Chronic Cadmium Exposure Stimulates SDF- Expression in an ERα Dependent Manner.doc

1、 ChronicCadmiumExposureStimulatesSDF-1ExpressioninanERaDependentMannerEsmeraldaPonce,NatalieB.Aquino,MaggieC.Louie*DepartmentofNaturalSciencesandMathematics,DominicanUniversityofCalifornia,SanRafael,California,UnitedStatesofAmericaAbstractCadmiumisanomnipotentenvironmentalcontaminantassociatedwithth

2、edevelopmentofbreastcancer.Studiessuggestthatcadmiumfunctionsasanendocrinedisruptor,mimickingtheactionsofestrogeninbreastcancercellsandactivatingthereceptortopromotecellgrowth.Althoughacutecadmiumexposureisknowntopromoteestrogenreceptor-mediatedgeneexpressionassociatedwithgrowth,theconsequenceofchro

3、niccadmiumexposureisunclear.Sinceheavymetalsareknown to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims toinvestigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell linechronicallyexposedto1027MC

4、dCl2servesasourmodelsystem.Datasuggestthatprolongedcadmiumexposuresresultinthedevelopmentofmoreaggressivecancerphenotypesincreasedcellgrowth,migrationandinvasion.Theresultsfromthis study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering themolecular

5、 interactions between ERa, c-jun and c-fos. This study provides a mechanistic link between chronic cadmiumexposureandERaanddemonstratesthatprolonged,low-levelcadmiumexposurecontributestobreastcancerprogression.Citation:PonceE,AquinoNB,LouieMC(2013)ChronicCadmiumExposureStimulatesSDF-1ExpressioninanE

6、RaDependentManner.PLoSONE8(8):e72639.doi:10.1371/journal.pone.0072639Editor:MasaruKatoh,NationalCancerCenter,JapanReceivedApril19,2013;AcceptedJuly11,2013;PublishedAugust28,2013Copyright: 2013 Ponce etal. This is an open-access article distributed under the terms of the Creative Commons Attribution

7、License, which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.Funding:ThisworkwassupportedinpartbygrantsfromtheNationalScienceFoundation(1039728toMCL)andtheNationalCancerInstitute(CA121983-02toMCLandCA121983-02S1toEP).Thefundershadnorolei

8、nstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:Introductionneoplastic growth in the mammary gland 2,4. Animal studieshavealsoshownthatcadmiumexposurecanincre

9、asethedensityofthemammaryglandsaswellasinducechangesintheliningofthe uterus, all of which are early signs of hormone-relatedtumorigenesis4,6,7.Breastcancerisoneofthemostcommonmalignanciesafflictingwomen in the United States, and it has the second highestmortalityrateassociatedwithanycancer.Themajori

10、tyofbreastcancers initially develop as hormone-dependent, with estrogenreceptorsexpressedinapproximately70%ofbreastcancercases.The presence or absence of ERa is a key determinant of theprognosis of the disease, in addition to determining whether thecancer will respond to hormone therapy or not. ERa-

11、positivebreast cancers are often hormone-responsive and are typicallytreated with antiestrogens like tamoxifen. However, hormone-dependent breast cancer frequently progresses into more malig-nant cancer phenotypes that are often hormone-independent. Inmanycases,theestrogenreceptor(ERa)isstillpresent

12、,buttheroleof ERa in hormone refractory breast tumorigenesis and itsunderlying mechanism are unclear. A potential mechanisminvolvesmetalloestrogensheavymetalsthatfunctionasendocrinedisruptorsandmimictheactionsofestrogen.Cadmium has also been shown to promote ER-dependentbreastcancercellproliferation

13、,potentiallythroughtheactivationof the ER signaling pathway 3,5,8,9. Results from our lab andothers have suggested that cells treated with cadmium expresshigher levels of ER target genes, including cyclin D1, c-myc,progesteronereceptor,andcathepsin-D3,5,8.Althoughcadmi-umisknowntobindtoERa,themechan

14、ismbehindER-mediatedgene expression and subsequent breast cancer cell growth is notcompletely established. Results from our previous study showedthat cadmium promotes the nuclear localization of ERa andenhancesthebindingofERatotargetgenepromoters8.Itwasalso demonstrated that cadmium potentiates the

15、interactionbetween ERa and c-jun, a member of the AP-1 family oftranscriptionfactors.Among the metalloestrogens, cadmium is the best character-ized. Cadmium is derived from various industrial sources,including present and former mining activities, production ofalloys,batteries,fertilizers,pigments,a

16、ndcombustionby-products.Human exposure results from the consumption of contaminatedwaterandfoodorinhalationofcigarettesmokeandcontaminatedfumes. Cadmium exposure has long been associated with thedevelopment of breast cancer 15, but the mechanism ofcadmiumsactiononbreastcancerremainselusive.Recentstu

17、dieshave suggested that cadmium may function as an endocrinedisruptor to perturb the normal hormonal cycle and promoteAlthoughthereisabetterunderstandingofhowacutecadmiumexposure activates ERa and mediates the expression of genesassociated with cell growth, it is unclear how chronic exposure ofcadmi

18、ummayaffectbreastcancerdevelopmentand/orER-targetgeneexpression.Previousstudiesonprostatecancerandsarcomashave suggested that chronic exposure to cadmium is associatedwith the development of more malignant tumors 1012.Furthermore,cadmiumhasbeendetectedinbreasttumortissues1,1316. While healthy indivi

19、duals had measurable cadmiumlevels in their mammary glands, significantly higher levels ofPLOSONE | 1August2013 | Volume 8 | Issue 8 | e72639 ChronicCadmiumExposurePromotesSDF-1Expressioncadmium were found in patients with breast cancer 1,1316.Theeffectsofcadmiuminthesetumorsandwhethe

20、ritspresenceresults in further progression of the disease are unknown,underscoringtheneedforchronicexposurestudies.MigrationandInvasionAssayA modified Boyden chamber assay was used to quantitativelyassessthecellsmigrationandinvasionabilities.Forthemigrationassay, cells were hormone-deprived, and 5.0

21、6104 cells wereThis study aims to investigate the chronic effects of cadmiumexposure onbreast cancerprogression. We developed a series ofcadmium exposed-breast cancer cell lines to study the effects ofchronic cadmium exposure on breast cancer progression. Thisstudyprovidesseverallinesofevidencesugge

22、stingthatprolongedcadmiumexposureresultsinmoreaggressivecancerphenotypesincreased cell growth, migration and invasion. Our results alsoshowforthefirsttimethatcellschronicallyexposedtocadmiumexpress higher levels of SDF-1 in an ERa-dependent manner.Furthermore, data from this study also suggest that

23、prolongedcadmium exposure alters the molecular dynamics between ERaand c-jun/c-fos to mediate direct changes in the expression ofcancer-promotinggeneslikeSDF-1.seeded in the upper chamber over polycarbonate membraneinserts and allowed to migrate toward the lower chamber filledwith DMEM+10% FBS+1% P/

24、S. The cells were incubated for1218hours,fixedinformalin,andstainedwithcrystalviolet.Thenumber of cells that migrated to the underside of the filter wascountedintriplicate.Fortheinvasionassay,aCytoSelectTMCellInvasion Assay Kit (Cell Biolabs, Inc, San Diego, CA) was usedaccording to the manufacturer

25、s protocol. Briefly, 5.06104hormone-deprived cells were seeded in the upper chamber andthecellsthatinvadedthroughthematrigelwerefixed,stainedandcountedaspreviouslydescribed.WesternBlotCellswerelysedin1%SDS-HEPESbufferandthetotalproteinconcentrationwasnormalizedusingtheBio-RadDcProteinAssaykit(Bio-Ra

26、d,Hercules,CA).ProteinswereseparatedusingSDS-polyacrylamide gel electrophoresis and transferred to polyvinyli-dene fluoride (PVDF) membranes (Millipore, Billerica, MA).Protein expression was monitored using protein-specific antibod-ies:a-ERa(NeoMarkers,Fremont,CA),a-cyclinD1(SantaCruzBiotechnology,

27、Santa Cruz, CA), a-c-myc (Santa Cruz Biotech-nology),a-cyclinA(SantaCruzBiotechnology),a-cyclinE(SantaCruzBiotechnology),a-cdk2(SantaCruzBiotechnology),a-cdk4(BDTransductionLaboratories,SanJose,CA),a-p21(SantaCruzMaterialsandMethodsCellCultureMCF7 cells were obtained from the American Type CultureCo

28、llection(ATCC,Manassas,VA).MCF7-Cdwasdevelopedbyexposing parental MCF7 cells to 1027 M CdCl2 for over 6months. Parental MCF7 and MCF7-Cd were maintained inDulbeccosModifiedEagleMedium(DMEM)supplementedwith10% Fetal Bovine Serum (FBS; Hyclone, Logan, UT) and 1%penicillinandstreptomycin(P/S).Bothcelll

29、inesweresubculturedevery34days.Biotechnology), a-p-27 (Santa Cruz Biotechnology), a-CDH1(SantaCruzBiotechnology),a-b-catenin(CellSignalingTechnol-ogy,Danvers,MA),a-GAPDH(CellSignalingTechnology).GrowthAssay1.56105 MCF7 and MCF7-Cd (MCF7-Cd4, Cd7, and Cd12)cellswereplatedinsix-wellplatesusinghormoned

30、epletedmedia.Cellswerecountedintriplicateevery2daysusingacellcounter(Nexcelom Bioscience LLC, Lawerence, MA). Data pointsrepresentthreeindependentexperiments.SDF-1ELISACellswereplatedatadensityof16105cellsperwellin6-wellplates.Cellculturemediawascollectedafter48hoursandstromalcell-derived factor-1 (

31、SDF-1) levels were measured with enzyme-linkedimmunosorbentassays(ELISA)SDF-1kit(RayBiotech,Inc,Norcross, GA) according to the manufacturers instructions.Briefly,100mLofsamplewasaddedto96-wellplatescontainingimmobilizedantibodiesspecificforhumanSDF-1andallowedtoincubateovernightwithshakingat4uC.Well

32、swerethenwashedand a biotinylated anti-human SDF-1 antibody was added,followed by another wash and subsequently by the addition ofHRP-conjugatedstreptavidin.Followingafinalwash,tetramethyl-benzidine(TMB)substrateforcolordevelopmentwasaddedandsampleswerereadat450nmaftertheadditionofastopsolution.Deri

33、vationofCadmiumCellLinesTo derive clonal cell lines chronically exposed to cadmium,MCF7cellswerefirstexposedto1027MCdCl2forover6monthsin order to mimic chronic exposures. Chronically exposed cellswere plated on soft agar to allow single cells to develop intocolonies. Briefly, a 1% agar solution made

34、 with complete media(DMEM+10%FBS+1%P/S)wasusedtocreatethebottomlayer.Serialdilutionsofsinglecellsuspensionsweremixedwithenoughcompletemediatomakea0.6%topagarthatwasaddedoverthe1% bottom agar layer and allowed to solidify for 30 minutes atroomtemperaturefollowedbyincubationat37uC.Whencoloniessizes re

35、ached about 100 cells or more, they were individuallyremovedfromsoftagarunderasepticconditionsandresuspendedincompletemediatopermitfurtherexpansion.siRNATransfection16105 cells were plated in 6 well plates and transfected withsiRNA(SantaCruzBiotechnology)targetingeitherERa,c-junorc-fos using siRNA t

36、ransfection reagents (Santa Cruz Biotechnol-ogy).AscrambledsiRNAwastransfectedasacontrol.Fivehoursafter transfection, the medium was changed to DMEM mediumcontaining10%FBSand1%P/S.Cellswereharvested48hourslater for gene and/or protein expression analysis using semi-quantitativereversetranscription-p

37、olymerasechainreactions(RT-PCR) or quantitative PCR (qPCR), and Western blot analysis,respectively.ScratchWoundAssayApproximately 2.06104 cells were plated in 6-well plates andallowed to grow to approximately 7580% of confluency inDMEM. A wound or scratch was created with a 200mLmicropipette tip and

38、 washed twice with 1X PBS. Cells wereallowed to migrate for 3 and cells were visualized under 5Xobjective with a 10X ocular magnification (with a final magnifi-cation of50X). At least three frames ofeach well werecaptureddigitally atday 0and day 3. Images presented in the figures arerepresentative o

39、f three independent experiments done in tripli-cates.ReverseTranscriptasePolymeraseChainReaction(RT-PCR)Total RNA was isolated from cells using TRI-Reagent (ZymoResearchCorporation,Irvine,CA)andcolumnsfromtheDirect-PLOSONE | 2August2013 | Volume 8 | Issue 8 | e72639 ChronicCadmiumExpo

40、surePromotesSDF-1Expressionzol RNA MiniPrep kit (Zymo Research) according to themanufacturers protocol. Three micrograms (mg) of total RNAwere used for the reverse transcription reaction with oligo-dT18primersandMoloneyMurineLukemiaReverseTranscriptase(M-MLV RT, Promega, Madison,WI). Gene expression

41、 was moni-tored using semi-quantitative PCR for 28 cycles. PCR productswereseparatedbyagarosegelelectrophoresis,andvisualizedusingChemi-Doc (Biorad). Gene expression was quantified using genespecificprimerswithquantitativereal-timePCRwithSYBRgreen(SA Biosciences, Valencia, CA). All primers were synt

42、hesized byIntegrated DNA Technologies, Inc. (IDT; San Diego, CA). RT-PCRPrimerSequences:U6RNA-ChIPF :GAGGGCCTATTTCCCATGATTCU6RNA-ChIPR:GAATTTGCGTGTCATCCTTGCLuciferaseReporterAssayThe reporter gene assay was performed by sequentiallytransfecting MCF7 cells with siRNA targeting ERa (Santa CruzBiotechn

43、ology) followed by the SDF1 promoter-Luc reporterplasmid. Five hours after siRNA transfection, the secondtransfectionwiththeSDF-1LucreporterplasmidalongwiththepRL-SV40Renillaluciferaseplasmid(Promega)wasaccomplishedusing Fugene HD (Promega) according to the manufacturersprotocol.Themediumwaschangedt

44、oDMEMmediacontaining10% FBS and penicillin and streptomycin, and cells wereharvested 48 hours post-transfection and analyzed using a dualluciferase assay kit (Promega). All reporter gene assays wereperformed inquadruplicate, withtheentire experiment repeatedatleastthreetimes.SDF-1-Lucwaspurchasedfro

45、mGenecopoeia(Rockville,MD).SDF-1FCACTTTAGCTTCGGGTCAATG:GTCAGCCTGAGCTACAGATGCSDF-1R:ERaF:ATGACCCTCCACACCAAAGCATACTGGCCAATCTTTCTCTGCCAERaR:c-mycF:CTCCACACATCAGCACAACTGTTTCCGCAACAAGTCCTCTc-mycR:cycD1F: AATGTGTGCAGAAGGAGGTCGAGGGCGGATTGGAAATGAAcycD1R:ResultsGAPDHF: GAAATCCCATCACCATCTTCCAG GAPDHR:ATGAGTCC

46、TTCCACGATACCAAAGWe previously found that acute cadmium exposures increasegrowthratesofthreeERa-positivebreastcancercelllinesMCF7,T47D and ZR75-1 and increase the expression of genesassociatedwithgrowth8.Theobservationthatacutecadmiumexposure stimulates breast cancer cell proliferation led us toquest

47、iontheeffectsofchroniccadmiumexposureonbreastcancerprogression. To understand how chronic cadmium exposureaffects the progression of ER-positive breast cancer, a cell linechronically exposed to cadmium was developed by exposingMCF7cellstolowconcentrationsofcadmium(1027M)foroversixmonths.Thegrowthpro

48、pertiesofcellschronicallyexposedtocadmium(MCF7-Cd)wereanalyzedinthepresenceorabsenceofcadmium and compared to parental MCF7 cells. Approximately1.56104cellswereplatedin6-wellplatesunderhormone-deprivedCoimmunoprecipitation(CoIP)AssayCellswereplatedin10cmtissuecultureplatesandharvested48hours later.

49、Cells were lysed in 0.5% NP-40 lysis buffer andsonicated three times for 30 seconds. Cell lysates were immuno-precipitated with a-ERa, a-c-fos, a-c-jun or normal rabbit IgG(Santa Cruz Biotechnology) for 2 hours, followed by incubationwith either protein A or G agarose beads for 1 hour. Proteincomple

50、xeswereseparatedwithSDS-PAGEfollowedbyWesternblot analysis with ERa, c-jun, c-fos, and normal rabbit IgGantibodies.ChromatinImmunoprecipitation(ChIP)Assays27Cellswereplatedin10cmplatesandharvestedbyfixationwithformaldehyde. Cells were lysed and sonicated in lysis buffer tofragment the chromatin into

51、 12kb fragments. Chromatinmixtures were immunoprecipitated with antibodies specific forERa, c-fos, and c-jun. Antibody complexes were purified usingprotein-A agarose beads (Pierce, Rockford, IL). After DNA-proteincomplexeswerereversedcross-linked(65uCfor46hours),DNAfragmentswerepurifiedusingQIAQuick

52、columns(Qiagen,Germantown, MD). Occupancy at specific promoters weredetermined with PCR using promoter specific primers. For there-ChIP assay, the protein A-antibody complexes from the firstChIP assay were extracted with 200ml of 10mM DTT for 30minutesat37uC.Afterthesupernatantwasdiluted10Xwithre-Ch

53、IPbuffer(20mMTris-HCl,pH8.1,2mMEDTA,150mMNaCl, and 0.1% Triton X-100), the chromatin mixture was re-ChIPedwithasecondantibody:a-ER,a-c-jun,a-c-fosornormalrabbit IgG, (Santa Cruz Biotechnology). This step was repeatedonemoretimeforthetriple-ChIPassayandthere-ChIPedDNA-proteincomplexeswereimmunoprecip

54、itatedwithathirdantibody(c-fos or rabbit IgG). Following, the purification of the finalantibodycomplexeswithproteinAbeads,theDNAwasreverse-cross-linkedfromtheproteinandpurifiedasdescribedabove.ChIPPrimerSequences:conditionsandtreatedwith10 Mcadmiumchloride(CdCl2 )ormock treated with phosphate buffer

55、ed saline (PBS). Cell growthwas monitored 2, 4 and 6 days after treatment. The MCF7-CdcellsdisplayedasignificantlyfastergrowthratethantheparentalMCF7cellsinthepresenceandabsenceofcadmium(Fig.1A).Metastatic phenotypes including the ability to migrate andinvade through extracellular matrix were also e

56、valuated. Theability of cells chronically exposed to cadmium to migrate wasanalyzed and compared to parental MCF7 cells using a scratch-wound assay (Fig. 1B). Cells were plated in a 6-well plate andallowedtoreach7080%confluence;ascratchwascreatedusingap200pipettetip.CellswererinsedwithPBSandmigratio

57、nwasobserved3dayslater.Resultsinfigure1BsuggestthatMCF7-CdcellsdisplayagreaterabilitytomigrateincomparisontoparentalMCF7cells.Toconfirmthequalitativeobservationsinfigure1B,a modified Boyden Chamber assay was used to quantify thedifference in cell migration between MCF7 and MCF7-Cd cells(Fig. 1C). In this assay, 56104 cells (MCF7 or MCF7-Cd) wereplatedintheupperchamberandallowedtomigratefor16hoursthrough an 8mM polycarbonate membrane. Cells that have theabili