功能上调控乳腺癌的转移

1、论文中英文摘要作者姓名： 王艳论文题目 ：LSD1 是 NuRD 复合体的一个亚基 , 功能上调控乳腺癌的转移作者简介 ：王艳，女， 1982 年 11 月出生， 2001 年 9 月进入北京大学医学部八年制基础医学专业学习， 2006 年 9 月师从于北京大学尚永丰教授， 于 2009 年 7月获博士学位。中文摘要LSD1 是第一个被发现的组蛋白去甲基化酶，属于以FAD 为辅酶的单胺氧化酶，能够催化 H3 K4me2 和 H3 K4me1 多肽去甲基化反应。 LSD1 广泛调控基因的转录，并与多种肿瘤的发生发展高度相关。 LSD1 与癌症之间的潜在联系目前被理解为多种肿瘤中组蛋白 H3K4的

2、甲基化水平下降和 H3K9 的水平上升的现象相关。虽然有少量报导说LSD1 可以激活基因的转录，但是目前更普遍认为 LSD1 对基因转录起到抑制作用。由于H3 K4me2 是公认的转录激活标志， LSD1 催化其去甲基化的活性便抑制了基因的转录。迄今，LSD1 在众多转录抑制复合体中被发现，如 CoREST 复合体 , CtBP 复合体和一系列 HDAC 复合体等等。在表观遗传治疗的大好前景下， LSD1 越来越显示出了它作为一个潜在治疗靶点的优越性。运用 anti-FLAG 亲和层析联合质谱的方法，我们首次报导组蛋白去甲基化酶LSD1 可以与组蛋白去乙酰化复合体NuRD 相互作用。 NuRD

3、 复合体是一个多亚基的复合体，包括ATP酶部分和组蛋白去乙酰化（HDAC ）酶部分，广泛参与基因转录抑制。高效液相色谱实验显示，HeLa 细胞中天然存在的 LSD1 多出现在高出其单体 110kDa 很多的 669-1000 kDa 的组分中，且与 NuRD 复合体的常见组分 MTA ， HDAC 等大致共同洗脱。更重要的是， NuRD 复合体的洗脱谱中的组蛋白去乙酰化 HDAC 活性范围与 LSD1 的组蛋白去甲基化 HDM 活性范围大致重合。利用免疫共沉淀实验的方法，我们在 HeLa，MCF-7 和 MDA-MB-231 细胞系中均证明 LSD1 可以与 NuRD 复合体所有的组分在体内相

4、互作用，说明 LSD1 是 NuRD 复合体的一个亚基。HDAC 酶活性和 HDM 酶活性检测结果显示， NuRD 复合体组分中拥有使小牛胸腺组蛋白或 HeLa 细胞单核小体的 H3 K4me2 和 H3 K4me1 显著下降的去组蛋白去甲基化酶活性， 且此活性可被 LSD1 的特异性抑制剂 Pargeline 抑制，也可被 LSD1 免疫清除实验所清除，说明 LSD1 是 NuRD 复合体的一个功能亚基，通过其 H3K4 去甲基化酶活性协同 NuRD 复合体抑制基因的转录。GST Pull-down 实验结果证明 LSD1 可以在体外直接与NuRD 复合体亚基之一的MTA 蛋白特异的相互作用

5、，却不与其他的 NuRD 复合体组分直接相互作用。而进一步分段截短体的 GST Pull-down 实验结果证明 LSD1 的 Tower 结构域是和 MTA 分子的 SANT 结构域是介导 LSD1 与三个 MTA 分子直接相互作用的结构域。 而利用重组蛋白进行的体外去甲基化实验则揭示，当纯化的 LSD1 单独存在时，虽可以催化组蛋白进行去甲基化反应，却无法完成对单核小体 H3K4 的去甲基化；而加入体外纯化的重组 MTA2 之后， LSD1 便可以单核小体为底物使之去甲基化了， 说明 NuRD 复合体中的 MTA 分子可以作为联系 LSD1 与染色质高级结构的桥梁。利用先进的染色质免疫共沉

6、淀-DNA 选择和连接（ChIP-DSL) 技术我们得到了 LSD1/NuRD复合体全基因组转录调控的可能的下游靶基因。这些靶基因多分布于TGF 通路，细胞连接，细胞粘附， MAPK 信号转导和细胞周期等通路，这些通路在细胞生长，生存，迁移，侵袭和转移中起到非常重要的作用。 其中，TGFB1，EGFR，RHOA 等都是上皮 -间质细胞转化（EMT ）和肿瘤转移密切相关的基因 , 提示 LSD1/NuRD 复合体与肿瘤的转移密切相关。 在利用实时定量 PCR 验证了芯片的结果之后，我们用传统的 ChIP 方法证实 LSD1， MTA3 和 Mi-2 都在 MCF-7 细胞中与 TGFB1 的启动

7、子相结合。而之后的连续 ChIP 实验亦证明，LSD1/MTA3/Mi-2存在于同一个蛋白复合体中并都结合在 TGFB1 的启动子上。这些实验的结果不但证明 TGFB1 是 LSD1/MTA3/NuRD 复合体的下游靶基因，且也同样进一步印证了 LSD1 是 NuRD 复合体的一个内在亚基。TGF 1 被认为是上皮 -间质细胞转化（ EMT ）的关键调控分子之一。鉴于TGF 1 在乳腺癌细胞的 EMT 以及侵袭和转移等过程中均发挥重要的促进作用， 其作为 LSD1/NuRD 复合体的靶基因出现提示 LSD1 很可能参与调控乳腺癌的侵袭和转移。利用转移小室实验我们发现正常 LSD1 过表达组与对

8、照相比， 乳腺癌 MDA-MB-231 细胞的侵袭潜力下降了 3 倍左右；而缺失与 NuRD 复合体 MTA 分子直接相互作用的 Tower 结构域的 LSD1 过表达组与对照组相比，则没有什么明显的变化。另一方面， LSD1 沉默组与其对照组相比 MDA-MB-231 细胞的侵袭力增加了约 5 倍左右。而且， LSD1 过表达而 Mi-2 沉默组与前述单纯过表达 LSD1 组的结果相比， MDA-MB-231 细胞侵袭力下降的效果被明显减弱了，说明 MDA-MB-231 细胞侵袭力的变化主要是通过 LSD1 与 NuRD 复合体结合后来实现的。通过加入外源 TGF 1 的“挽救”实验证明，

9、LSD1 沉默之后引起的 231 细胞侵袭力增强的作用可被 TGF 1 I 型受体的 ATP 酶拮抗剂 SB431542 所 “挽救 ” ，说明 TGF 1 信号通路在 LSD1 介导的抑制乳腺癌MDA-MB-231细胞侵袭力中起到极为关键的作用。通过活体动物成像实验我们发现，在腹部第四个乳腺脂肪垫注射组中，LSD1 过表达或 LSD1 沉默并不影响 MDA-MB-231 细胞在接种的乳腺脂肪垫部位的原位生长和进入血液循环，同时转移灶信号的分析结果表明 LSD1 过表达组与对照相组比乳腺癌细胞的转移力明显下降，而 LSD1 沉默组与对照组相比肺部转移灶信号明显增强；在尾静脉注射组中，LSD1

10、过表达组的肺部转移灶明显被抑制而LSD1 沉默组的肺转移信号明显增强；同样的，心脏注射组的骨转移灶中LSD1 过表达组的后肢骨转移灶明显被抑制而LSD1 沉默组中后肢骨转移灶信号明显增强。 实验结果清楚的表明， LSD1 过表达可抑制乳腺细胞的转移而LSD1 沉默可增强乳腺癌细胞在SCID 小鼠体内的转移，揭示LSD1 可抑制体内乳腺癌细胞的转移。为了更加深入的研究LSD1 在乳腺癌发生发展中的作用，及验证LSD1 与 TGF 1 的相关性及其病理意义，我们收集了65 个乳腺癌病人的病理样本，其中30 个样本是癌与癌旁组织配对的样本。通过分析30 个癌与癌旁组织配对的样本中LSD1 与 TGF

11、 1 的转录水平，我们发现 LSD1 在癌灶中的转录水平明显低于癌旁组织，并且与TGF 1 的转录负相关。这些结果表明 LSD1 参与抑制乳腺癌转移且验证了TGF 1 是 LSD1 的下游靶基因。综上所述，我们的研究表明 LSD1 是一个 NuRD 复合体的成员，首次将组蛋白去乙酰化和组蛋白去甲基化这两种重要的组蛋白修饰联系起来。由于 LSD1 的加入， NuRD 复合体在之前的染色质重塑 ATP 酶和组蛋白去乙酰化酶的两种活性的基础上又增加了组蛋白去甲基化酶的活性，揭示了组蛋白去乙酰化和组蛋白去甲基化这两种重要的组蛋白修饰在染色质重塑中相互协调作用的机理，对认识表观遗传调控的分子机制具有开创

12、性的理论意义。我们利用先进的染色质免疫共沉淀 -DNA 选择和连接（ ChIP-DSL) 技术发现上述 LSD1/NuRD 复合体调控一系列以 TGFB1（转化生长因子 1）为代表的在上皮 -间质细胞转换 (EMT) 中起关键作用的基因。由于 EMT 是癌症发生转移的关键步骤，且 TGF 1 在乳腺癌细胞的 EMT 以及侵袭和转移等过程中均发挥重要的促进作用，因此我们首先发现的 LSD1/NuRD 复合体对 TGF 1 的调控具有着极为重要的病理生理学意义。 在进一步研究探索中， 我们发现 LSD1 体内体外均能抑制乳腺癌的侵袭和转移，而且通过对人乳腺癌病例样本的分析表明，癌灶中 LSD1 水

13、平与正常癌旁组织相比明显下调且与 TGF 1 的水平显著负相关 , 从而首次证明 LSD1 这一表观遗传调控因子在抑制乳腺癌转移中有着非常重要的作用。该研究显示 LSD1 能够抑制乳腺癌的转移，为乳腺癌转移的干预提供了新的可能的分子靶点。诚然，我们的研究结果仅仅是表观遗传学调控乳腺癌转移机制的冰山一角，我们将继续致力于研究包括乳腺癌在内的影响人民健康的重大疾病。关键词：LSD1, MTA2, NuRD 复合体 , TGF 1, 乳腺癌转移LSD1 is a bona fide Subunit of the NuRD Complex and Targets theMetastasis Progr

14、ams in Breast CancerWang YanABSTRACTLysine-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 LS

15、D1 exerts pathway-specific activity in animal 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

16、and enrichment of H3-K9 methylation are 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 e

17、ffort to better understand the mechanistic 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 an

18、cestral MTA protein. Mass spectrometric 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

19、 blotting analysis, suggesting that LSD1 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 extracti

20、on and size exclusion approach. The result 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 p

21、eak centered between 669 and 1000 kDa. 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 p

22、rofiles of the NuRD complex and LSD1 were 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 end

23、ogenous proteins. Immunoprecipitation(IP) with antibodies against LSD1 followed by immunoblotting(IB) with antibodies against the NuRD complex proteinsdemonstrated that LSD1 co-immunoprecipitated with all of the NuRD components. Reciprocally, IP with antibodies against the components of the NuRD com

24、plex and IB with antibodies against LSD1 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 physi

25、cal association and to examine the functional 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 compl

26、ex possessedan enzymatic activity that led to a significant decreasein 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 nuc

27、leosomal substrates, whereas no apparent 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 L

28、SD1.In order to determine the molecular 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/translatedindividual components of the NuRD complex. These experiments revealed that LSD1 interacts directly wi

29、th MTA1, MTA2 and MTA3, but not with 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

30、domain of LSD1 and the SANT domain 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

31、 the demethylation reaction endowed 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

32、NuRD complex and to explore the 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

33、promoters targeted by the LSD1/NuRD 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

34、 involved in cell growth, survival, 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

35、-PCR analysis in MCF-7 cells under 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 theTGFB1 p

36、romoter in MCF-7 cells. In addition, 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 LSD

37、1 is physically associated with and is an integral component of the NuRD complexin vivo.The identification of the key regulators in epithelial-to-mesenchymal transitions, such as TGF 1, as targets of LSD1/NuRD complexes and the well-documented roles of TGF 1 in breast cancer metastasissuggest that L

38、SD1 may also function in breast 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 theinvasive potential of these cells wa

39、s investigated using transwell 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 potentia

40、l of MDA-MB-231 cells could be rescued by addition of exogenous TGF 1 and the invasion-promoting effect of LSD1 knockdown could be effectively inhibited by SB-431542, an ATP analog inhibitor of the TGF type receptor kinase. These results suggest a critical role of the TGF 1 signaling pathway in medi

41、ating the effect of LSD1 on the invasive 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 metastasisin vivo, MDA-MB-231 cells that had been engineered to stablyexpress fireflyluciferase were infected

42、with lentivirues carrying LSD1 cDNA or LSD1-specificsiRNA. The effect of the gain-of-function and loss-of-function of LSD1 on spontaneous lungmetastasis, on seeding lung metastasis, and on seeding bone metastasis of MDA-MB-231-Luctumors was assessed in immunocompromisedSCID mice by orthotopic implan

43、tation, intravenousinjection, and intracardiac injection, respectively. The results of these experiments indicate thatLSD1 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 LSD1suppresses

44、 the metastatic potential of breast cancerin vivo.In order to further support the role of LSD1 in breast cancer as well as to substantiate the functional link between LSD1 and TGF 1 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 i