arraywww

RESEARCHOpenAccessSmallmoleculeantagonistofthebonemorphogeneticproteintypeIreceptorssuppressesgrowthandexpressionofId1andId3inlungcancercellsexpressingOct4ornestinElaineLangenfeld1,MalikDeen2,EmmanuelZachariah4andJohnLangenfeld3,4*AbstractBackground:Bonemorphogeneticproteins(BMP)areembryonicmorphogensthatareaberrantlyexpressedinlungcancer.
BMPsmediatecellfatedecisionsandself-renewalofstemcells,throughtranscriptionregulationofinhibitorofdifferentiationprotein/DNAbindingproteins(Id1-3).
InhibitionofBMPsignalingdecreasesgrowthandinducescelldeathoflungcancercellslinesbydownregulatingtheexpressionofIdproteins.
ItisnotknownwhethertheBMPsignalingcascaderegulatesgrowthandtheexpressionofIdproteinsoflungcancercellsexpressingthestemcellmarkersOct4and/ornestin.
Methods:LungcancercellsexpressingOct4ornestinwereisolatedfromlungcancercelllinesbystablytransfectingtheOct4promoterornestinpromoterexpressionvectorsthatinduceexpressionofthegreenfluorescentproteinreporter.
Results:OurstudiessuggestthatlungcancercellsexpressingOct4ornestinaredifferentcellpopulations.
MicroarrayandquantitativeRT-PCRdemonstratedthattheexpressionofspecificstemcellmarkersweredifferentbetweenisolatedOct4andnestincells.
BoththeOct4andnestinpopulationsweremoretumorigenicthancontrolsbuthistologicallytheywerequitedifferent.
TheisolatedOct4andnestincellsalsorespondeddifferentlytoinhibitionofBMPsignaling.
BlockadeofBMPsignalingwiththeBMPreceptorantagonistDMH2causedsignificantgrowthinhibitionofboththeOct4andnestincellpopulationsbutonlyincreasedcelldeathinthenestinpopulation.
DMH2alsoinducedtheexpressionofnestinintheOct4populationbutnotinthenestincells.
WealsoshowthatBMPsignalingisanimportantregulatorofId1andId3inboththeOct4andnestincellpopulations.
Furthermore,weshowthatNeuNisfrequentlyexpressedinNSCLCandprovideevidencesuggestingthatOct4cellsgiverisetocancercellsexpressingnestinand/orNeuN.
Conclusion:Thesestudiesshowthatalthoughbiologicallydifferent,BMPsignalingisgrowthpromotingincancercellsexpressingOct4ornestin.
InhibitionofBMPsignalingdecreasesexpressionofIdproteinsandsuppressesgrowthofcancercellsexpressingOct4orNestin.
SmallmoleculeantagonistsoftheBMPtypeIreceptorsrepresentpotentialnoveldrugstotargetthepopulationofcancercellsexpressingstemcellmarkers.
Keywords:Oct4,Nestin,NueN,BMP,Antagonist,Id1,Id3,Cellgrowth,Celldeath*Correspondence:langenje@rwjms.
rutgers.
edu3DivisionofThoracicSurgery,Rutgers-RobertWoodJohnsonMedicalSchool,OneRobertWoodJohnsonPlace,P.
O.
Box19,NewBrunswick,NJ08903-0019,USA4Rutgers-CancerInstituteofNewJersey,NewBrunswick,NJ08903-0019,USAFulllistofauthorinformationisavailableattheendofthearticle2013Langenfeldetal.
;licenseeBioMedCentralLtd.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Langenfeldetal.
MolecularCancer2013,12:129http://www.
molecular-cancer.
com/content/12/1/129IntroductionLungcanceristheleadingcauseofcancerdeathsintheworld.
Morepatientsdiefromlungcancerthanbreast,colon,prostate,andkidneycancercombined.
Approxi-mately85%ofpatientsdiagnosedwithlungcancerwilldiefromtheirdisease.
Lungcancersinitiallyrespondingtochemotherapeuticagentswilleventuallydevelopre-sistancetotherapy.
TheexpressionofstemmarkersOct4and/ornestinincancercellsisassociatedwithresistancetochemotherapeuticagentsleadingtotreat-mentfailures[1-5].
Cancerstemcells(CSC)havebeendefinedasraretumorcellswiththecapacitytoself-renewalandinitiatetumorgrowthinmousexenograftsthathistologicallyre-capitulatetheprimarytumor[6,7].
CSCarereportedtobemoreresistanttochemotherapyagentsandthein-ductionofapoptosiscomparedtootherpopulationsofcellswithinthesametumor[8-11].
Self-renewalandchemotherapyresistanceincancer-initiatingcellsisme-diatethroughtheexpressionofinhibitorofdifferenti-ation/DNAbindingproteinsId1andId3[12-14].
CD44andCD133antigensarecommonlyusedtoiso-lateCSCfromlungandothercarcinomas[7,11,15-19].
IsolatedCD44andCD133cancercellsalsoexpressstemcellregulatorsOct4,Sox2,nanog,andnestin[11,20-23].
Oct4istransientlyexpressedduringearlydevelopmentinpluripotentstemcellsandisrequiredforself-renewal[24].
Nestinisamarkerofneuralprogenitorcellsandisfrequentlyexpressedincancercellsofnon-smallcelllungcarcinomas[21,25-27].
AlthoughseveralstudieshaveshownCD44+andCD133+cellsinitiatetumorgrowthatasignificantlylowernumberofcellscomparedtothenegativepopulations,CD44-andCD133-popula-tionshavealsobeenreportedbetumorinitiatingcellsinsomestudies[17,28].
Thesestudiessuggestthatfurthercharacterizationofspecificpopulationofcancercellsmaybeneeded.
Self-renewalisanessentialmechanismrequiredforstemcellstomaintainlong-termpopulatingcells.
Bonemorpho-geneticproteins2and4(BMP2/4)mediateself-renewalofembryonicstemsbystimulatingtheexpressionofId1[29].
BMPssignalthroughtransmembraneserine/kinasescom-posedoftypeI(alk2,alk3,andalk6)andtypeIIreceptors.
TheBMPreceptorcomplexphosphorylatessmad-1/5,whichthenactivatesresponseelementsontheId1,Id2,andId3promoters[30,31].
DownregulationoftypeIBMPre-ceptorswithsiRNAandselectivesmallmoleculeantago-nistsdecreasesthephosphorylationofsmad-1/5causingadecreaseinexpressionofId,Id2,andId3inlungcancercelllines[32].
TheinhibitionofBMPtypeIreceptorsalsoin-ducescelldeathandcausessignificantgrowthinhibitionoflungcancercelllines,whichismediatedthroughthedown-regulationofIdproteins[32].
TheroleoftheBMPsignal-ingcascaderegulatingtheexpressionofIdproteinsandgrowthofcancercellsexpressingOct4ornestinisnotknown.
WefurtherdelineatetheheterogeneityoflungcancerbyshowingthatOct4,nestin,andNeunareexpressedinlungcancercelllinesandprimarylungtumors.
Weiso-latedfromlungcancercelllines,cellsthatexpressOct4ornestin.
OurstudiessuggestthatOct4andnestinex-pressingcancercellsareadifferentpopulationoftumor-initiatingcells.
InhibitionofBMPsignalingwiththeselectiveantagonistDMH2causedadecreaseintheexpressionofId1/Id3andinducedsignificantgrowthinhibitionofcancercellsexpressingOct4ornestin.
BlockadeofBMPsignalingwithsmallmoleculeantago-nistsofthetypeIBMPreceptorsrepresentsapotentialmeanstoregulatethegrowthoflungcancercellsex-pressingstemcellmarkers.
MaterialsandmethodsCellcultureTheA549andH1229lungcancercelllineswerecul-turedinDulbecco'sModifiedEagle'smedium(DMEM,SigmaAldrich,StLouis,MO,USA)with5%fetalbovineserum(FBS)[33].
ThelungcancercelllinesH157,H727,U1752,andH358,andH865wereculturedin90%RPMIand10%FCS.
ThecelllineswereobtainedfromATCCandfromMalcolmBrock,JohnHopkinsUniversity.
ExpressionvectorsTheOct4promoter/EGFPplasmidvectorwasagiftfromWeiCui(RoslinInstitute,Midiothian,UK[34].
Thenestinpromoter/EGFPwasobtainedfromRohanHumphrey(LaJolla,CA).
TheSM22promoter/lucifer-aseexpressionvectorwasobtainedfromJulianSolway(UniversityofChicago,ChicagoIL)[35].
TheSM22pro-moterwasclonedintothepAcGFP1–1expressionvec-torattheXhoI/HindIIIsites(Clontech,PaloAlto,CA).
Cellsweretransfectedusingelectroporationandthense-lectedwithneomycin.
ControlcellsweretransfectedwithpcDNA3.
1vector(Invitrogen)expressingEGFP(Clontech).
HumantumorsamplesHumanlungtumortissuesampleswereobtainedfromtheRutgersCancerInstituteofNewJersey(CINJ)afterapprovalbytheinstitutionalreviewboardandethicscommitteeoftheRutgersRobertWoodJohnsonMed-icalSchool.
Protocolapprovalnumber,0220013730.
Thereviewcommitteewaivedtheneedforconsentsincenopatientidentifierswereused.
CelldeathassayCellswereplatedin6wellplatesat106cellsperwellandtreatedwith1μMDMSOor1μMDMH2forLangenfeldetal.
MolecularCancer2013,12:129Page2of15http://www.
molecular-cancer.
com/content/12/1/12948hours.
Adherentandfloatingcellswereharvestedandincubatedwith0.
1mg/mlofethidiumbromide.
Im-mediatelyafterstainingapproximately100cellswerecountedandthepercentageofcellsthattookupeth-idiumbromidewasdetermined.
CellcountsCellswereplatedinto6wellplatesat105cellsperwellandtreatedwith1μMDMSOor1μMDMH2for7days.
Thecellsweredetachedwithtrypsin,stainedwithtrypanblue,andthenumberoflivecellscountedusingahemacytometer.
ImmunoflourescentimagingImmunofluorescentimagingwasperformedonbothnon-adherentandadherentcellsaspreviouslydescribed[36].
Cellsweretrypsinizedandimmunofluorescentim-agingperformedorplacedintocloningchambers(NuncLab-Tek,Rochester,NY).
Briefly,cellswerefixedwith3.
7%formaldehyde,permeabilizedwith0.
5%TritonX,andblockedwith1%BSA/PBS.
Cellswereincubatedwithprimaryantibodiesin1XPBS/1%BSAatroomtemperatureforonehour.
AppropriateAlexFluor488,568,or647(Invitrogen/MolecularProbes)conjugatedsecondaryantibodieswereused.
Thesecondaryantibodywasaddedforonehouratroomtemperature.
Controlsweretreatedinthesamemannerbutdidnotreceiveprimaryantibody.
Inallnegativecontrolssamplestherewasnofluorescentsignal.
Primaryantibodiesusedwererabbitanti-Oct4(SantaCruz,SantaCruz,CA),rabbitanti-humannestin(Chemicon),mouseanti-humannestin(Chemicon),andmouseanti-NeuN(Chemicon,Temecula,CA).
FluorescentimageswerecapturedusingaNikonEclipseTE300invertedepifluorescentmicroscopeandaCoolSnapblackandwhitedigitalcamera.
IPLabimagingsoftwarewasusedtoassignpseudo-colortoeachchannel.
Immunohistochemistry(IHC)IHCwasperformedonformalin-fixedparaffin-embed-dedprimaryNSCLCandtumorxenograftsinmice.
Antibodiesusedweremouseanti-Oct4A(CellMarque,Rocklin,CA),mouseanti-humannestin(Chemicon),mouseanti-NeuN(Chemicon,Temecula,CA),andmouseanti-smoothmuscleactin(SMA)(clone1A4)(Sigma,St.
Louis,MI).
IHCwasperformedon5μmtissuesections.
DetectionofOct4andNeuNonprimaryNSCLCusedTris-EDTAantigenretrievalusingVantanaBenchmarkXTautomatedIHCsystem.
SeminomawasusedasapositivecontrolforOct4andnormalbrainforNeuN.
Fordetectionofnestin,NeuN,andSMAantigenretrievalwasperformedusingTargetRetrievalSolution(DakoCytomation,Carpentaria,CA).
Onthesesamples,theBiomoduleIHCStainingKit(Invitrogen)wasusedasperthemanufacture'sinstructions.
IHConcelllineswasperformedbyplatingcellsonglasscoverslips,fix-ingin4%paraformaldehydefor10minutes,incubatingwithprimaryOct4antibodyfor1hour,andusingthebiomoduleIHCstainingkitfordetection.
QuantificationofgeneexpressionRNAwasextractedusingtheRNeasykitasperthemanu-facturer'sinstructions(Qiagen,Valencia,CA).
DNAasewasusedtoremoveanyDNAcontamination.
cDNAwasgener-atedusingAdvantageRTforPCRkit(BDBiosciences-Clontech,PaloAlto,CA).
QuantitativePCRwasperformedwiththeStratageneMx3005preal-timethermalcycler(AgilentTechnologies)withpredesignedandvalidatedTaq-Mangeneexpressionassaysaccordingtothemanufac-turer'sspecifications(LifeTechnologies,GrandIsland,NY).
Referencenumbersusedare:GAPDH(Hs99999905_m1),actin(99999903_m1),ACVRL1(alk1)(Hs00163543_m1),ACVR1A(alk2)(Hs00153836_m1),BMR1A(alk3)(Hs00831730_s1),BMPR1B(alk6)(Hs00176144_m1),Pou3f1(Hs00538614_s1)CD133(Hs01009250_m1),UBE2Q1(Hs01079904_m1),Pank3(Hs00388176_g1),andSel1L(Hs01071406_m1),NegativecontrolincludedallreagentsexceptcDNA.
ExpressionwasnormalizedtoGAPDHusingtheformula2ΔCT.
SYBERGreenwasusedtodetectdouble-strandedDNAforthefollowingprimers.
Nestin(F)5′-GCC-CTG-ACC-ACT-CCA-GTT-TA-3′(R)5′-GGA-GTC-CTG-GAT-TTC-CTT-CC-3′,Sox-2(F)5′-CAT-CAC-CCA-CAG-CAA-ATG-AC-3′(R)5′-TGC-AAA-GCT-CCT-ACC-GTA-CC-3′.
Oct4Aspecificprimerswere(F)5′-TCC-CTT-CGC-AAG-CCC-TCA-T-3′and(R)5′-TGA-CGG-TGC-AGG-GCT-CCG-GGG-AGG-CCC-CAT-C-3′.
Oct4primersspanningthefirstintronwere(set2)(F)5′-GAA-GCT-GGA-GAA-GGA-GAA-GC-3′.
(R)5′-GCC-GGT-TAC-AGA-ACC-ACA-CT-3′.
PCRproductswererunonagel,cDNApurified,andsequenced(GENEWIZ,SouthPlainfield,NJ).
GenomiccontaminationwasexaminedbyquantitativePCRofRNAsamples.
Negativecontrolin-cludedallreagentsexceptcDNA.
TransientgeneknockdownSilencerSelectValidatedsiRNAwasusedtoknockdownexpressionofOct4(LifeTechnologies,GrandIsland,NY),IDnumberS10871.
SilencerSelectNegativeCon-trolsiRNA(4390843)wasusedtoconfirmspecificityofknockdown.
OnemillionH1299cellsweretransfectedwith30nMsiRNAwiththeNucleofectorII(AmaxaBiosystems,Gaitherburg,MD)usingthemanufacture'sNucleofectorkitT.
Optimizationwasperformedusingtheenhancedgreenfluorescentreporter(EGFP)(Clon-tech)expressedinthepcDNA3.
1vector(Invitrogen),whichshowedapproximately80%ofthecellsweretransfectedusingthistransfectionprotocol.
Fourty-eightLangenfeldetal.
MolecularCancer2013,12:129Page3of15http://www.
molecular-cancer.
com/content/12/1/129hoursaftertransfectiontheexpressionofOct4expres-sionwasexaminedbyquantitativePCRandWesternblotanalysis.
MicroarrayByFACS,106cellsexpressinghighlevelsofGFPwereiso-latedfromH1299cellsstablyexpressingtheOct4pro-moter/GFPorNestinpromoter/GFPreportervectors.
After24hourstotalRNAwasisolatedusingRNeasyMiniKitasdescribedbythemanufacturer(Qiagen).
DNAsetreatedRNAconcentrationwasmeasuredusingNanoDrop1000spectrophotometer(ThermoScientific)andthequal-itywasanalyzedwithBioanalyzer2100(Agilent).
SpottedmicroarrayswereusedtoidentifydifferentiallyexpressedgenesbetweentheOct4/GFPandNestin/GFPcells.
AfterreversetranscriptionwithSuperScriptII,cDNAwastran-scribedandthesampleswerelabeledwithCy3,andhybrid-izedtohumanonearrayversion4.
2(HOA4.
2)DNAmicroarrays(PhalanxBiotech)containing30,968featuresprobingforapproximately20,230uniquegenes,accordingtostandardproceduresfollowedattheFunctionalGenom-icsoftheCancerInstituteofNewJersey.
MicroarrayswerescannedwiththeGenePix4000BScanner(AxonInstru-ments).
TheGeneExpressionOmnibus(GEO)numberforthemicroarraydataisGSE49281.
FlowcytometryFlourescenceactivatedcellsorting(FACS)analysiswasperformedusingaBeckmanCoulterEpicsXL.
Cellsort-ingwasperformedusingMoFloXDPcellssorter(Beckman,Coulter).
Celllinesstablytransfectedwithex-pressionvectorsweresortedforcellswithhighexpres-sionofGFPornoGFPexpression.
PostsortingFACSanalysiswasusedtoconfirmexpression.
ForFACSana-lysis,theprimaryantibodymouseanti-humanCD44(BPParmingen,SanDiego,CA)wasaddedtocellsonicefor60minutes.
Secondaryantibodieswereaddedfor60mi-nutesonice.
Controlcellsweretreatedwithsecondaryantibodyonly.
IsolatingcellsfromtumorsTumorxenograftsfrommiceweremincedandtreatedwith"digestionbuffer"(10mlHBSS,50mgcollagenasepowder,200μl2.
5%trypsin,50μl1MCaCl2,50μlDNAse).
Fetalbovineserum(FBS)wasaddedandsam-pleswerepassedthrougha100-micronfilter.
Cellswerecentrifugedandsuspendedin3mlofredbloodcelllysisbuffer(0.
15Mammoniumchloride,7mMpotassiumbicarbonate,0.
09mMtetrasodiumEDTA)for10mi-nutes.
ByFACS,theGFP(+)cellswerethenisolated.
WesternblotanalysisTotalcellularproteinwaspreparedusingRIPAbuffercontainingaproteaseinhibitorcocktailandproteinconcentrationwasmeasuredusingtheBCAassayasdescribed[37].
Inbrief,proteinwasanalyzedbySDS-PAGE,transferredtonitrocellulose(SchleicherandSchuell,Keene,NH).
Afterblocking,theblotswereincu-batedovernightat4°CwiththeappropriateprimaryantibodyinTris-bufferedsalinewith1%Tween(TBST)and5%non-fatmilk.
Secondaryantibodieswereappliedfor1houratroomtemperature.
Specificproteinsweredetectedusingtheenhancedchemiluminescencesystem(Amersham,ArlingtonHeights,IL).
Theprimaryanti-bodiesthatwereusedwererabbitmonoclonalanti-pSmad1/5/8(CellsignalingTechnology,DanversMA)rabbitanti-actin,anaffinityisolatedantigenspecificanti-body(Sigma,SaintLouis,MO),rabbitmonoclonalanti-Id1,rabbitmonoclonalanti-Id3(Calbioreagents,SanMateo,CA),rabbitanti-Oct4(SantaCruz,SantaCruz,CA),mouseanti-humannestin(Chemicon),andmouseanti-NeuN(Chemicon,Temecula,CA).
DifferentiationofsinglecellsByFACS,theGFP(+)andGFP()cellswereisolatedfromOct4/GFPandNestin/GFPcelllinesandone-hundredcellsplacedintocloningchamberscontainingcellculturemedium(NuncLab-Tek,Rochester,NY)[33].
Cellswereculturedinregularculturemediaforap-proximately14daysuntilcoloniesformed.
Immuno-fluorescentimagingwasthenperformedasdescribedabove.
StatisticalanalysisTocomparetwogroups,astudentt-testwasused.
Dif-ferenceswithPvalues≤.
05wereconsideredstatisticallysignificant.
ResultsExpressionofOct4inlungcancercelllinesOct4hastwoalternativelysplicedvariantsthatcodetheOct4AandOct4Bisoforms.
Oct4Aregulatesself-renewalofstemcells[38,39]andthefunctionofOct4Bisnotknown.
ToevaluateOct4Aexpressioninourlungcancercelllines,quantitativeRT-PCRwasperformed.
Aseminoma(sem),wasusedasapositivecontrol.
APCRproductwasamplifiedunder34(29–34)cyclesinallcelllineswithnoproductinthenegativecontrol.
Sequen-cingconfirmedthattheamplifiedproductwasOct4Aandnotapseudogene.
TocontrolforthepresenceofgenomiccontaminationRNAsamplesweretreatedwithDNaseandPCRperformed.
Thisshowedeithernoproductoramplificationatahighcyclenumber(38–39cycles).
Inaddition,toensurecDNAwasbeingamp-lifiedandnotgenomicDNA,primersrecognizingexon1andexon2wereused(primerset2).
QuantitativeRT-PCRamplifiedtheexpected420basepairproductatlessthan34cyclesinallcellslines(Figure1A).
Langenfeldetal.
MolecularCancer2013,12:129Page4of15http://www.
molecular-cancer.
com/content/12/1/129Westernblotanalysis,usinganantibodyrecognizingOct4A,demonstratedbandsat72kdand40kdintheseminomaandinall6lungcancercelllinesexamined(Figure1Ba).
TheexpectedsizeofOct4Aisapproxi-mately40kd.
Westernblotanalysisusingonlythesec-ondaryantibodyrevealedafaintbandat72kdintheseminoma(Figure1Bb),suggestingthatthiscouldbeanon-specificband.
WesternblotanalysisshowedthatknockdownofOct4usingsiRNAtargetingOct4Ashowedadecreaseinthe40Kdbandbutnotanon-specific24Kdband(Figure1C).
QuantitativePCRalsoshowedareductionintheexpressionofOct4followingsilencingofOct4withsiRNA(Additionalfile1:FigureS1).
ImmunohistochemistrydemonstratedthatOct4isFigure1Oct4A,nestin,andNeuNareexpressedinlungcancercelllines.
(A)PCRproductofquantitativePCRonlungcancercelllinesusingprimerset2showingOct4Aexpression.
(B-a)WesternblotanalysisusingantibodyrecognizingOct4A(40kd).
(B-b)Westernblotusingonlysecondaryantibody(C)WesternblotanalysisofOct4showingsiRNAknockdownofOct4.
(D)ImmunoflourscentimagingofH1299cellsshowingnuclearexpressionofOct4(blackarrowsshowpositivecells).
(E)RT-PCRdemonstratingnestinexpressioninlungcancercelllinesshowninFigure1A(lanes1–6).
Lane8,isactincontrol,lane7negativecontrol.
(F)Westernblotanalysisshowingnestinexpressioninlungcancercelllines.
(G)ImmunoflourscentimagingofH1299cellsshowingexpressionofnestinincytoplasmicfilaments(40x).
(H)ImmunoflourescentimagingandcorrespondingphasecontrastimageofH1299cellsshowingnestinisexpressedonlyinasubsetofcells(20x).
Whitearrowsshowimmunopositivecells.
(I)WesternblotanalysisshowingtheexpressionofNeuNinlungcancercelllines.
(J)ImmunoflourescentimagingandcorrespondingphasecontrastimageshowingnuclearexpressionofNeuNoccursinasubsetofcells(20x).
(K)FACSanalysisforCD44expressiononH1299cells.
(a)controlcellsstainedwithphycoerythrin(PE)secondaryantibodyarewithoutfluorescence.
(b)Greaterthan99%ofcellsstainedwithanti-CD44andPEsecondarydemonstratedfluorescence.
Langenfeldetal.
MolecularCancer2013,12:129Page5of15http://www.
molecular-cancer.
com/content/12/1/129expressedinthenucleusinapproximately16%ofthecellswithinthecelllines(Figure1D).
ExpressionofnestininlungcancercelllinesByquantitativeRT-PCR,nestinwasexpressedinall6lungcancercelllinesexamined(Figure1E).
Amplifica-tionoccurredunder34cycles(26–34)inallofthecelllines.
SequencingofthePCRproductconfirmedthatnestinwasamplified.
Westernblotanalysisfornestinshowedstrongexpressioninallthelungcancercelllinesexamined(Figure1F).
Immunofluorescentimagingoflungcancercelllinesshowedthatnestinisexpressedinthecytoplasmfila-ments(Figure1G).
Thiswasconfirmedusingbothmonoclonalandpolyclonalanti-humanNestinanti-bodies.
Nestinisexpressedonlyinasubsetofthecells(Figure1H),whichwasapproximately20%withineachcellline.
NeuNexpressioninlungcancercelllinesSincenestinisamarkerofneuralcellstypes,weexam-inedwhetherlungcancercellsexpressNeuN(NeuronalNuclei).
NeuNisaproteindetectedinmatureneurons[27].
MonoclonalantibodiesdetectingNeuNhaveshownthatNeuNisnotexpressedinneuralprogenitorsex-pressingnestin[40].
WesternblotanalysisusingthemonoclonalNeuNantibodyshowedanapproximately70Kdbandinallthelungcancercelllinesstudied(Figure1I).
ImmunoflourescentimagingshowednuclearexpressionofNeuNinthelungcancercelllines(Figure1J).
SimilartoOct4andNestinexpression,NeuNwasexpressedinapproximately15%ofcellsineachcellline(Figure1J).
CD44expressioninlungcancercelllinesByFACSanalysis,over99%ofA549andH1299cellsexpressedCD44(Figure1Kanddatanotshown).
There-fore,inA549andH1299lungcancercelllines,CD44doesnotappeartorepresentaspecificpopulationofcells.
Oct4,nestin,andNeuNexpressioninprimaryNSCLCToaccesswhethertheheterogeneityidentifiedinthelungcancercelllinesoccursinprimarylungcancer,weexaminebyimmunohistochemistry(IHC)theexpressionofOct4A,Nestin,andNeuNinNSCLC.
PriorstudieshavereportedthatOct4andNestinareexpressedinNSCLC[20,41].
Usingamonoclonalantibodyrecogniz-ingOct4Awould,weshowednuclearexpressionofOct4Ainaseminoma(Figure2A).
Oct4Awasexpressedin11ofthe12NSCLCexamined.
NuclearexpressionOct4AwasseeninoneNSCLC(Figure2C)whilethecytoplasmicexpressionwasdemonstratedin10tumors(Figure2D).
Cellcountsshowedonly1-3%ofthecancercellsexpressedOct4A.
Nestinwasexpressedinfranklymalignantcancercellsin15of20(75%)NSCLC(Figure2F),whichisconsistentwithpriorreports[41].
ThepercentageofcancercellsexpressingNestinwasfromingmorphologicalfeaturesofneuro-endocrinedifferentiation,13of18(72%)NSCLCexpressedNeuN(Figure2H).
Thepercentageofcellsex-pressingNeuNwassignificantlyhigherthanthatofNes-tin(Additionalfile2:TableS1).
Incomparison,theFigure2Immunohistochemical(IHC)stainingforOct4,Nestin,andNeuNinprimaryNSCLC.
Shownarerepresentativeimages.
(B,E,G)Negativecontrols.
Positivecellsarestainedbrown.
(A)SeminomawasusedaspositivecontrolforOct4.
(C)showsnuclearstainingincancercellsforOct4and(D)showscytoplasmicexpressionofOct4.
(F-H)CancercellsexpressingNestinorNeuNexpression.
(aa)MagnifiedimageofcellsexpressingNeuN.
(I-J)ChromograninandsynaptophysinwerenotexpressedintheNSCLCshownabove.
Langenfeldetal.
MolecularCancer2013,12:129Page6of15http://www.
molecular-cancer.
com/content/12/1/129tumorsthatexpressedthyroidtranscription1(TTF-1)nearlyallthecancercellswereimmunopositive(Additionalfile2:TableS1).
TodeterminewhetherNeuNand/orNestinimmunopositivecellsareofneuro-endocrineorneurallineages,IHCfortheneuroendo-crinemarkersynaptophysinandchromograninwasperformed.
Only1ofthe10tumorspositiveNeuNand/orNestinexpressedsynaptophysin(Figure2I)andnoneexpressedchromogranin(Figure2J).
Thisdatasug-gestthatcancercellsexpressingNeuNorNestinareofaneurallineage.
IsolationofOct4/GFPandnestin/GFPcellsA549,H1299,andU1752celllineswerestablytrans-fectedwithanexpressionvectorcontainingpromotersofOct4ornestinthatregulatestheexpressionofGFP.
HumanembryonicstemcellsexpressingthisOct4/GFPreporterintransgenicmicewereshowntobepluripo-tent[34].
SomaticcellsdidnotactivatethisexogenousOct4promoterconstruct[42].
Lungcancercellsex-pressinghighlevelsofGFPwereobtainedinall3celllines(Figure3A).
TheGFP(+)cellsweresortedbyFACSandplacedintocellculture.
Afterapproximately6weeks,thepercentageofcellsexpressingGFPde-creasedtoapproximately50%and50%becameGFP().
ByFACS,theGFP(+)andGFP()populationswereagainisolatedtoover99%purity(Figure3A).
Quantita-tiveRT-PCRshowedthatOc4andSox2expressionwas4to5-foldhigherintheGFP(+)cellsincomparisontotheGFP()cells(Figure3B).
Immunofluorescentim-agingshowedthattheGFP(+)populationexpressedOct4(Figure3C).
All3celllinestransfectedwiththeNestin/GFPreporteralsoshowedstrongGFPexpression(Figure3D).
TheGFPpositivecellswereisolatedbyFACSandplatedintocellculture.
Afterapproximately4weekstheGFP+andGFP-cellswereisolated.
QuantitativePCRdemonstrateda4.
5foldhigherexpressionofNestinintheGFP+cellsincom-parisontotheGFP()cells(Figure3E).
Dualimmuno-fluorscentimagingshowedthatNeuNwasnotexpressedinNestin/GFPcells,suggestingthatNeuNandNestinrepre-sentdifferentcellpopulations(Figure3F).
ExpressionprofilesaredifferentbetweentheOct4/GFPandNestin/GFPcellsToexaminedifferencesinexpressionbetweentheOct4/GFPandNestin/GFPcells,theGFP(+)cellswereisolatedtoover99%puritybyFACS.
QuantitativePCRdemon-stratedafive-foldhigherexpressionofnestinintheNestin/GFPcellscomparedtoOct4/GFPcells(Figure3G).
Therewastwo-foldhigherexpressionofOct4andCD133intheOct4/GFPcellscomparedtoNestin/GFPcells(Figure3G).
Microarrayanalysisshowedthattherewere603genesthatweredifferentlyexpressedby>2foldbetweenGFPcellsisolatedfromtheOct4/GFPandNestin/GFPcells.
Byquan-titativePCR,weexaminedselectedgenesthathada4foldorhigherdifferenceinexpressionandwererelatedtocan-cergrowthand/orstemness.
Pou3f1,whichisexpressedinneuralprogenitorscells[43],wasexpressedinNestin/GFPcellsbutnotOct4/GFPcells(Figure3H).
Sel1L,regulatesself-fatedecisions[44]andenhancestumorprogression[45]wasexpressedover3foldmoreinOct4/GFPcellsthantheNestin/GFPcells(Figure3H).
UBE2q1andPank3areregulatorsofcellularmetabolismandenhancecellgrowth[46,47]werealsoconfirmedtohaveagreaterthan3foldhigherexpressionintheOct4/GFPcellscomparedtoNes-tin/GFPcells(Figure3H).
ThelevelofexpressionoftheBMPtypeIreceptorsdif-fersbetweenpluripotentstemcellsandstemcellprogeni-tors.
Alk3(BMPRIA)isexpressedatmuchhigherlevelinpluripotentstemcellscomparedtoAlk6(BMPRIB)[48].
Alk6levelsincreaseinsomestemcellprogenitors.
Tofur-therassessdifferencesbetweentheOct4/GFPandNestin/GFPcells,theleveloftheBMPtypeIreceptorsalk2,alk3,andalk6wasexaminedbyquantitativeRT-PCR.
GFP(+)isolatedfromOct4/GFPcellsshoweda26foldhigherex-pressionofalk3comparedGFP(+)cellsisolatedfromNes-tin/GFPcells(Figure3I).
Alk2andalk6wereexpressed2.
5and5foldhigherrespectivelyintheOct4/GFPcellscom-paredtotheNestin/GFPcells(Figure3I).
Oct4/GFPandNestin/GFPcellsaretumorinitiatingcellsByFACS,theGFP+cellswereisolatedfromtheH1299,A549,andU1752cellsstablyexpressingtheOct4/GFPortheNestin/GFPreporters.
Controlswerecellsstablyex-pressingGFPbyaconstitutivelyactiveCMVpromoter(Vector/GFP)andOct4/GFP+cellsthatlostGFPexpres-sion(GFP-)afterisolation.
Celllineswerealsostablytrans-fectedwithanexpressionvectorcontainingthesmoothmusclepromoter,Sm22,thatdrivesGFPexpression.
OnemillionSm22/GFPcellsdidnotformtumorsinmiceafter4monthswhile106Oct4/GFPandNestin/GFPdid(Figure4A).
OnehundredthousandVector/GFP(0of3)andGFP()(0of3)fromtheH1299,A549,andU1759celllinesdidnotformtumorsafter4months,whiletheNestin/GFPcells(3of3)andOct4/GFP(2of3)developedtumorswithin2weeks(Figure4B-D).
OnehundredthousandSm22/GFPcellsfromA549andU1759cellsdidnotformtumorsafter4months.
InallthreecelllinestheNestin/GFPgrewfasterthantheOct4/GFPcells.
OnehundredthousandOct4/GFPcellsisolatedfromtheU1752cellsdidnotinitiatetumorgrowth(Figure4D).
TumorinitiationwasslowerintheU1752cellstaking12weeksfortheNestin/GFPcellstoformatumor.
When105Oct4/GFPcellsderivedfromU1752cellswereco-injectedwithMatrigelatumorformedwithin3weekswhiletheGFP()cellsdidnotdevelopatumorafter4months.
Therefore3of3Oct4/GFPandLangenfeldetal.
MolecularCancer2013,12:129Page7of15http://www.
molecular-cancer.
com/content/12/1/129Nestin/GFPcellsdemonstratedabilitytoinitiatetumorgrowthgreaterthanVector/GFP,Sm22/GFP,andGFP()controls.
TenOct4/GFPcellsformedtumorscomparedthe104Vector/GFPcells(Additionalfile3:TableS2).
One-hundredthousandOct4/GFPandNestin/GFPcellsiso-latedfromtheA549andH1299celllinesre-establishedtumorgrowthfollowingre-injectionintomice(Figure4Fanddatanotshown).
TheNestin/GFP(+)cellsagainformedtumorsthatgrewfasterthantheOct4/GFP(+)cells(Figure4F).
HistologicallytheOct4/GFPandnestin/GFPtumorsaredifferentHematoxylinandEosinstainingdemonstratedthattheOct4/GFPandNestin/GFPtumorsrecapitulatedadenocar-cinomasofthelungbutthedegreeofdifferentiationwasdifferent.
TheOct4/GFP(+)cellsisolatedfromA549andFigure3IsolationoflungcancercellsexpressingOct4ornestin.
LungcancercelllineswerestablyexpressedwithOct4ornestinpromotersthatdriveGFPexpression(Oct4/GFPandNestin/GFP).
(A)H1299cellshowingGFPexpressionafterstabletransfectionwithOct4/GFPreporter.
ByFACS,GFP(+)cellsweresortedfromtheOct4/GFPcellsandplacedintocellculture.
After6weekscellsweresortedfortheGFP(+)andGFP()populations.
(A)FACSanalysisshows>99%purityofGFP(+)andGFP()cellpopulations.
(B)QuantitativeRT-PCRshowssignificantlyhigherexpressionofOct4andSox2inGFP(+)cellscomparedtoGFP()cells(n=3).
(C)ImmunoflourescentimagingshowingOct4/GFP(+)cellsstainforOct4.
(D)TheGFP(+)cellsweresortedfromH1299cellsstablyexpressingNestin/GFPreporterandplacedintocellculture.
Afterapproximately8weeks,theGFP(+)andGFP()cellswereisolatedbyFACS.
(E)QuantitativeRT-PCRshowshigherexpressionofnestininGFP(+)cellscomparedtoGFP()cells.
(F)DualimmunoflourescentimageshowsNeuN(red)isnotexpressedinNestin/GFP(+)(green)cells.
(G-I)GFP(+)cellswereisolatedfromOct4/GFPandNestin/GFPcells.
QuantitativeRT-PCRdemonstratesdifferentialexpressionofstemregulationsandBMPtypeIreceptorsbetweenOct4/GFPandNestin/GFPcells(n=2).
Langenfeldetal.
MolecularCancer2013,12:129Page8of15http://www.
molecular-cancer.
com/content/12/1/129H1299cellsdevelopedtumorsthatweremoredifferenti-ated,formingglandularstructuresresemblingacini(Figure5A,D).
TheNestin/GFPdevelopedpoorlydifferenti-atedtumorswithnoglandformation(Figure5B,E).
TheOct4/GFPtumorsalsohadalargeamountofstromalcellssurroundingtheacini,whichstainforsmoothmuscleactin(SMA)demonstratingthattheywereeithersmoothmusclecellsormyofibroblasts(Figure5H).
TumorsfromtheNes-tin/GFPcellsshowedlittleexpressionofSMA(Figure5I).
OnemillionunselectedA549andH1299cellsstablyex-pressingGFP(Vector/GFP)developedpoorlydifferentiatedtumorswithlittlestromaltissue(Figure5C,F).
BMPsignalinginOct4/GFPandNestin/GFPcellsTheselectiveantagonistofthetypeIBMPreceptorDMH2causessignificantgrowthsuppressionanda3-foldincreaseincelldeathofunselectedH1299andA549cells,whichinvolvesthedownregulationofId1andId3[32].
ToassesswhetherBMPsignalingcascadeisactiveincancercellsexpressingstemcellmarkers,theOct4/GFP,Nestin/GFP,andGFP()cellsweretreatedwiththeDMH2.
WesternblotanalysisdemonstratedthatDMH2causedasignificantreductionintheexpres-sionoftheBMPtranscriptionfactorpSmad1/5anditsdirectdownstreamtargetsId1andId3inOct4/GFP,Figure4Oct4/GFPandNestin/GFPcellsinitiatetumorgrowth.
(A-F)StudiesshowdifferencesintumorinitiationofdifferentcellpopulationsisolatedfromH1299,A549,andU1752celllines.
(A)ByFACS,106GFP(+)cellswereisolatedfromH1299cellsstablyexpressingOct4/GFP,Nestin/GFP,orSM22/GFPexpressionvectorsandinjectedsubcutaneouslyintonudemice.
(B)105GFP(+)cellsisolatedformVector/GFPandOct4/GFPcellswereinjectedintonudemice.
GFP()cellswerealsoisolatedfromOct4/GFP(+)cellsthatbecameGFP()afterbeingincellculturefor6weeks.
(C-D)ByFACS,105GFP(+)wereisolatedfromA549andU1752cellsstablyexpressingVector/GFP,Oct4/GFP,Nestin/GFP,SM22/GFPexpressionvectorsandinjectedsubcutaneouslyintonudemice.
GFP()cellswereisolatedfromOct4/GFPcells.
(E)105GFP(+)andGFP()cellsisolatedfromU1752Oct4/GFPcellswereinjectedintonudemicewithMatrigel.
ThesestudiesshowthatOct4/GFPandNestin/GFPcellsaremoretumorigenicthanVector/GFP,GFP(),andSM22cells,whichdidnotformtumorsatthesecellconcentrations.
Inaddition,Nestin/GFPcellsgrowfasterthanOct4/GFPcells.
(F)104GFP(+)cellsisolatedfromOct4/GFPandNestin/GFPtumorsre-establishedtumorafterrepeatedinjectionsintonudemice.
Langenfeldetal.
MolecularCancer2013,12:129Page9of15http://www.
molecular-cancer.
com/content/12/1/129Nestin/GFP,andGFP()cells(Figure6A).
DMH2causedsignificantgrowthinhibitionofOct4/GFP,Nes-tin/GFP,andGFP()cells(6B).
InhibitionofBMPsig-nalingcausedasignificantlygreaterinductionofcelldeathintheNestin/GFPcellscomparedtotheOct4/GFP(Figure6C).
SinceBMPsignalinginhibitsneuraldifferentiationofembryonicstemcells[49,50],weexam-inedwhetherDMH2alteredtheexpressionofnestininOct4/GFPand/orNestin/GFPcells.
DMH2inducedasignificantincreaseintheexpressionofnestinintheOct4/GFPcellswithasmalldecreaseinexpressionintheNestin/GFPcells(Figure6D).
DMH2didnotcauseasignificantchangeintheexpressionofOct4ineithercellline(datanotshown).
Oct4/GFPcellsgivesrisetocellsexpressingnestinandNeuNThedownregulationofId1inembryonicstemcellspro-motesdifferentiation[29].
ThedownregulationofId1andtheinductionofnestinintheOct4/GFPcellsfol-lowinginhibitionofBMPsignalingsuggestedthattheOct4/GFPcellsmightundergocellulardifferentiation.
Toassessdifferentiation,tumorsformedfromtheOct4/GFPcellswereexaminedfortheexpressionofnestinandNeuN.
ByIHC,approximately3%ofthecancercellsfromtheOct4/GFPtumorsexpressedNestinandNeuN(Figure7).
Thenestin(+)cellslocalizedtotheperipheryofthetumoraciniandtheNeuN(+)cellswereidenti-fiedinthecenteroftheacini(Figure7).
Sincethenestin(+)andNeuN(+)cellswereidentifiedintwodifferentregionsofthetumorsuggeststhattheyaretwoseparatecellpopulations.
Tofurtherassessdifferentiation,coloniesformedfromasingleOct4/GFPcellwereexaminedfortheexpressionofnestinorNeuN.
ByFACS,theGFP(+)andGFP()cellswereisolatedfromtheH1299Oct4/GFPcellsand100cellsplatedontoglasscoverslips.
Microscopycon-firmedthatcellsadheredtothecoverslipsassinglecells.
Aftertwoweekscolonieswereexaminedbyimmu-noflourescentimagingandthepercentageofcoloniesstainingforeitherNeuNornestinweredetermined.
ThemajorityofthecellswithineachcolonylostGFPexpres-sion(Figure7G).
AllthecoloniesfromtheGFP(+)cellsstainedforNeuNand96%stainedfornestin(Figure7andAdditionalfile4:TablesS3andAdditionalfile5:TableS4).
ThecoloniesfromtheGFP()cellsstainedforNeuNin52%andnestinin40%(Additionalfile4:TablesS3andAdditionalfile5:TableS4).
DualFigure5HistologicallythetumorsformedfromOct4/GFPandNestin/GFPcellsaredifferent.
HematoxilinandEosin(H&E)stainingwasperformedontumorsformedfromGFP(+)cellsisolatedfromOct4/GFPandNestin/GFPofA549andH1299celllines(A-F).
Tumorsformedfrom106Vector/GFPcellswereusedasacontrol.
(A,D)Oct4/GFPcellsformedmoredifferentiatedtumorswithacinisurroundedbylargeamountsofstromaltissue.
Blackarrowsmarkstromaltissue.
(B,E)TheNestin/GFPand(C,F)Vector/GFPtumorswerepoorlydifferentiatedwithminimalstromaltissue.
(H)IHCshowthatthestromaltissuefoundinOct4/GFPtumorsstainforsmoothmuscleactin(SMA).
(I)VerylittleSMAwasexpressedintheNestn/GFPtumors.
Langenfeldetal.
MolecularCancer2013,12:129Page10of15http://www.
molecular-cancer.
com/content/12/1/129immunoflourescentimagingshowedthatcellsexpressingNeuNornestinnolongerexpressedGFP(Figure7H-J).
OnlysmallpercentageofthecellswithineachcolonyexpressedeitherNeuNornestin(Figure7H-J).
SinceonlyminorityofthecellswithinacolonyexpressedNeuNornestin,suggeststhattheirexpressionoccurslateinthecolonyformation.
Together,thesedatasup-portthatOct4/GFPcellsgiverisetocellsthatexpressNeuNorNestin.
DiscussionCD133+andCD44+cellsarereportedtorepresent"cancerstemcells"inlungcarcinomas,whichhavealsobeenshowntoexpressOct4and/ornestin[11,20,21].
WeprovideevidencethatlungcancercellsexpressingOct4ornestinaredifferentcellpopulations.
Thelevelofexpressionofnestin,BMPreceptors,andotherstemcellregulatorsaredifferentiallyexpressedbetweentheOct4/GFPandNestin/GFPcells.
Wealsodemonstratebio-logicaldifferencesbetweentheOct4/GFPandNestin/GFPcells.
TheNestin/GFPcellsgrewfasterinnudemicethanOct4/GFPcellsandformpoorlydifferentiatedtumors.
TheOct4cellsformedmoredifferentiatedtu-morsandhadamuchlargenumberofcellsexpressingsmoothmuscleactin.
TheresponsetoBMPreceptoran-tagonistalsodiffered.
DMH2inducedtheexpressionofnestinintheOct4/GFP+cellsbutnotintheNestin/GFP+cells.
InhibitionoftheBMPsignalingcascadealsocausedmorecelldeathintheNestin/GFPcellscom-paredtotheOct4/GFPcells.
WeshowthatCD44isexpressedinnearlyallcancercellsinourcelllinesandCD133isexpressedinbothOct4andnestincellpopulations.
OtherreportshavedemonstratedthatCD133+cancercellsalsoexpressOct4,nestin,nanog,andSox2[1,51].
Thelevelofex-pressionofOct4and/ornestinincancercellsmayFigure6InhibitionofBMPsignalingdecreasesId1andId3expressionandinhibitscellgrowthofNestin/GFP,Oct4/GFP,andGFP()cells.
(A)WesternblotanalysisofthedifferentcellpopulationsisolatedfromH1299cellstreatedwith1μMDMH2for48hours.
DMH2decreasespSmad1/5anditstranscriptionaltargetId1andId3inallcellpopulations.
(B)Cellsweretreatedwith1μMDMH2for7daysandlivecellscounted.
Datarepresentsthemeanof3independentexperimentsdepictedaspercentofvehiclecontrol.
(C)Oct4/GFPandNestin/GFPcellsweretreatedwith1μMDMH2for48hoursandthepercentcellsthattakeupEthidiumbromidecounted.
Datarepresentsthemeanof4independentexperimentsshownaspercentofvehiclecontrol.
SignificantcelldeathoccurredonlyintheNestin/GFPcells.
(D)QuantitativeRT-PCRfornestinofOct4/GFPandNestin/GFPcellstreatedwith1μMDMH2for48hours.
Datarepresentsthemeanofatleast3independentexperiments.
InhibitionofBMPsignalinginducednestinexpressioninOct4/GFPcellsbutnotNestin/GFPcells.
Langenfeldetal.
MolecularCancer2013,12:129Page11of15http://www.
molecular-cancer.
com/content/12/1/129inducespecificsurvivalmechanisms.
Knockdownofnes-tinwithsiRNAdecreasesmigrationandinvasivenessofpancreaticcancercelllines[52].
Nestinregulatessur-vivalandself-renewalofneuralstemcells[53].
PatientswithNSCLCexpressingnestindevelopedmoremetasta-sisandhadapoorersurvival[41].
KnockdownofOct4withsiRNAinCD133+lungcancercellsinducedapop-tosis,decreasedtumorigenicity,andincreasedsensitivitytochemotherapyandradiation[20].
OurdifferentiationassayssuggeststhatOct4cellsgiverisetocancercellsexpressingnestinandNeuN.
Furtherstudiesareneededtodetermineifahierarchalorganizationoccursin"cancerstemcells"andexaminethebiologyofotherpopulationofcellsfoundwithinlungcarcinomas.
BMP2andBMP4arehighlyconservedproteinsre-quiredfordevelopmentfrominsectstohumans.
BMPsignalingisnotactiveinadultlungtissuebutisreacti-vatedwithinflammationandcancer[54,55].
BMP2ishighlyoverexpressedin98%ofNSCLCwithlittleex-pressioninpairednormallungtissueandbenignlungtumors[55].
BMP-2signalingisassociatedwithpoorprognosisandtumorprogression[56,57].
BMPsignalinghasbeenshowntostimulatecancergrowth,survival,mi-gration,invasion,metastasis,andtumorangiogenesisofFigure7Oct4/GFP(+)cellsgiverisetocellsexpressingnestinandNeuN.
105GFP(+)cellswereisolatedfromOct4/GFPcellsfromA549andH1299cellsandwereinjectedsubcutaneouslyintonudemice.
IHCwasperformedonthetumorsfornestinandNeuNfrom(A-C)A549Oct4/GFPand(D-F)H1299Oct4/GFPcells.
(A,D)Representnegativecontrols.
(B,E)Cancercellsexpressingnestin(blackarrow)werelocatedalongtheperipheryofthetumoracini(whitearrow).
(C,F)CancercellsexpressingNeuN(blackarrows)werelocatedtowardthecenterofthetumoracini.
(G-J)ByFACS,GFP(+)cellswereisolatedfromH1299Oct4/GFPcellsandsinglecellsgrownonglasscoverslipsfor2weeks.
ImmunoflourscentimagingwasperformedoncoloniesforexpressionofGFP,Nestin,orNeuN.
Shownarerepresentativeimagesofsinglecolonies.
(G)ImmunflourescentimageforGFPshowingonlyaportionofcellsinthecolonyexpressGFP.
Arrowsshowtheborderofthecolony.
(H)DualimmunoflourescentforNeuN(redwithwhitearrows)andGFP(green).
(I)Immunoflourescentimagingfornestin(red)and(J)itscorrespondingphasecontrastimage.
Whitearrowsshowcellsexpressingnestin.
Langenfeldetal.
MolecularCancer2013,12:129Page12of15http://www.
molecular-cancer.
com/content/12/1/129severaldifferenttumors[36,37,58-64].
WeshowthatpharmacologicalblockadeofBMPtypeIreceptorscausessignificantgrowthinhibitionoflungcancercellsexpressingOct4ornestin.
InhibitionofBMPsignalingalsocausedsignificantgrowthinhibitionandofnon-selectedcancercellsandGFP()cells,whichwerelesstumorigenic.
ThesedatasuggestthatBMPantagonistsaffectthegrowthofmorethanjusttheOct4andnestinpopulations.
Sincecancercellsexpressingstemcellmakersrepresentonlyasmallpercentageofthecancercells,therapeuticallytargetingtheothercellpopulationsislikelyneeded.
BMPreceptorantagonistsmediategrowthinhibitionoflungcancercellsbydownregulatingtheexpressionofIdproteins[32].
BMP2/4stimulatesself-renewalofem-bryonicstemcellsbyinducingtheexpressionofId1[29].
StudieshaveshownthatId1mediatesself-renewalof"cancerstemcells"andresistancetochemotherapy[12,13].
Withinhighgradegliomas,cancercellswithhighIdexpression(Id1-high)hadahighself-renewalcapacity[12].
CancercellswithlowexpressionofId1(Id1low)werehighlyproliferativewithlittleabilitytoself-renewal[12].
InhibitionofId1inId1-highcellsde-creasedself-renewalcapacityandinId1-lowcellsitde-creasedproliferation,suggestingthatIdproteinshavemorethanonebiologicalfunction.
SilencingofId1andId3togetherdecreasedself-renewalandincreasedsensi-tivitytochemotherapeuticsofcoloncancer-initiatingcells[14].
WeshowthatDMH2,asmallmoleculeantag-onistoftheBMPtypeIreceptors,effectivelydecreasesId1andId3expressioninlungcellsexpressingstemcellmarkers.
FuturestudiesareneededtodeterminewhetherBMPantagonistsenhancetheeffectivenessofchemotherapeuticsanddecreasesself-renewalofcancercellsexpressingstemcellmarkers.
ConclusionThesestudiesfurtherdelineatetheheterogeneityoflungcarcinomas.
Ourstudiessuggestthatcancercellsex-pressingthestemcellmarkersOct4andnestinrepresentuniquecellpopulations.
WeshowthatpharmacologicalblockadeoftheBMP/IdsignalingcascadewithsmallmoleculestargetingthetypeIBMPreceptorscausessig-nificantgrowthinhibitionofnon-selectedandcancercellsexpressingstemcellmarkers.
ThesestudiesprovidefurtherevidencethatBMPreceptorantagonistsrepre-sentnoveldrugsforthetreatmentofcancer.
AdditionalfilesAdditionalfile1:FigureS1.
QuantitativeRT-PCRshowingsiRNAdecreasesOct4expressioninH1299cells(n=3).
Additionalfile2:TableS1.
Thepercentagecancercellswithinprimarylungcarcinomasexpressingnestin,NeuN,orTTF-1byimmunohistochemistry.
Additionalfile3:TableS2.
Tumorformationfollowinginjectionofinjectionof10,000,100,and10cellsfromGFP(+)cellsisolatedfromOct4/GFPandVector/GFPcells.
Additionalfile4:TableS3.
ByFACS,theGFP(+)andGFP()cellsweresortedfromH1299Oct4/GFPcellsandplatedassinglecellsontoglasscoverslips.
After2weeks,colonieswerestainedfortheexpressionofnestinandthenumberofpositivecoloniescountedusingimmunoflourescentimaging.
Additionalfile5:TableS4.
ByFACS,theGFP(+)andGFP()cellsweresortedfromH1299Oct4/GFPcellsandplatedassinglecellsontoglasscoverslips.
After2weeks,colonieswerestainedfortheexpressionofNeuNandthenumberofpositivecoloniescountedusingimmunoflourescentimaging.
CompetinginterestsApatentapplicationwassubmittedfortheuseofBMPantagonistsforthetreatmentofcancer.
Therehavenotbeenanyroyaltiespaidoranticipatedinthenearfutureregardingthiswork.
Authors'contributionsELcarriedoutthemolecularbiologystudiesandassistedinterpretationofthedata.
MDcarriedoutandanalyzedimmunohistochemistrystudies.
EZperformedandinterpretedmicroarraystudies.
JLplannedexperimentaldesign,interpretedalldata,anddraftedmanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgmentsThisstudywassupportedbyagrantfromtheOfficeofPatentsandLicensing,Rutgers-RobertWoodJohnsonMedicalSchool.
WewouldliketothankCharlesHongfromVanderbiltUniversityforhissupporttothisprojectandforprovidingBMPantagonists.
Authordetails1DepartmentofSurgery,Rutgers-RobertWoodJohnsonMedicalSchool,OneRobertWoodJohnsonPlace,P.
O.
Box19,NewBrunswick,NJ08903-0019,USA.
2DepartmentofPathology,Rutgers-RobertWoodJohnsonMedicalSchool,OneRobertWoodJohnsonPlace,P.
O.
Box19,NewBrunswick,NJ08903-0019,USA.
3DivisionofThoracicSurgery,Rutgers-RobertWoodJohnsonMedicalSchool,OneRobertWoodJohnsonPlace,P.
O.
Box19,NewBrunswick,NJ08903-0019,USA.
4Rutgers-CancerInstituteofNewJersey,NewBrunswick,NJ08903-0019,USA.
Received:26July2013Accepted:17October2013Published:26October2013References1.
WangD,ZhuH,ZhuY,LiuY,ShenH,YinR,ZhangZ,SuZ:CD133(+)/CD44(+)/Oct4(+)/Nestin(+)stem-likecellsisolatedfromPanc-1celllinemaycontributetomulti-resistanceandmetastasisofpancreaticcancer.
ActaHistochem2013,115:349–356.
310.
1016/j.
acthis.
2012.
1009.
1007.
Epub2012Oct1011.
2.
WenK,FuZ,WuX,FengJ,ChenW,QianJ:Oct-4isrequiredforanantiapoptoticbehaviorofchemoresistantcolorectalcancercellsenrichedforcancerstemcells:effectsassociatedwithSTAT3/Survivin.
CancerLett2013,333:56–65.
10.
1016/j.
canlet.
2013.
1001.
1009.
Epub2013Jan1020.
3.
BourguignonLY,WongG,EarleC,ChenL:Hyaluronan-CD44v3interactionwithOct4-Sox2-NanogpromotesmiR-302expressionleadingtoself-renewal,clonalformation,andcisplatinresistanceincancerstemcellsfromheadandnecksquamouscellcarcinoma.
JBiolChem2012,287:32800–32824.
Epub32012Jul32830.
4.
QinQ,SunY,FeiM,ZhangJ,JiaY,GuM,XiaR,ChenS,DengA:ExpressionofputativestemmarkernestinandCD133inadvancedserousovariancancer.
Neoplasma2012,59:310–315.
310.
4149/neo_2012_4040.
Langenfeldetal.
MolecularCancer2013,12:129Page13of15http://www.
molecular-cancer.
com/content/12/1/1295.
ChenJ,LiY,YuTS,McKayRM,BurnsDK,KernieSG,ParadaLF:Arestrictedcellpopulationpropagatesglioblastomagrowthafterchemotherapy.
Nat2012,488:522–526.
510.
1038/nature11287.
6.
VisvaderJE,LindemanGJ:Cancerstemcellsinsolidtumours:accumulatingevidenceandunresolvedquestions.
NatRevCancer2008,8:755–768.
7.
Al-HajjM,WichaMS,Benito-HernandezA,MorrisonSJ,ClarkeMF:Prospectiveidentificationoftumorigenicbreastcancercells.
ProcNatlAcadSciUSA2003,100:3983–3988.
Epub2003Mar3910.
8.
TooleBP,SlomianyMG:Hyaluronan,CD44andEmmprin:partnersincancercellchemoresistance.
DrugResistUpdat2008,11:110–121.
110.
1016/j.
drup.
2008.
1004.
1002.
Epub2008May1019.
9.
LiuG,YuanX,ZengZ,TuniciP,NgH,AbdulkadirIR,LuL,IrvinD,BlackKL,YuJS:AnalysisofgeneexpressionandchemoresistanceofCD133+cancerstemcellsinglioblastoma.
MolCancer2006,5:67.
10.
IzumiyaM,KabashimaA,HiguchiH,IgarashiT,SakaiG,IizukaH,NakamuraS,AdachiM,HamamotoY,FunakoshiS,etal:Chemoresistanceisassociatedwithcancerstemcell-likepropertiesandepithelial-to-mesenchymaltransitioninpancreaticcancercells.
AnticancerRes2012,32:3847–3853.
11.
LeungEL,FiscusRR,TungJW,TinVP,ChengLC,SihoeAD,FinkLM,MaY,WongMP:Non-smallcelllungcancercellsexpressingCD44areenrichedforstemcell-likeproperties.
PLoSOne2010,5:e14062.
14010.
11371/journal.
pone.
0014062.
12.
BarrettLE,GranotZ,CokerC,IavaroneA,HambardzumyanD,HollandEC,NamHS,BenezraR:Self-renewaldoesnotpredicttumorgrowthpotentialinmousemodelsofhigh-gradeglioma.
CancerCell2012,21:11–24.
doi:10.
1016/j.
ccr.
2011.
1011.
1025.
13.
Ponz-SarviseM,NguewaPA,PajaresMJ,AgorretaJ,LozanoMD,RedradoM,PioR,BehrensC,WistubaII,Garcia-FrancoCE,etal:InhibitorofDifferentiation-1asaNovelPrognosticFactorinNSCLCPatientswithAdenocarcinomaHistologyandItsPotentialContributiontoTherapyResistance.
ClinCancerRes2011,17:4155–4166.
Epub2011May4153.
14.
O'BrienCA,KresoA,RyanP,HermansKG,GibsonL,WangY,TsatsanisA,GallingerS,DickJE:ID1andID3regulatetheself-renewalcapacityofhumancoloncancer-initiatingcellsthroughp21.
CancerCell2012,21:777–792.
710.
1016/j.
ccr.
2012.
1004.
1036.
15.
PrinceME,SivanandanR,KaczorowskiA,WolfGT,KaplanMJ,DalerbaP,WeissmanIL,ClarkeMF,AillesLE:Identificationofasubpopulationofcellswithcancerstemcellpropertiesinheadandnecksquamouscellcarcinoma.
ProcNatlAcadSciUSA2007,104:973–978.
Epub2007Jan2008.
16.
DalerbaP,DyllaSJ,ParkIK,LiuR,WangX,ChoRW,HoeyT,GurneyA,HuangEH,SimeoneDM,etal:Phenotypiccharacterizationofhumancolorectalcancerstemcells.
ProcNatlAcadSciUSA2007,104:10158–10163.
Epub12007Jun10154.
17.
ZhengX,ShenG,YangX,LiuW:MostC6cellsarecancerstemcells:evidencefromclonalandpopulationanalyses.
CancerRes2007,67:3691–3697.
18.
BertoliniG,RozL,PeregoP,TortoretoM,FontanellaE,GattiL,PratesiG,FabbriA,AndrianiF,TinelliS,etal:HighlytumorigeniclungcancerCD133+cellsdisplaystem-likefeaturesandaresparedbycisplatintreatment.
ProcNatlAcadSciUSA2009,106:16281–16286.
Epub12009Sep16210.
19.
SinghSK,HawkinsC,ClarkeID,SquireJA,BayaniJ,HideT,HenkelmanRM,CusimanoMD,DirksPB:Identificationofhumanbraintumourinitiatingcells.
Nat2004,432:396–401.
20.
ChenYC,HsuHS,ChenYW,TsaiTH,HowCK,WangCY,HungSC,ChangYL,TsaiML,LeeYY,etal:Oct-4expressionmaintainedcancerstem-likepropertiesinlungcancer-derivedCD133-positivecells.
PLoSONE2008,3:e2637.
21.
JanikovaM,SkardaJ,DziechciarkovaM,RadovaL,ChmelovaJ,KrejciV,SedlakovaE,ZapletalovaJ,LangovaK,KleinJ,etal:Identificationofcd133+/nestin+putativecancerstemcellsinnon-smallcelllungcancer.
BiomedPapMedFacUnivOlomoucCzechRepub2010,154:321–326.
22.
BasakSK,VeenaMS,OhS,HuangG,SrivatsanE,HuangM,SharmaS,BatraRK:Themalignantpleuraleffusionasamodeltoinvestigateintratumoralheterogeneityinlungcancer.
PLoSOne2009,4:e5884.
23.
LiuC,CaoX,ZhangY,XuH,ZhangR,WuY,LuP,JinF:Co-expressionofOct-4andNestininhumanbreastcancers.
MolBiolRep2012,39:5875–5881.
5810.
1007/s11033-11011-11398-11036.
Epub12011Dec11030.
24.
NicholsJ,ZevnikB,AnastassiadisK,NiwaH,Klewe-NebeniusD,ChambersI,ScholerH,SmithA:FormationofpluripotentstemcellsinthemammalianembryodependsonthePOUtranscriptionfactorOct4.
Cell1998,95:379–391.
25.
LendahlU,ZimmermanLB,McKayRD:CNSstemcellsexpressanewclassofintermediatefilamentprotein.
Cell1990,60:585–595.
26.
WieseC,RolletschekA,KaniaG,BlyszczukP,TarasovKV,TarasovaY,WerstoRP,BohelerKR,WobusAM:Nestinexpression–apropertyofmulti-lineageprogenitorcellsCellMolLifeSci2004,61:2510–2522.
27.
Espinosa-JeffreyA,Becker-CataniaSG,ZhaoPM,ColeR,EdmondJ,deVellisJ:SelectivespecificationofCNSstemcellsintooligodendroglialorneuronalcelllineage:cellcultureandtransplantstudies.
JNeurosciRes2002,69:810–825.
28.
OhSY,KangHJ,KimYS,KimH,LimYC:CD44-negativecellsinheadandnecksquamouscarcinomaalsohavestem-cellliketraits.
EurJCancer2013,49:272–280.
210.
1016/j.
ejca.
2012.
1006.
1004.
Epub2012Jul1016.
29.
YingQL,NicholsJ,ChambersI,SmithA:BMPinductionofIdproteinssuppressesdifferentiationandsustainsembryonicstemcellself-renewalincollaborationwithSTAT3.
Cell2003,115:281–292.
30.
HollnagelA,OehlmannV,HeymerJ,RutherU,NordheimA:Idgenesaredirecttargetsofbonemorphogeneticproteininductioninembryonicstemcells.
JBiolChem1999,274:19838–19845.
31.
KurookaH,NakahiroT,MoriK,SanoK,YokotaY:BMPsignalingisresponsibleforserum-inducedId2expression.
BiochemBiophysResCommun2012,420:281–287.
210.
1016/j.
bbrc.
2012.
1002.
1150.
Epub2012Mar1016.
32.
LangenfeldE,HongCC,LankeG,LangenfeldJ:BonemorphogeneticproteintypeIreceptorantagonistsdecreasegrowthandinducecelldeathoflungcancercelllines.
PLoSOne2013,8:e61256.
61210.
61371/journal.
pone.
0061256.
Print0062013.
33.
LangenfeldEM,KongY,LangenfeldJ:Bonemorphogeneticprotein-2-inducedtransformationinvolvestheactivationofmammaliantargetofrapamycin.
MolCancerRes2005,3:679–684.
34.
GerrardL,ZhaoD,ClarkAJ,CuiW:StablytransfectedhumanembryonicstemcellclonesexpressOCT4-specificgreenfluorescentproteinandmaintainself-renewalandpluripotency.
StemCells2005,23:124–133.
35.
SolwayJ,SeltzerJ,SamahaFF,KimS,AlgerLE,NiuQ,MorriseyEE,IpHS,ParmacekMS:Structureandexpressionofasmoothmusclecell-specificgene,SM22alpha.
JBiolChem1995,270:13460–13469.
36.
LangenfeldEM,KongY,LangenfeldJ:Bonemorphogeneticprotein2stimulationoftumorgrowthinvolvestheactivationofSmad-1/5.
Oncogene2006,25:685–692.
37.
LangenfeldEM,CalvanoSE,Abou-NuktaF,LowrySF,AmentaP,LangenfeldJ:Thematurebonemorphogeneticprotein-2isaberrantlyexpressedinnon-smallcelllungcarcinomasandstimulatestumorgrowthofA549cells.
Carcinog2003,24:1445–1454.
Epub2003Jun1419.
38.
KellnerS,KikyoN:TranscriptionalregulationoftheOct4gene,amastergeneforpluripotency.
HistolHistopathol2010,25:405–412.
39.
LiYQ:Masterstemcelltranscriptionfactorsandsignalingregulation.
CellReprogram2010,12:3–13.
40.
LeporeAC,HanSS,Tyler-PolszCJ,CaiJ,RaoMS,FischerI:Differentialfateofmultipotentandlineage-restrictedneuralprecursorsfollowingtransplantationintotheadultCNS.
NeuronGliaBiol2004,1:113–126.
41.
RyugeS,SatoY,WangGQ,MatsumotoT,JiangSX,KatonoK,InoueH,SatohY,MasudaN:Prognosticsignificanceofnestinexpressioninresectednon-smallcelllungcancer.
Chest2011,139:862–869.
Epub2010Sep2019.
42.
RedshawZ,StrainAJ:HumanhaematopoieticstemcellsexpressOct4pseudogenesandlacktheabilitytoinitiateOct4promoter-drivengeneexpression.
JNegatResultsBiomed2011,9:2.
43.
ElkabetzY,PanagiotakosG,AlShamyG,SocciND,TabarV,StuderL:HumanEScell-derivedneuralrosettesrevealafunctionallydistinctearlyneuralstemcellstage.
GenesDev2008,22:152–165.
110.
1101/gad.
1616208.
44.
BiunnoI,CattaneoM,OrlandiR,CantonC,BiagiottiL,FerreroS,BarberisM,PupaSM,ScarpaA,MenardS:SEL1Lamultifacetedproteinplayingaroleintumorprogression.
JCellPhysiol2006,208:23–38.
45.
FerreroS,FalleniM,CattaneoM,MalferrariG,CantonC,BiagiottiL,MaggioniM,NosottiM,CoggiG,BosariS,BiunnoI:SEL1Lexpressioninnon-smallcelllungcancer.
HumPathol2006,37:505–512.
Epub2006Mar2020.
46.
LeonardiR,ZhangYM,YunMK,ZhouR,ZengFY,LinW,CuiJ,ChenT,RockCO,WhiteSW,JackowskiS:Modulationofpantothenatekinase3activitybysmallmoleculesthatinteractwiththesubstrate/allostericregulatorydomain.
ChemBiol2010,17:892–902.
810.
1016/j.
chembiol.
2010.
1006.
1006.
47.
BanerjeeS,BrooksWS,CrawfordDF:InactivationoftheubiquitinconjugatingenzymeUBE2Q2causesaprophasearrestandenhancedLangenfeldetal.
MolecularCancer2013,12:129Page14of15http://www.
molecular-cancer.
com/content/12/1/129apoptosisinresponsetomicrotubuleinhibitingagents.
Oncogene2007,26:6509–6517.
Epub2007Apr6530.
48.
ZhangJ,NiuC,YeL,HuangH,HeX,TongWG,RossJ,HaugJ,JohnsonT,FengJQ,etal:Identificationofthehaematopoieticstemcellnicheandcontrolofthenichesize.
Nat2003,425:836–841.
49.
MizusekiK,SakamotoT,WatanabeK,MugurumaK,IkeyaM,NishiyamaA,ArakawaA,SuemoriH,NakatsujiN,KawasakiH,etal:Generationofneuralcrest-derivedperipheralneuronsandfloorplatecellsfrommouseandprimateembryonicstemcells.
ProcNatlAcadSciUSA2003,100:5828–5833.
Epub2003Apr5830.
50.
ZimmermanLB,DeJesus-EscobarJM,HarlandRM:TheSpemannorganizersignalnogginbindsandinactivatesbonemorphogeneticprotein4.
Cell1996,86:599–606.
51.
GuoY,LiuS,WangP,ZhaoS,WangF,BingL,ZhangY,LingEA,GaoJ,HaoA:Expressionprofileofembryonicstemcell-associatedgenesOct4,Sox2andNanoginhumangliomas.
Histopathol2011,59:763–775.
710.
1111/j.
1365-2559.
2011.
03993.
x.
52.
MatsudaY,NaitoZ,KawaharaK,NakazawaN,KorcM,IshiwataT:Nestinisanoveltargetforsuppressingpancreaticcancercellmigration,invasionandmetastasis.
Cancer2011,11:512–523.
Epub2011.
53.
ParkD,XiangAP,MaoFF,ZhangL,DiCG,LiuXM,ShaoY,MaBF,LeeJH,HaKS,etal:Nestinisrequiredfortheproperself-renewalofneuralstemcells.
StemCells2010,28:2162–2171.
54.
SountoulidisA,StavropoulosA,GiaglisS,ApostolouE,MonteiroR,deSousaLopesSMC,ChenH,StrippBR,MummeryC,AndreakosE,SiderasP:ActivationoftheCanonicalBoneMorphogeneticProtein(BMP)PathwayduringLungMorphogenesisandAdultLungTissueRepair.
PLoSOne2012,7:e41460.
Epub42012Aug41420.
55.
LangenfeldEM,BojnowskiJ,PeroneJ,LangenfeldJ:Expressionofbonemorphogeneticproteinsinhumanlungcarcinomas.
AnnThoracSurg2005,80:1028–1032.
56.
LePageC,PuiffeML,MeunierL,ZietarskaM,deLadurantayeM,ToninPN,ProvencherD,Mes-MassonAM:BMP-2signalinginovariancanceranditsassociationwithpoorprognosis.
JOvarianRes2009,2:4.
57.
ParkY,KangMH,SeoHY,ParkJM,ChoiCW,KimYH,KimIS,KimJS,OhSC:Bonemorphogeneticprotein-2levelsareelevatedinthepatientswithgastriccancerandcorrelatewithdiseaseprogression.
MedOncol2010,27:1192–1199.
58.
FongYC,LiTM,WuCM,HsuSF,KaoST,ChenRJ,LinCC,LiuSC,WuCL,TangCH:BMP-2increasesmigrationofhumanchondrosarcomacellsviaPI3K/Aktpathway.
JCellPhysiol2008,217:846–855.
59.
RothhammerT,BatailleF,SprussT,EissnerG,BosserhoffAK:FunctionalimplicationofBMP4expressiononangiogenesisinmalignantmelanoma.
Oncogene2007,26:4158–4170.
Epub2006Dec4118.
60.
QiuH,YangB,PeiZC,ZhangZ,DingK:WSS25inhibitsgrowthofxenograftedhepatocellularcancercellsinnudemicebydisruptingangiogenesisviablockingbonemorphogeneticprotein(BMP)/Smad/Id1signaling.
JBiolChem2010,285:32638–32646.
Epub32010Aug32632.
61.
LaiTH,FongYC,FuWM,YangRS,TangCH:Osteoblasts-derivedBMP-2enhancesthemotilityofprostatecancercellsviaactivationofintegrins.
Prostate2008,68:1341–1353.
62.
ClementJH,RaidaM,SangerJ,BicknellR,LiuJ,NaumannA,GeyerA,WaldauA,HortschanskyP,SchmidtA,etal:Bonemorphogeneticprotein2(BMP-2)inducesinvitroinvasionandinvivohormoneindependentgrowthofbreastcarcinomacells.
IntJOncol2005,27:401–407.
63.
RaidaM,ClementJH,AmeriK,HanC,LeekRD,HarrisAL:Expressionofbonemorphogeneticprotein2inbreastcancercellsinhibitshypoxiccelldeath.
IntJOncol2005,26:1465–1470.
64.
RaidaM,ClementJH,LeekRD,AmeriK,BicknellR,NiederwieserD,HarrisAL:Bonemorphogeneticprotein2(BMP-2)andinductionoftumorangiogenesis.
JCancerResClinOncol2005,131:741–750.
Epub2005Nov2001.
doi:10.
1186/1476-4598-12-129Citethisarticleas:Langenfeldetal.
:SmallmoleculeantagonistofthebonemorphogeneticproteintypeIreceptorssuppressesgrowthandexpressionofId1andId3inlungcancercellsexpressingOct4ornestin.
MolecularCancer201312:129.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
biomedcentral.
com/submitLangenfeldetal.
MolecularCancer2013,12:129Page15of15http://www.
molecular-cancer.
com/content/12/1/129