttestwasusedtocalculatethe p-valuesassociatedwithfinding536prostategenes obtainedduringthisstudy(whichincludes85/97candidategenes,frompreviousdifferentialexpressionstudy [36]and451candidategenesthatarecorrespondingto CisregulatorySNPs,thataresignificantlyassociatedwith the85candidategenes)withinaannotatednetworkfrom IngenuityKnowledgeBase.RT-PCRvalidationinnovelpanelofprostatecancerlinesRT-PCRassaywasdoneusinga7500FASTReal-Time ABISystem. Briefly,totalRNAfromeachculturedprostatecellline wasextracted,separately,withRNAzolB(Tel-TestInc., Friedswood,Tx)accordingtothemanufacturer sprotocol andquantifiedwithNucleicAcidQuantitationKit(NBI, Plymouth,MN).TotalRNA(1ug)wasreversetranscribed intocDNAwithRT2FirststrandKit(SABiosciences/A QiagenCompany))and1.10ofthereverse-transcribed productfromeachsamplewasusedforPCRtoamplify ABCD3gene,usingaRT2qPCRPrimerAssayforHuman ABCD3(SABiosciences/AQiagenCompany)TheexpressionofGAPDHwasusedasaninternalcontrol/housekeepinggene.ExperimentalconditionsfortheABCD3 genewasoptimizedtoanalyzetheamplifiedproductin thelinearrangeofamplificationbyadjustingamplification cyclesforeachsetofprimers.Theexpectedbandsize(bp) sizeofthePCRproductwas83,asdescribedbyvendor (SABiosciences).Novelprostatecancercelllines:descriptionNon-malignant(RC-77N/E)andmalignant(RC-77T/E) prostatecellswerederivedfromanAfricanAmerican prostatecancerpatientandbothareandrogensensitive [45].RC-77N/Ecellswereis olatedfrompathologicalReams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page2of10

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normalcells,whileRC-77T/Ewerederivedfromstage T3tumor.BothcelllinesareculturedinKeratinocyte Serum-FreeMedium(KGM)LifeTechnologies, Gaithersburg,Md.,USA),supplementedwithbovine pituitaryextract(BPE),recombinantepidermalgrowth factor(rEGF),1%(v/v)penicillin-streptomycin-neomycin(PSN)antibioticmixturesand1%(v/v)amphotericin B(KGM)(LifeTechnologies, Gaithersburg,MD,USA). MDA-2PC-2B,alsoderivedfromanAfricanAmerican patientareandrogendependent,metastaticandareculturedinF12Kmedium.DU-145,acelllineoriginally derivedfromabrainmetastasisofahumanprostate adenocarcinoma[46]retainstheandrogenindependence oftheoriginaltumoranddoesnotexpressafunctional AR[47].ThiscelllinehasbothLHRH-Randepidermal growthfactorreceptors(EGFR)andproducestheEGFR ligands,transforminggrowthfactor a (TGFa )andEGF [48,49].Utilizingestablishe dprotocols,DU-145cells weretransfectedbyretroviral-containingEGFRconstructs[50].Thewild-type(WT)EGFRconstructisa full-lengthcDNAderivedfromaplacentalcDNA library.CellsexpressingWTEGFRatlevelsthatescape downregulationdemonstrateenhancedinvasivenessin vitro[51].LNCaPcellswerederivedfromalymphnode metastasis[52].TheCaucasianLNCaP,C42-Bprostate cancercelllinesweremaintainedinT-mediumaspreviouslydescribed.ResultsOriginofthe97prostatecandidategenesTodetectforgene-geneinteractionsinAAprostate tumorsinAfricanAmeri canmales,weusedacDNA microarraygenelistobtainedfromapilotprojectcDNA microarraycomparisonstudyofprostatetumorgene expressioninAAandCA[36].Toobtaindifferentially expressedgeneorthegenelist,weusedfoursnapfrozen tumorsandfoursnapfrozennon-tumormatchedcontrols, each,fromAAandCA.AlltumorshadaGleasonscore ofsix.Geneexpressionprofilesweremeasuredforeachof themicrodissectedCaPtumorsamplesusingAffymetrix U133Ahumanarraysasdescribedin[36].Eachofthe8 prostatetumorsand8matchedcontrolsunderwentsingle hybridizationandwasarrayed individually(i.e.samples werenotpooled).DatafromthemicroarrayCELfiles wereuploadedtoR-Bioconductorforanalysis[53].We pairednormalAAtissuetotumorAAandpairednormal CAtotumorCAtogenerateforcasepairedt-testsfor eachracegroup;genelistsofdifferentiallyexpressedgenes inAATumorvs.AAcontrolsandofCATumorvs.CA controlsweregenerated.Welookedfordifferentially expressedgenesthatmetthefilteringcriteriaofa4.0-fold changeandap<0.0001.NeitherthecomparisonofAA tumortoAAcontrolsnorthecomparisonCAtumorsto CAcontrolsyieldedgenesthatmetbothpartsofour filteringcriteria.However,whenwelookedattheratioof CAtumor/CAnormaltoAAtumor/AAnormal(casematchedratios-racegroupt estsforspecificexpression trends)wefound97statisticallysignificant,differentially expressedgeneswith4-foldorgreaterfoldchangeand p<0.0001.Itwasnecessarytoratiotheratiostocontrolfor thehighdegreeofgeneticvariationinAAtumorandAA non-tumorsamples.ScandatabaseSNPandexpressiontranscriptlevel associationresultsAfteruploadingour85/97prostatecandidategeneswith appropriateHUGOgenesymbolsandqueryingforSNPs inour85/97significantlyassociatedwithSNPsinthe HapMapYoruba(YRI)population;approximately,26527 genotype-phenotypeassociationswereobtainedwitha p-value<10-3,ofwhich17542/26527associationshada p-value<10-4(datanotshown) SNPsandexpressiontranscr iptlevels(eQTL)associationsresultsidentifiedtwogene-geneassociations.Associationresultsinlymphoblastoidcelllinesshowedthat expressionofRanGAP1genewhichisakeyregulatorof theRANGTP/GDPcycle,locatedonchromosome22, maybeinvolvedwithseveralSNPsinABCD3genewhich isATP-Bindingcassette,subfamilymemberthatislocated onchromosome1(Shownasencircleddotsonfarleftin Figure1).Inaddition,expressionofSTXBP2genewhich isasyntaxin-bindingprote inthatislocatedonchromosome19maybeinvolvedwitharegiononchromosome 12.Thechromosome12regionconsistsoftransmembraneandtetratricopeptiderepeatcontaining2(TMTC2) genethatisapproximately400kbawayfromtheregion whereSTXBP2geneisassociated(shownasencircled dotsonfarrightofFigure1).Genome-wideresultsalso showedthattherewere1167 cis interactions(where expressiongeneandSNParelocatedonthesamechromosome)outof26527associationswithap-value<10-3. Mostofthe cis -regulatoryassociationswerefoundinproteincodingregions.CaPcandidategenesfoundinERK,MapK,NFKBpathwaysPathwayanalysiswasperformedinanattempttodefine biologicalrelationshipsamongcandidategenesidentified duringourstudyusingthegenesthatareinvolvedwith thedownstreameffectsofSNPsalongwiththe85/97 candidateprostatecancergenesasdescribedinmethods.IngenuityPathwayAnalysis(IPA)wasusedtoperformthepathwayanalysis.T hissoftwareconsistsofa curateddatabaseandseveralanalysistoolstodetermine theprobabilityoffindingasetofgeneswithinannotated pathwayornetworkannotati on.Resultsshowedahigh probabilityforfindingourcandidategenesinthreenetworkhubscenteredonERK,MAPKandNFkBpathways(ShowninFigures2,3and4,respectively).TheseReams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page3of10

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12345678910111213 14 1516 17 18 19 20 21 229 8 7 6 5 4 C hromosome Number Figure1 GWASPlotofGene-GeneAssociationsillustratesp-values(shownony-axis)ofSNPforgenevariantsfoundonchromosomes1-22(xaxis).Thex-axisshowsChromosomes1thru22.EachdotrepresentsgenevariantsorSNPs.Inthecircleddotstotheextremeleftpositioned aboveChromosome1(x-axis),thedotwiththehighestp-valuerepresentsanABCD3genevariantwithadefinedrs#thatisstronglyinteracting withRANGAP1toinfluenceCaPtumorsinAfricanAmerican.AllofthedotsinthecirclerevealanassociationofSNPssurroundingABCD3gene withbasalgeneexpressionofRanGAP1.ThisvariationingeneexpressionRanGAP1mightbeinfluencedbytheSNPsinABCD3Similarlyinthe circledpointstothefarright,positionedabovechromosome12(x-axis),thedotwiththehighestp-valuerepresentstheTMTC2genevariant withadefinedrs#,thatstronglyinteractswithSTXBP2.AllthedotsinthecirclerepresentanassociationofSNPssurroundingTMTC2genewith basalgeneexpressionofSTXBP2. Figure2 IngenuityPathwayAnalysisidentified ERK astheTopnetworksignalingHubwheretheshadedshapesindicatethecandidategenes fromthepresentstudy. RANGAP1 isfoundinthe ERK pathway.Dottedlineindicatesanindirectcellularinteractionandsolidlinesindicatea physicalinteractionbetweengenes.GenesareidentifiedwiththeirHUGOsymbol.Dottedlineindicatesanindirectcellularinteractionandsolid linesindicateaphysicalinteraction(actsonorinhibits)betweengenes.Differentshapes(diamond,circleorrectangles)ofthenodesrepresent functionalclassificationofthegenes. Reams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page4of10

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top threenetworkswithpvaluesof<0.05basedon Fisher sexacttestwereassociatedwithgeneticdisorder, cellulardevelopment,celldeathandcellsignaling. Directinteractionsbetweenthegenesinthenetwork pathwaysareindicatedbysolidlinesandindirectrelationshipsareindicatedbydashedlines.Theshaded genesrepresentour536candidategenesidentifiedin ourassociationstudyasdescribedinmethodsection(85 candidategenes+451cis-regulatorygenesthatare associatedwiththe85candidategenes).Diamond shapesrepresentenzymes,ovalrepresenttranscription regulators.Squares:cytokinesandtriangles:kinases.(For fullexplanationofshapesandthefunctionalclassificationtheyrepresentgoto http://www.springerimages. com/Images/LifeScien ces/1-10.1007_s12014-0109053-0-2),ABCD3genehighlyexpressedinaametastaticprostate cancerlinesOurassociationstudyof85candidategeneswithgenome-wideSNPsinHapMapYRIlymphoblastoidcell lineshasrevealedanassociationofSNPssurrounding ABCD3genewithbasalgeneexpressionofRanGAP1 usingdataobtainedfromSCANdatabase(Figure1).This variationinexpressionlevelsofRanGAP1mightbeinfluencedbytheSNPsinABCD3.Toconfirmourresults obtainedduringtheassociationstudy,wetestedABCD3 expressioninclinicallyrelevantcelllines.Verificationof Figure3 IngenuityPathwayAnalysisidentified MapK assecondTopnetworksignalingHubwheretheshadedshapesindicatethecandidate genesfromcurrentstudy.Dottedlineindicatesanindirectcellularinteractionandsolidlinesindicateaphysicalinteractionbetweengenes. MoleculesareidentifiedwiththeirHUGOsymbol.Differentshapes(diamond,circleorrectangles)ofthenodesrepresentfunctionalclassification ofthegenesshown. Reams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page5of10

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ABCD3innovelAA/CAprostatecancercelllines revealedanincreaseinexpressionwithincreasedmetastasisacrossanovelpanelofAfricanAmericanandCaucasianprostatecancerpairedcelllines(Figure5).The malignant RC-77T/E cellsisolatedfromAAshowed2foldincreasedexpressioncomparedtonon-malignant RC-77N/Ematchedpair(Fi gure5).Themetastatic, androgendependent MDA-2PC-2Bcellline derived fromAA)exhibiteda10-foldABCD3expression(Figure 5).PreviouslywehavedemonstratedthatDU-145WT (EGFRoverexpressing)cellsexhibitincreasedinvasivenessandmetastasisbothinvitroandinvivo[49].Therefore,weexaminedABCD3geneexpressioninthe DU-145WTcellandinnon-transfectedDU-145cells. DU-145WTcellsshoweda4-foldincreaseinexpression relativetoDU-145prostatecelllines(Figure5).Asimilar patternofexpressionwasobservedintheandrogenindependentmetastaticC4-2BcellsderivedfromCaucasian androgendependentLNCaPcells,thusprovidingfirm evidenceofincreasedABCD3geneexpressionwith increasedprostatecancerprogressioninAAtumors (Figure5).Discussion/conclusionToaddresstheunderlyinggeneticcauseofprostatecancer burdeninAfricanAmericans, wepreviously obtaineda cohortofnormaltumorpairedsamplesfromAfrican AmericanandCaucasianmen,andlookedfordifferential geneexpressionwithineachgroup.Utilizingastrictfilteringcriteria,weobservedover97differentiallyexpressed genesinourAfricanAmericanvs.Caucasiansampleset [36].Tofurthernarrowthelisttogenesofutmostimportanceinprostatediseaseonset,weusedagenome-wide associationapproachthatallowedustorapidlyscanfor SNPsinthegenomeofhealthyYorubaipopulation(YorubaifromIbaden,Nigeria)thatmightpossiblybeassociated Figure4 IngenuityPathwayAnalysisidentified NFKB astheTopNetworksignalingHubwheretheshadedshapesindicatethecandidategenes fromthepresentstudy.Dottedlineindicatesanindirectcellularinteractionandsolidlinesindicateaphysicalinteraction(.I.e.binding)between genes.MoleculesareidentifiedwiththeirHUGOsymbol.Differentshapes(diamond,circleorrectangles)ofthenodesrepresentfunctional classificationofthegenesshown. Reams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page6of10

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withour85/97prostatecandidategenes.Weutilizedthe HapMapdatabaseofYBI,Nigerianlymphoblastoidcell lines,sincetherearealimitednumberofSNPdatabases containingAfricanAmericanpatients.Mostimportantly theYorubaipopulationratherthantheCaucasian(CEU) populationwasutilizedbecausethecloseancestralties [54]betweenWestAfricanAfricansandAfricanAmericanswouldincreaseourchancesoffindingsimilargenetic variantsassociatedwithprostatediseaseinmenofAfrican descent(NigeriansandAfricanAmericans).Approximately536geneswereidentifiedinourassociationstudy asdescribedinmethodssection(85candidategenes+451 cis-regulatorygenes)thatareassociatedwiththe85/97 candidategenes.Hereinwereportthatasignificantnumberofdirectgene-geneinteractionswerefound,however themostsignificantinteractionswereobservedonchromosome1andchromosome12.Giventheimportanceof thesechromosomesinprostatedisease,itwasappropriate toinvestigateABCD3geneinvolvementinAfricanAmericanprostatecancer.Ouranalysisofgene-geneinteractions onchromosome1and12revealedthatABCD3/RanGAP1 andSTXBPandTMTC2geneshowedthestrongestassociations.OfthesetheABCD3/RanGAP1geneswerepredictedtohavethemostsignificantinteractions.Assuch neitherABCD3orRanGAP1expressionhasbeenimplicatedinprostatecancer,ther eforewechosetofocuson ABCD3.The A TPb inding c assettegenesconsistofvarioussubfamilies,aretypical lyexpressedinbothnormal andcancercells.Theirfunctionshavebeenimplicatedin acquiredMultidrugResistance,MDR,incancercelllines. Forexample,recentreportshaveshownthatMCF-7/ AdVp3000cellsthatwerederivedbyselectionforgrowth inthepresenceofdoxorubicin,exhibita459-foldoverexpressionofABCC3relativetotheparentalcellline[55]. Asimilarsituationinprostatecancercelllineshasbeen observedasMDR1/Pgp/ABCB1andmultidrugresistanceassociatedprotein-1(MRP1/ABCC1),withthehalfABC transporter,breastcancerresistanceproteinBCRP/ ABCG2,isabletoselectivelyisolatetheputativeprostate stemcellsfromtheprostatetissuemicroenvironment throughconstitutiveeffluxofandrogenandprotectsthe putativetumorstemcellsfromandrogendeprivation, hypoxia,oradjuvantchemotherapy[56].Thesefindings aresupportedbyunpublisheddata,fromYateslaboratory, thatsuggestthatfluorescence-activatedcellsorting,FACS isolationofthe(SP)cells,s electiveforfunctionalABC transporterpumps,havehigherinvivotumorigenicity comparedtoothercellsurfacemarkers(unpublished data).Thus,itispossiblethatABCD3couldpossiblycontributetoaggressiveprostatecancer. MultipleABCfamilygeneshavebeenimplicatedtoplay aroleinchemoresistanceandprogressionofprostateand breastcancer,howevergiventhelargenumberoffamily subtypesonlyafewhavebeenassociatedwithprostate cancerprogressiontoaggre ssivedisease.SinceABCD3 geneexpressionhasnotbeenidentifiedpreviouslyinprostatecancer,weverifiedthisincommonlyutilizedprostate cancercelllines,aswellasinanormalandprimarytumor celllinepairderivedfromanAfricanAmericanprostate cancerpatient[45,57].Asinprostatepatientsamples, ABCD3wasconsistentlyoverexpressedintheRC-77N/E/ RC-77T/E,LNCaP/C4-2Bmodels,withMDA-PC-2Bcells exhibitingthehighestexpression(10-fold).Thisdatais consistentwiththeexpressionofothermembersofthe ABCgenefamilythathaveb eenimplicatedinprostate cancer,andservesasproof-of-principleevidencethat ABCD3overexpressionisindeedcorrelatedwithprostate cancerprogression.ThatMDA-2PC-2Bcellsexhibitedthe highestexpressionlevels,furtherimplicatesarolefor ABCD3inAfricanAmericanprostatecancers. TofurthersubstantiatethelinkofABCD3withother cellsignalingmoleculesthat contributetoprostatecancer,weutilizedanindirectinsilicoingenuitypathway analysis.ABCD3,showedahighprobabilityofbeing foundwithinthreegrowthf actorinitiatednetworkhubs involvingERK,MAPKandNFkBproteins.TheERK MAPKhasbeenimplicatedinanumberofpathophysiologicaleventsincludingandrogenreceptorsignaling[58] andtheepithelial-to-mesenchymal(EMT)[59]that occursascancercellsacquirethepropertytometastasize. Thatweobserveda4foldincreaseinABCD3expression inanEGFRoverexpressingDU-145WTcelllinecomparedtonon-transfectedDU-145cells,highlightsaputativenovelregulatorofABCD3.EGFRisoverexpressedin Figure5 ExpressionofABCD3inpanelofpairedprostatecancer celllines.(A)qRT-PCRofnon-malignantAfricanAmericanRC-77N/E wascomparedtomalignantRC-77T/Ecells,DU-145wascompared toDU-145WT(EGFRoverexpressing),LnCaPwascomparedtoC42B,andallsampleswerecomparedtoAfricanAmericanMDA-PC-2b cells.Resultsshownisrepresentativeofexperimentsperformedin triplicate. Reams etal InfectiousAgentsandCancer 2011, 6 (Suppl2):S1 http://www.infectagentscancer.com/content/6/S2/S1 Page7of10

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AfricanAmericanProstatepatients[60]andarobust activatorofMAPKERKinnormalandcancercell[61]. Furthermoreoverexpressionissufficienttoincrease proliferation,invasionrelatedEMT,andmetastasis [49,62,63].Thus,itappearsthatABDC3isanovelprostatecancerassociatedgene,thatcould,inpart,beregulatedbyEGFRsignaling. AlthoughfurtherstudiestoinvestigatetheABCD3/RanGAP1relationshipneedtobeconducted,ourresults clearlyillustratetheutilityofhighdensitySNPanalysis,in conjunctionwithappropriatecelllinesthatrepresentthe clinicalconditions,toidentifyregulatorygenesinprostate cancer.Althoughamorein-depthanalysisoftheexact roleofABCD3insucheventsascellproliferation,and chemoresistanceiswarrantedandareunderway.The resultsofthisstudyprovidearationaleforuseofDU-145/ DU-145WTandMDA-PC-2Basculturemodelstostudy molecularmechanismsassociatedwiththehealthdisparity inAfricanAmericanprostatecancerpatients.LimitationofstudyThe97membercandidategenelistwasderivedfroma limitednumberofprostatetumorsamplesandmatched controlfromAAandCA.Nonetheless,approximately, 26527genotype-phenotypeassociationswereobtained withap-value<10-3,ofwhich17542/26527associations hasap-value<10-4.TheassociationswiththehighestPvaluesshowedtwodistinctassociation;oneofwhichhas ledustothinkthattheABCgenefamilyplaysan importantroleinprostatecanceraggressivenessand chemoresistance.Acknowledgements WeacknowledgeDoDGrant#W81XWH-04-1-03-26andNIH/NCRR/RCMIGrant #SG12RR0020-27toR.R.R;MayoClinicDeptofMedicalInformaticsforsupport servicestoK.R.K;Grant#G12RR03059-21A1toC.Y;RCMICancerClusterfor providednetworkingopportunitiesamongitsinvestigators(R.R.R.andC.Y.). SpecialthankstoKarunyaKandimallaforintroducingR.R.R.toK.R.K. Thisarticlehasbeenpublishedaspartof InfectiousAgentsandCancer Volume6Supplement2,2011:ProceedingsoftheFirstBiennialConference ontheScienceofGlobalProstateCancerDisparitiesinBlackMen.Thefull contentsofthesupplementareavailableonlineathttp://www. infectagentscancer.com/supplements/6/S2. Authordetails1CollegeofPharmacyandPharmaceuticalSciences,FloridaA&MUniversity, Tallahassee,Florida,USA.2DepartmentofMedicalInformatics,MayoClinic CollegeofMedicine,Rochester,MN,USA.3DeptofBiologyandCenterfor CancerResearch,TuskegeeUniversity,Tuskegee,Alabama,USA.4Collegeof Pharmacy,UniversityofFlorida,Gainesville,Florida,USA.5ProstateDisease Center,DepartmentofUrology,UniversityofFlorida,Gainesville,Florida, USA. Authors contributions Allauthorsreadandapprovedthefinalmanuscript. 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