功能上調(diào)控乳腺癌的轉(zhuǎn)移

1、論文中英文摘要作者姓名：王艷論文題目：LSD1是NuRD復(fù)合體的一個亞基，功能上調(diào)控乳腺癌的轉(zhuǎn)移作者簡介：王艷，女，1982年11月出生，2001年9月進入北京大學(xué)醫(yī)學(xué)部八年制基礎(chǔ)醫(yī)學(xué)專業(yè)學(xué)習(xí)，2006年9月師從于北京大學(xué)尚永豐教授，于2009年7月獲博士學(xué)位。中文摘要LSD1是第一個被發(fā)現(xiàn)的組蛋白去甲基化酶，屬于以FAD為輔酶的單胺氧化酶，能夠催化H3K4me2和H3K4me1多肽去甲基化反應(yīng)。LSD1廣泛調(diào)控基因的轉(zhuǎn)錄，并與多種月中瘤的發(fā)生發(fā)展高度相關(guān)。LSD1與癌癥之間的潛在聯(lián)系目前被理解為多種月中瘤中組蛋白H3K4的甲基化水平下降和H3K9的水平上升的現(xiàn)象相關(guān)。雖然有少量報導(dǎo)說LSD1

2、可以激活基因的轉(zhuǎn)錄，但是目前更普遍認(rèn)為LSD1對基因轉(zhuǎn)錄起到抑制作用。由于H3K4me2是公認(rèn)的轉(zhuǎn)錄激活標(biāo)志，LSD1催化其去甲基化的活性便抑制了基因的轉(zhuǎn)錄。迄今，LSD1在眾多轉(zhuǎn)錄抑制復(fù)合體中被發(fā)現(xiàn)，如CoREST復(fù)合體，CtBP復(fù)合體和一系列HDAC復(fù)合體等等。在表觀遺傳治療的大好前景下，LSD1越來越顯示出了它作為一個潛在治療靶點的優(yōu)越性。運用anti-FLAG親和層析聯(lián)合質(zhì)譜的方法，我們首次報導(dǎo)組蛋白去甲基化酶LSD1可以與組蛋白去乙?；瘡?fù)合體NuRD相互作用。NuRD復(fù)合體是一個多亞基的復(fù)合體，包括ATP酶部分和組蛋白去乙?；℉DAC）酶部分，廣泛參與基因轉(zhuǎn)錄抑制。高效液相色譜實驗

3、顯示，HeLa細胞中天然存在的LSD1多出現(xiàn)在高出其單體110kDa很多的669-1000kDa的組分中，且與NuRD復(fù)合體的常見組分MTA,HDAC等大致共同洗脫。更重要的是，NuRD復(fù)合體的洗脫譜中的組蛋白去乙?；疕DAC活性范圍與LSD1的組蛋白去甲基化HDM活性范圍大致重合。利用免疫共沉淀實驗的方法，我們在HeLa,MCF-7和MDA-MB-231細胞系中均證明LSD1可以與NuRD復(fù)合體所有的組分在體內(nèi)相互作用，說明LSD1是NuRD復(fù)合體的一個亞基。HDAC酶活性和HDM酶活性檢測結(jié)果顯示，NuRD復(fù)合體組分中擁有使小牛胸腺組蛋白或HeLa細胞單核小體的H3K4me2和H3K4me

4、1顯著下降的去組蛋白去甲基化酶活性，且此活性可被LSD1的特異性抑制劑Pargeline抑制，也可被LSD1免疫消除實驗所清除，說明LSD1是NuRD復(fù)合體的一個功能亞基，通過其H3K4去甲基化酶活性協(xié)同NuRD復(fù)合體抑制基因的轉(zhuǎn)錄。GSTPull-down實驗結(jié)果證明LSD1可以在體外直接與NuRD復(fù)合體亞基之一的MTA蛋白特異的相互作用，卻不與其他的NuRD復(fù)合體組分直接相互作用。而進一步分段截短體的GSTPull-down實驗結(jié)果證明LSD1的Tower結(jié)構(gòu)域是和MTA分子的SANT結(jié)構(gòu)域是介導(dǎo)LSD1與三個MTA分子直接相互作用的結(jié)構(gòu)域。而利用重組蛋白進行的體外去甲基化實驗則揭示，當(dāng)純

5、化的LSD1單獨存在時，雖可以催化組蛋白進行去甲基化反應(yīng)，卻無法完成對單核小體H3K4的去甲基化；而加入體外純化的重組MTA2之后，LSD1便可以單核小體為底物使之去甲基化了，說明NuRD復(fù)合體中的MTA分子可以作為聯(lián)系LSD1與染色質(zhì)高級結(jié)構(gòu)的橋梁。利用先進的染色質(zhì)免疫共沉淀-DNA選擇和連接（ChIP-DSL）技術(shù)我們得到了LSD1/NuRD復(fù)合體全基因組轉(zhuǎn)錄調(diào)控的可能的下游靶基因。這些靶基因多分布于TGFP通路，細胞連接，細胞粘附，MAPK信號轉(zhuǎn)導(dǎo)和細胞周期等通路，這些通路在細胞生長，生存，遷移，侵襲和轉(zhuǎn)移中起到非常重要的作用。其中，TGFB1,EGFR,RHOA等都是上皮-間質(zhì)細胞轉(zhuǎn)化

6、（EMT）和月中瘤轉(zhuǎn)移密切相關(guān)的基因,提示LSD1/NuRD復(fù)合體與月中瘤的轉(zhuǎn)移密切相關(guān)。在利用實時定量PCR驗證了芯片的結(jié)果之后，我們用傳統(tǒng)的ChIP方法證實LSD1,MTA3和Mi-2者B在MCF-7細胞中與TGFB1的啟動子相結(jié)合。而之后的連續(xù)ChIP實驗亦證明，LSD1/MTA3/Mi-2存在于同一個蛋白復(fù)合體中并都結(jié)合在TGFB1的啟動子上。這些實驗的結(jié)果不但證明TGFB1是LSD1/MTA3/NuRD復(fù)合體的下游靶基因，且也同樣進一步印證了LSD1是NuRD復(fù)合體的一個內(nèi)在亞基。TGFP1被認(rèn)為是上皮-間質(zhì)細胞轉(zhuǎn)化（EMT）的關(guān)鍵調(diào)控分子之一。鑒于TGFP1在乳腺癌細胞的EMT以及

7、侵襲和轉(zhuǎn)移等過程中均發(fā)揮重要的促進作用，其作為LSD1/NuRD復(fù)合體的靶基因出現(xiàn)提示LSD1很可能參與調(diào)控乳腺癌的侵襲和轉(zhuǎn)移。利用轉(zhuǎn)移小室實驗我們發(fā)現(xiàn)正常LSD1過表達組與對照相比，孚L腺癌MDA-MB-231細胞的侵襲潛力下降了3倍左右；而缺失與NuRD復(fù)合體MTA分子直接相互作用的Tower結(jié)構(gòu)域的LSD1過表達組與對照組相比，則沒有什么明顯的變化。另一方面，LSD1沉默組與其對照組相比MDA-MB-231細胞的侵襲力增加了約5倍左右。而且，LSD1過表達而Mi-2沉默組與前述單純過表達LSD1組的結(jié)果相比，MDA-MB-231細胞侵襲力下降的效果被明顯減弱了，說明MDA-MB-231細

8、胞侵襲力的變化主要是通過LSD1與NuRD復(fù)合體結(jié)合后來實現(xiàn)的。通過加入外源TGFP1的挽救”實驗證明，LSD1沉默之后引起的231細胞侵襲力增強的作用可被TGFP1I型受體的ATP酶拮抗劑SB431542所挽救"，說明TGFP1信號通路在LSD1介導(dǎo)的抑制乳腺癌MDA-MB-231細胞侵襲力中起到極為關(guān)鍵的作用。通過活體動物成像實驗我們發(fā)現(xiàn)，在腹部第四個乳腺脂肪墊注射組中，LSD1過表達或LSD1沉默并不影響MDA-MB-231細胞在接種的乳腺脂肪墊部位的原位生長和進入血液循環(huán)，同時轉(zhuǎn)移灶信號的分析結(jié)果表明LSD1過表達組與對照相組比乳腺癌細胞的轉(zhuǎn)移力明顯下降，而LSD1沉默組與對

9、照組相比肺部轉(zhuǎn)移灶信號明顯增強；在尾靜脈注射組中，LSD1過表達組的肺部轉(zhuǎn)移灶明顯被抑制而LSD1沉默組的肺轉(zhuǎn)移信號明顯增強；同樣的，心臟注射組的骨轉(zhuǎn)移灶中LSD1過表達組的后肢骨轉(zhuǎn)移灶明顯被抑制而LSD1沉默組中后肢骨轉(zhuǎn)移灶信號明顯增強。實驗結(jié)果清楚的表明，LSD1過表達可抑制乳腺細胞的轉(zhuǎn)移而LSD1沉默可增強乳腺癌細胞在SCID小鼠體內(nèi)的轉(zhuǎn)移，揭示LSD1可抑制體內(nèi)乳腺癌細胞的轉(zhuǎn)移。為了更加深入的研究LSD1在乳腺癌發(fā)生發(fā)展中的作用，及驗證LSD1與TGFP1的相關(guān)性及其病理意義，我們收集了65個乳腺癌病人的病理樣本，其中30個樣本是癌與癌旁組織配對的樣本。通過分析30個癌與癌旁組織配對的

10、樣本中LSD1與TGFP1的轉(zhuǎn)錄水平，我們發(fā)現(xiàn)LSD1在癌灶中的轉(zhuǎn)錄水平明顯低于癌旁組織，并且與TGFP1的轉(zhuǎn)錄負(fù)相關(guān)。這些結(jié)果表明LSD1參與抑制乳腺癌轉(zhuǎn)移且驗證了TGFP1是LSD1的下游靶基因。綜上所述，我們的研究表明LSD1是一個NuRD復(fù)合體的成員，首次將組蛋白去乙?；徒M蛋白去甲基化這兩種重要的組蛋白修飾聯(lián)系起來。由于LSD1的加入，NuRD復(fù)合體在之前的染色質(zhì)重塑ATP酶和組蛋白去乙?；傅膬煞N活性的基礎(chǔ)上又增加了組蛋白去甲基化酶的活性，揭示了組蛋白去乙酰化和組蛋白去甲基化這兩種重要的組蛋白修飾在染色質(zhì)重塑中相互協(xié)調(diào)作用的機理，對認(rèn)識表觀遺傳調(diào)控的分子機制具有開創(chuàng)性的理論意義。我

11、們利用先進的染色質(zhì)免疫共沉淀-DNA選擇和連接（ChIP-DSL）技術(shù)發(fā)現(xiàn)上述LSD1/NuRD復(fù)合體調(diào)控一系列以TGFB1（轉(zhuǎn)化生長因子P1）為代表的在上皮-間質(zhì)細胞轉(zhuǎn)換（EMT）中起關(guān)鍵作用的基因。由于EMT是癌癥發(fā)生轉(zhuǎn)移的關(guān)鍵步驟，且TGFP1在乳腺癌細胞的EMT以及侵襲和轉(zhuǎn)移等過程中均發(fā)揮重要的促進作用，因此我們首先發(fā)現(xiàn)的LSD1/NuRD復(fù)合體對TGFP1的調(diào)控具有著極為重要的病理生理學(xué)意義。在進一步研究探索中，我們發(fā)現(xiàn)LSD1體內(nèi)體外均能抑制乳腺癌的侵襲和轉(zhuǎn)移，而且通過對人乳腺癌病例樣本的分析表明，癌灶中LSD1水平與正常癌旁組織相比明顯下調(diào)且與TGFP1的水平顯著負(fù)相關(guān)，從而首次

12、證明LSD1這一表觀遺傳調(diào)控因子在抑制乳腺癌轉(zhuǎn)移中有著非常重要的作用。該研究顯示LSD1能夠抑制乳腺癌的轉(zhuǎn)移，為乳腺癌轉(zhuǎn)移的干預(yù)提供了新的可能的分子靶點。誠然，我們的研究結(jié)果僅僅是表觀遺傳學(xué)調(diào)控乳腺癌轉(zhuǎn)移機制的冰山一角，我們將繼續(xù)致力于研究包括乳腺癌在內(nèi)的影響人民健康的重大疾病。關(guān)鍵詞：LSD1,MTA2,NuRD復(fù)合體,TGFP1,乳腺癌轉(zhuǎn)移LSD1isabonafideSubunitoftheNuRDComplexandTargetstheMetastasisProgramsinBreastCancerWangYanABSTRACTLysine-specificdemethylase1(L

13、SD1)wasthefirsthistonedemethylaseidentifiedthatcatalyzestheremovalofmono-anddi-methylationmarksonhistoneH3-K4.DespitethepotentialbroadactionofLSD1intranscriptionregulation,recentstudiesindicatethatLSD1exertspathway-specificactivityinanimaldevelopmentandhavelinkedLSD1toseveralhigh-riskcancers,implyin

14、gcomplicatedmechanisticactionsofthisseeminglysimpleenzyme.ThepotentiallinkbetweencancerandLSD1activityisunderscoredbytheobservationthatlossofH3-K4methylationandenrichmentofH3-K9methylationareassociatedwithseveraltypesoftumors.Indeed,withintheframeworkoftheso-calledepigenetictherapies,thereisagrowing

15、interestinLSD1asapotentialdrugtarget.Molecularcarcinogenesishasbeentheprimaryresearchfocusinthislaboratory.Inanefforttobetterunderstandthemechanisticrolesofthemetastasistumorantigen(MTA),asubunitoftheNuRDcomplex,incancermetastasis,weemployedaffinitypurificationandmassspectrometrytoidentifytheprotein

16、sthatareassociatedwithMTA2,thephylogeneticallyclosestrelativetotheancestralMTAprotein.MassspectrometricanalysisindicatethatMTA2co-purifiedwithMi-2,HDAC1,HDAC2,RbAp46,RbAp48,andMBD3,allofwhicharecomponentsoftheNuRDcomplex,aswellaswithLSD1.ThepresenceofLSD1intheMTA2/NuRDcomplexwasfurtherconfirmedwithi

17、tsantibodiesbyWesternblottinganalysis,suggestingthatLSD1isassociatedwiththeNuRDcomplexinvivo.TofurthershowthatLSD1isassociatedwiththeNuRDcomplexinvivo,proteinfractionationexperimentswerecarriedoutbyfastproteinliquidchromatography(FPLC)withSuperose6columnsandahighsaltextractionandsizeexclusionapproac

18、h.TheresultindicatesthatnativeLSD1fromHeLacellswaselutedwithanapparentmolecularmassmuchgreaterthanthatofthemonomericprotein;LSD1immunoreactivitywasdetectedinchromatographicfractionsfromtheSuperose6columnwitharelativelysymmetricalpeakcenteredbetween669and1000kDa.Significantly,theelutionpatternofLSD1l

19、argelyoverlappedwiththatoftheNuRDcomplexproteinsincludingMTA2,HDAC1,HDAC2,andRbAp46/48,furthersupportingtheideathatLSD1isassociatedwiththeNuRDcomplexinvivo.Moreover,thechromatographicprofilesoftheNuRDcomplexandLSD1werecompatiblewiththeirassociatedenzymaticactivities.Toconfirmtheinvivointeractionbetw

20、eenLSD1andtheNuRDcomplex,totalproteinsfromHeLacellswereextracted,andco-immunoprecipitationexperimentswereperformedwithantibodiesdetectingtheendogenousproteins.Immunoprecipitation(IP)withantibodiesagainstLSD1followedbyimmunoblotting(IB)withantibodiesagainsttheNuRDcomplexproteinsdemonstratedthatLSD1co

21、-immunoprecipitatedwithalloftheNuRDcomponents.Reciprocally,IPwithantibodiesagainstthecomponentsoftheNuRDcomplexandIBwithantibodiesagainstLSD1alsorevealedthatthecomponentsoftheNuRDcomplexco-immunoprecipitatedwithLSD1.Inaddition,theassociationbetweenLSD1andtheNuRDcomplexwasalsodetectedinhumanbreastcar

22、cinomaMCF-7cellsandMDA-MB-231cells.TofurtherinvestigatethephysicalassociationandtoexaminethefunctionalconnectionbetweenLSD1andtheNuRDcomplex,theMTA2-containingproteincomplexwasimmunoprecipitatedfromHeLacellsstablyexpressingFLAG-MTA2withtheanti-FLAGantibodyandanalyzedforenzymaticactivities.Asexpected

23、,theMTA2-containingcomplexpossessedanenzymaticactivitythatledtoasignificantdecreaseintheacetylationlevelofH3.Remarkably,however,theimmunoprecipitatesalsocontainedastrongdemethylaseactivityfordi-methylH3-K4andanevidentdemethylaseactivityformono-methylH3-K4onbothbulkhistonesandthenucleosomalsubstrates

24、,whereasnoapparenteffectonthedi-methylofH3-K9wasdetected.Furthermore,thedemethylationactivityoftheimmunoprecipitatesondi-methylH3-K4couldbeeffectivelyinhibitedbypargyline,aninhibitorspecificformonoamineoxidasessuchasLSD1,orimmunodepletionofLSD1.InordertodeterminethemolecularbasisfortheinteractionofL

25、SD1withtheNuRDcomplex,GSTpull-downassayswereconductedusingGST-fusedLSD1constructandinvitrotranscribed/translatedindividualcomponentsoftheNuRDcomplex.TheseexperimentsrevealedthatLSD1interactsdirectlywithMTA1,MTA2andMTA3,butnotwiththeothercomponentsoftheNuRDcomplexthatwetested.Inordertomaptheinteracti

26、oninterfaceofLSD1withthemembersoftheMTAfamily,GSTpull-downassayswereperformedwithGST-fusedLSD1andMTAdomain-constructs.TheresultsindicatedthattheTowerdomainofLSD1andtheSANTdomainoftheMTAproteinsareresponsibleforthedirectinteractionbetweenthem.Furthermore,histonedemethylationassaysonisolatedmononucleo

27、someswithrecombinantproteinsshowedthat,whilerecombinantLSD1alonewasunabletodemethylateH3K4,additionofMTA2tothedemethylationreactionendowedtheabilityofrecombinantLSD1todemethylatenucleosomalsubstrates,supportingtheideathattheMTAproteinsintheNuRDcomplexfunctiontobridgeLSD1tothechromatinstructure.Inord

28、ertofurtherinvestigatethefunctionalassociationbetweenLSD1andtheNuRDcomplexandtoexplorethebiologicalsignificanceofthisassociation,weanalyzedthegenome-widetranscriptionaltargetsoftheLSD1/NuRDcomplexesusingtheChromatinImmunoPrecipitation-DNASelectionandLigation(ChIP-DSL)approach.Theseexperimentsidentif

29、iedatotalof1,153differentpromoterstargetedbytheLSD1/NuRDcomplexes.Thegeneswerethenclassifiedintocellularsignalingpathways.Interestingly,analysisofthetargetsoftheLSD1/NuRDcomplexesidentifiedsignalingpathwaysincludingTGF:,cellcommunication,focaladhesion,MAPK,andcellcyclethatarecriticallyinvolvedincell

30、growth,survival,migration,andinvasion.Thegenesinthesepathwaysinclude,amongothers,TGFB1,EGFR,RHOA,ANGPTL4,LAMININALPHA4,COLLAGENVIandENDOTHELIN-1thatareknowntobeimplicatedinepithelial-to-mesenchymaltransitionand/ormetastasis.Real-timequantitativeRT-PCRanalysisinMCF-7cellsunderLSD1knockdownofthemRNAex

31、pressionofselectedgenes,whichrepresenteachofthepathways,confirmedtheChIP-DSLexperiments.Later,theChIP-DSLexperimentswerefurthersubstantiatedbyconventionalChIPtodemonstratethatLSD1andMTA3co-occupytheTGFB1promoterinMCF-7cells.Inaddition,sequentialChIPorChIP/Re-ChIPconfirmedthatLSD1,MTA3,andMi-2existin

32、thesameproteincomplexontheTGFB1promoter.Takentogether,theseexperimentsnotonlysupporttheideathatTGFB1istargetedbytheLSD1/MTA3/NuRDcomplexbutalsoconfirmthatLSD1isphysicallyassociatedwithandisanintegralcomponentoftheNuRDcomplexnvivo.Theidentificationofthekeyregulatorsinepithelial-to-mesenchymaltransiti

33、ons,suchasTGF-1,astargetsofLSD1/NuRDcomplexesandthewell-documentedrolesofTGF-1inbreastcancermetastasissuggestthatLSD1mayalsofunctioninbreastcancerinvasionandmetastasis.Therefore,wefirstinvestigatedtheeffectofLSD1onthecellularbehaviourofbreastcancercellsinvitro.Forthispurpose,theimpactofthegain-of-fu

34、nctionandloss-of-functionofLSD1ontheinvasivepotentialofthesecellswasinvestigatedusingtranswellinvasionassays.TheseexperimentsshowthatwhileoverexpressionofLSD1resultedinmorethan3-folddecreaseincellinvasion,LSD1knockdownledtoincreasedcellinvasionabout5-fold.Moreover,theinhibitoryeffectofLSD1overexpres

35、sionontheinvasivepotentialofMDA-MB-231cellscouldberescuedbyadditionofexogenousTGFi11andtheinvasion-promotingeffectofLSD1knockdowncouldbeeffectivelyinhibitedbySB-431542,anATPanaloginhibitoroftheTGFI-typereceptorkinase.TheseresultssuggestacriticalroleoftheTGFsignalingpathwayinmediatingtheeffectofLSD1o

36、ntheinvasivepotentialofMDA-MB-231cells.Inordertofurtherstudytheinvasion-inhibitoryeffectofLSD1andtoinvestigateitspossibleroleinbreastcancermetastasisinvivo,MDA-MB-231cellsthathadbeenengineeredtostablyexpressfireflyluciferasewereinfectedwithlentiviruescarryingLSD1cDNAorLSD1-specificsiRNA.Theeffectoft

37、hegain-of-functionandloss-of-functionofLSD1onspontaneouslungmetastasis,onseedinglungmetastasis,andonseedingbonemetastasisofMDA-MB-231-LuctumorswasassessedinimmunocompromisedSCIDmicebyorthotopicimplantation,intravenousinjection,andintracardiacinjection,respectively.Theresultsoftheseexperimentsindicat

38、ethatLSD1overexpressionsuppressedthemetastaticspreadofMDA-MB-231tumorsandLSD1knockdownenhancedthemetastaticspreadofthetumorsinSCIDmice,suggestingthatLSD1suppressesthemetastaticpotentialofbreastcancervivo.InordertofurthersupporttheroleofLSD1inbreastcanceraswellastosubstantiatethefunctionallinkbetweenLSD1andTGF1andextendthephysiologicalrelevanceofthislink,wecollected65breasttumorsamples,ofwhich30includedadjacentnormaltissue,frombreastcancerpatients.Theresultsrevealedastatisticallysignifican