論文中英文摘要

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(gè)亞基, 功能上調(diào)控乳腺癌的轉(zhuǎn)移作者簡(jiǎn)介：王艷，女，1982年11月出生，2001年9月進(jìn)入北京大學(xué)醫(yī)學(xué)部八年制基礎(chǔ)醫(yī)學(xué)專(zhuān)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(gè)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H3 K4me2和H3 K4me1 多肽去甲基化反應(yīng)。LSD1廣泛調(diào)控基因的轉(zhuǎn)錄，并與多種腫瘤的發(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腫瘤中組蛋白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(bào)導(dǎo)

2、說(shuō)LSD1可以激活基因的轉(zhuǎn)錄，但是目前更普遍認(rèn)為L(zhǎng)SD1對(duì)基因轉(zhuǎn)錄起到抑制作用。由于H3 K4me2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(lái)越顯示出了它作為一個(gè)潛在治療靶點(diǎn)的優(yōu)越性。運(yùn)用anti-FLAG親和層析聯(lián)合質(zhì)譜的方法，我們首次報(bào)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(gè)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

3、效液相色譜實(shí)驗(yàn)顯示，HeLa細(xì)胞中天然存在的LSD1多出現(xiàn)在高出其單體110kDa很多的669-1000 kDa的組分中，且與 NuRD 復(fù)合體的常見(jiàn)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(shí)驗(yàn)的方法，我們?cè)贖eLa，MCF-7和MDA-MB-231細(xì)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(shuō)明LSD1是NuRD復(fù)合體的一個(gè)亞基。 HDAC酶活性和HDM酶活性檢測(cè)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(xì)胞單核小體的H3

4、 K4me2和H3 K4me1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免疫清除實(shí)驗(yàn)所清除，說(shuō)明LSD1是NuRD復(fù)合體的一個(gè)功能亞基，通過(guò)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。GST Pull-down實(shí)驗(yàn)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(jìn)一步分段截短體的GST Pull-down實(shí)驗(yàn)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(gè)MTA分子直接相互作用的結(jié)構(gòu)域。而利用重組蛋白進(jìn)

5、行的體外去甲基化實(shí)驗(yàn)則揭示，當(dāng)純化的LSD1單獨(dú)存在時(shí)，雖可以催化組蛋白進(jìn)行去甲基化反應(yīng)，卻無(wú)法完成對(duì)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(shuō)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(jí)結(jié)構(gòu)的橋梁。利用先進(jìn)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TGF通路，細(xì)胞連接，細(xì)胞粘附，MAPK信號(hào)轉(zhuǎn)導(dǎo)和細(xì)胞周期等通路，這些通路在細(xì)胞生長(zhǎng)，生存，遷移，侵襲和轉(zhuǎn)移中起到非常重要的作用。其中，TGFB1，EGFR，

6、RHOA等都是上皮-間質(zhì)細(xì)胞轉(zhuǎn)化（EMT）和腫瘤轉(zhuǎn)移密切相關(guān)的基因, 提示LSD1/NuRD復(fù)合體與腫瘤的轉(zhuǎn)移密切相關(guān)。在利用實(shí)時(shí)定量PCR驗(yàn)證了芯片的結(jié)果之后，我們用傳統(tǒng)的ChIP方法證實(shí)LSD1，MTA3和Mi-2都在MCF-7細(xì)胞中與TGFB1的啟動(dòng)子相結(jié)合。而之后的連續(xù)ChIP實(shí)驗(yàn)亦證明，LSD1/MTA3/Mi-2存在于同一個(gè)蛋白復(fù)合體中并都結(jié)合在TGFB1的啟動(dòng)子上。這些實(shí)驗(yàn)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(jìn)一步印證了LSD1是NuRD復(fù)合體的一個(gè)內(nèi)在亞基。TGF1被認(rèn)為是上皮-間質(zhì)細(xì)胞轉(zhuǎn)化（EMT）的關(guān)鍵調(diào)控分子之一。鑒于TGF1

7、在乳腺癌細(xì)胞的EMT以及侵襲和轉(zhuǎn)移等過(guò)程中均發(fā)揮重要的促進(jìn)作用，其作為L(zhǎng)SD1/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(shí)驗(yàn)我們發(fā)現(xiàn)正常LSD1過(guò)表達(dá)組與對(duì)照相比，乳腺癌MDA-MB-231細(xì)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(guò)表達(dá)組與對(duì)照組相比，則沒(méi)有什么明顯的變化。另一方面，LSD1沉默組與其對(duì)照組相比MDA-MB-231細(xì)胞的侵襲力增加了約5倍左右。而且，LSD1過(guò)表達(dá)而Mi-2沉默組與前述單純過(guò)表達(dá)LSD1組的結(jié)果相比，MDA-MB-231細(xì)胞侵襲力下降的效果被明顯減弱了，說(shuō)明

8、MDA-MB-231細(xì)胞侵襲力的變化主要是通過(guò)LSD1與NuRD復(fù)合體結(jié)合后來(lái)實(shí)現(xiàn)的。通過(guò)加入外源TGF1的“挽救”實(shí)驗(yàn)證明，LSD1沉默之后引起的231細(xì)胞侵襲力增強(qiáng)的作用可被TGF1 I型受體的ATP酶拮抗劑SB431542所 “挽救” ，說(shuō)明TGF1信號(hào)通路在LSD1介導(dǎo)的抑制乳腺癌MDA-MB-231細(xì)胞侵襲力中起到極為關(guān)鍵的作用。通過(guò)活體動(dòng)物成像實(shí)驗(yàn)我們發(fā)現(xiàn)，在腹部第四個(gè)乳腺脂肪墊注射組中，LSD1過(guò)表達(dá)或LSD1沉默并不影響MDA-MB-231細(xì)胞在接種的乳腺脂肪墊部位的原位生長(zhǎng)和進(jìn)入血液循環(huán)，同時(shí)轉(zhuǎn)移灶信號(hào)的分析結(jié)果表明LSD1過(guò)表達(dá)組與對(duì)照相組比乳腺癌細(xì)胞的轉(zhuǎn)移力明顯下降，而L

9、SD1沉默組與對(duì)照組相比肺部轉(zhuǎn)移灶信號(hào)明顯增強(qiáng)；在尾靜脈注射組中，LSD1過(guò)表達(dá)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(hào)明顯增強(qiáng)；同樣的，心臟注射組的骨轉(zhuǎn)移灶中LSD1過(guò)表達(dá)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(hào)明顯增強(qiáng)。實(shí)驗(yàn)結(jié)果清楚的表明，LSD1過(guò)表達(dá)可抑制乳腺細(xì)胞的轉(zhuǎn)移而LSD1沉默可增強(qiáng)乳腺癌細(xì)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(xì)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(yàn)證LSD1與TGF1的相關(guān)性及其病理意義，我們收集了65個(gè)乳腺癌病人的病理樣本，其中30個(gè)樣本是癌與癌旁組織配對(duì)的樣本。通過(guò)分析30個(gè)癌與

10、癌旁組織配對(duì)的樣本中LSD1與TGF1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TGF1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(yàn)證了TGF1是LSD1的下游靶基因。綜上所述，我們的研究表明LSD1是一個(gè)NuRD復(fù)合體的成員，首次將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聯(lián)系起來(lái)。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(jī)理，對(duì)認(rèn)識(shí)表觀遺傳調(diào)控的分子機(jī)制具有開(kāi)創(chuàng)性的理論

11、意義。我們利用先進(jìn)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(zhǎng)因子1）為代表的在上皮-間質(zhì)細(xì)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TGF1在乳腺癌細(xì)胞的EMT以及侵襲和轉(zhuǎn)移等過(guò)程中均發(fā)揮重要的促進(jìn)作用，因此我們首先發(fā)現(xiàn)的LSD1/NuRD復(fù)合體對(duì)TGF1的調(diào)控具有著極為重要的病理生理學(xué)意義。 在進(jìn)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(guò)對(duì)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TGF1的水平顯著負(fù)相關(guān), 從而

12、首次證明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(diǎn)。誠(chéng)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(jī)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, TGF1, 乳腺癌轉(zhuǎn)移LSD1 is a bona fide Subunit of the NuRD Complex and Targets the Metastasis Programs in Breast CancerWang Yan ABSTRACTLysi

13、ne-specific demethylase 1 (LSD1) was the first histone demethylase identified that catalyzes the removal of mono- and di-methylation marks on histone H3-K4. Despite the potential broad action of LSD1 in transcription regulation, recent studies indicate that LSD1 exerts pathway-specific activity in a

14、nimal development and have linked LSD1 to several high-risk cancers, implying complicated mechanistic actions of this seemingly simple enzyme. The potential link between cancer and LSD1 activity is underscored by the observation that loss of H3-K4 methylation and enrichment of H3-K9 methylation are

15、associated with several types of tumors. Indeed, within the framework of the so-called epigenetic therapies, there is a growing interest in LSD1 as a potential drug target.Molecular carcinogenesis has been the primary research focus in this laboratory. In an effort to better understand the mechanist

16、ic roles of the metastasis tumor antigen (MTA), a subunit of the NuRD complex, in cancer metastasis, we employed affinity purification and mass spectrometry to identify the proteins that are associated with MTA2, the phylogenetically closest relative to the ancestral MTA protein. Mass spectrometric

17、analysis indicate that MTA2 co-purified with Mi-2, HDAC1, HDAC2, RbAp46, RbAp48, and MBD3, all of which are components of the NuRD complex, as well as with LSD1. The presence of LSD1 in the MTA2/NuRD complex was further confirmed with its antibodies by Western blotting analysis, suggesting that LSD1

18、 is associated with the NuRD complex in vivo. To further show that LSD1 is associated with the NuRD complex in vivo, protein fractionation experiments were carried out by fast protein liquid chromatography (FPLC) with Superose 6 columns and a high salt extraction and size exclusion approach. The res

19、ult indicates that native LSD1 from HeLa cells was eluted with an apparent molecular mass much greater than that of the monomeric protein; LSD1 immunoreactivity was detected in chromatographic fractions from the Superose 6 column with a relatively symmetrical peak centered between 669 and 1000 kDa.

20、Significantly, the elution pattern of LSD1 largely overlapped with that of the NuRD complex proteins including MTA2, HDAC1, HDAC2, and RbAp46/48, further supporting the idea that LSD1 is associated with the NuRD complex in vivo. Moreover, the chromatographic profiles of the NuRD complex and LSD1 wer

21、e compatible with their associated enzymatic activities. To confirm the in vivo interaction between LSD1 and the NuRD complex, total proteins from HeLa cells were extracted, and co-immunoprecipitation experiments were performed with antibodies detecting the endogenous proteins. Immunoprecipitation(I

22、P) with antibodies against LSD1 followed by immunoblotting(IB) with antibodies against the NuRD complex proteins demonstrated that LSD1 co-immunoprecipitated with all of the NuRD components. Reciprocally, IP with antibodies against the components of the NuRD complex and IB with antibodies against LS

23、D1 also revealed that the components of the NuRD complex co-immunoprecipitated with LSD1. In addition, the association between LSD1 and the NuRD complex was also detected in human breast carcinoma MCF-7 cells and MDA-MB-231 cells.To further investigate the physical association and to examine the fun

24、ctional connection between LSD1 and the NuRD complex, the MTA2-containing protein complex was immunoprecipitated from HeLa cells stably expressing FLAG-MTA2 with the anti-FLAG antibody and analyzed for enzymatic activities. As expected, the MTA2-containing complex possessed an enzymatic activity tha

25、t led to a significant decrease in the acetylation level of H3. Remarkably, however, the immunoprecipitates also contained a strong demethylase activity for di-methyl H3-K4 and an evident demethylase activity for mono-methyl H3-K4 on both bulk histones and the nucleosomal substrates, whereas no appa

26、rent effect on the di-methyl of H3-K9 was detected. Furthermore, the demethylation activity of the immunoprecipitates on di-methyl H3-K4 could be effectively inhibited by pargyline, an inhibitor specific for monoamine oxidases such as LSD1, or immunodepletion of LSD1. In order to determine the molec

27、ular basis for the interaction of LSD1 with the NuRD complex, GST pull-down assays were conducted using GST-fused LSD1 construct and in vitro transcribed/translated individual components of the NuRD complex. These experiments revealed that LSD1 interacts directly with MTA1, MTA2 and MTA3, but not wi

28、th the other components of the NuRD complex that we tested. In order to map the interaction interface of LSD1 with the members of the MTA family, GST pull-down assays were performed with GST-fused LSD1 and MTA domain-constructs. The results indicated that the Tower domain of LSD1 and the SANT domain

29、 of the MTA proteins are responsible for the direct interaction between them. Furthermore, histone demethylation assays on isolated mononucleosomes with recombinant proteins showed that, while recombinant LSD1 alone was unable to demethylate H3K4, addition of MTA2 to the demethylation reaction endow

30、ed the ability of recombinant LSD1 to demethylate nucleosomal substrates, supporting the idea that the MTA proteins in the NuRD complex function to bridge LSD1 to the chromatin structure. In order to further investigate the functional association between LSD1 and the NuRD complex and to explore the

31、biological significance of this association, we analyzed the genome-wide transcriptional targets of the LSD1/NuRD complexes using the Chromatin ImmunoPrecipitation-DNA Selection and Ligation (ChIP-DSL) approach. These experiments identified a total of 1,153 different promoters targeted by the LSD1/N

32、uRD complexes. The genes were then classified into cellular signaling pathways. Interestingly, analysis of the targets of the LSD1/NuRD complexes identified signaling pathways including TGF, cell communication, focal adhesion, MAPK, and cell cycle that are critically involved in cell growth, surviva

33、l, migration, and invasion. The genes in these pathways include, among others, TGFB1, EGFR, RHOA, ANGPTL4, LAMININ ALPHA 4, COLLAGEN VI and ENDOTHELIN-1 that are known to be implicated in epithelial-to-mesenchymal transition and/or metastasis.Real-time quantitative RT-PCR analysis in MCF-7 cells und

34、er LSD1 knockdown of the mRNA expression of selected genes, which represent each of the pathways, confirmed the ChIP-DSL experiments. Later, the ChIP-DSL experiments were further substantiated by conventional ChIP to demonstrate that LSD1 and MTA3 co-occupy the TGFB1 promoter in MCF-7 cells. In addi

35、tion, sequential ChIP or ChIP/Re-ChIP confirmed that LSD1, MTA3, and Mi-2 exist in the same protein complex on the TGFB1 promoter. Taken together, these experiments not only support the idea that TGFB1 is targeted by the LSD1/MTA3/NuRD complex but also confirm that LSD1 is physically associated with

36、 and is an integral component of the NuRD complex in vivo.The identification of the key regulators in epithelial-to-mesenchymal transitions, such as TGF1, as targets of LSD1/NuRD complexes and the well-documented roles of TGF1 in breast cancer metastasis suggest that LSD1 may also function in breast

37、 cancer invasion and metastasis. Therefore, we first investigated the effect of LSD1 on the cellular behaviour of breast cancer cells in vitro. For this purpose, the impact of the gain-of-function and loss-of-function of LSD1 on the invasive potential of these cells was investigated using transwell

38、invasion assays. These experiments show that while overexpression of LSD1 resulted in more than 3-fold decrease in cell invasion, LSD1 knockdown led to increased cell invasion about 5-fold. Moreover, the inhibitory effect of LSD1 overexpression on the invasive potential of MDA-MB-231 cells could be

39、rescued by addition of exogenous TGF1 and the invasion-promoting effect of LSD1 knockdown could be effectively inhibited by SB-431542, an ATP analog inhibitor of the TGFtype receptor kinase. These results suggest a critical role of the TGF1 signaling pathway in mediating the effect of LSD1 on the in

40、vasive potential of MDA-MB-231 cells. In order to further study the invasion-inhibitory effect of LSD1 and to investigate its possible role in breast cancer metastasis in vivo, MDA-MB-231 cells that had been engineered to stably express firefly luciferase were infected with lentivirues carrying LSD1

41、 cDNA or LSD1-specific siRNA. The effect of the gain-of-function and loss-of-function of LSD1 on spontaneous lung metastasis, on seeding lung metastasis, and on seeding bone metastasis of MDA-MB-231-Luc tumors was assessed in immunocompromised SCID mice by orthotopic implantation, intravenous inject

42、ion, and intracardiac injection, respectively. The results of these experiments indicate that LSD1 overexpression suppressed the metastatic spread of MDA-MB-231 tumors and LSD1 knockdown enhanced the metastatic spread of the tumors in SCID mice, suggesting that LSD1 suppresses the metastatic potenti

43、al of breast cancer in vivo. In order to further support the role of LSD1 in breast cancer as well as to substantiate the functional link between LSD1 and TGF1 and extend the physiological relevance of this link, we collected 65 breast tumor samples, of which 30 included adjacent normal tissue, from breast cancer patients. The results revealed a statistically significant decrease in LSD1 expression in tumors compared to the adjacent