功能上调控乳腺癌的转移

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，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。

TGFβ1被认为是上皮-间质细胞转化（EMT）的关键调控分子之一。

鉴于TGFβ1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TGFβ1的“挽救”实验证明，LSD1沉默之后引起的231细胞侵袭力增强的作用可被TGFβ1 I型受体的ATP 酶拮抗剂SB431542所“挽救”，说明TGFβ1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TGFβ1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TGFβ1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TGFβ1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TGFβ1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β1）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TGFβ1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TGFβ1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水平与正常癌旁组织相比明显下调且与TGFβ1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 Programs 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 LSD1 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 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 effort 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 ancestral 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 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 extraction 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 peak 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 profiles 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 endogenous 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 complex 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 physical 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 complex possessed an enzymatic activity that 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 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 LSD1.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/translated individual components of the NuRD complex. These experiments revealed that LSD1 interacts directly with 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 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 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 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 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 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-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 the TGFB1 promoter 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 LSD1 is physically associated with and is an integral component of the NuRD complex in vivo.The identification of the key regulators in epithelial-to-mesenchymal transitions, such as TGFβ1, as targets of LSD1/NuRD complexes and the well-documented roles of TGFβ1 in breast cancer metastasis suggest that LSD1 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 the invasive potential of these cells was 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 potential of MDA-MB-231 cells could be rescued by addition of exogenous TGFβ1 and the invasion-promoting effect of LSD1 knockdown could be effectively inhibited by SB-431542, an ATP analog inhibitor of the TGFβ type I receptor kinase. These results suggest a critical role of the TGFβ1 signaling pathway in mediating 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 metastasis in vivo, MDA-MB-231 cells that had been engineered to stably express firefly luciferase were infected with lentivirues carrying LSD1 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 injection, 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 potential 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 TGFβ1 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 LSD1expression in tumors compared to the adjacent normal mammary tissue and a significant negative correlation between LSD1 and TGFB1expression in these samples. These data are consistent with a role of LSD1 in suppressing breast cancer metastasis and support TGFβ1 as a downstream effector of LSD1.In conclusion, our group for the first time reported that LSD1 is an integral component of the Mi-2/ NuRD complex. Transcriptional target analysis by ChIP-DSL revealed that the LSD1/NuRD complexes target several cellular signaling pathways including TGFβ1 signaling pathway that are critically involved in cell proliferation, survival, and epithelial-to-mesenchymal transition. We demonstrated that LSD1 inhibits the invasion of breast cancer cells in vitro and suppresses breast cancer metastatic potential in vivo. We found that LSD1 is down-regulated in breast carcinomas and that its level of expression is negatively correlated with that of TGFβ1. Our data indicate that LSD1 is a bona fide subunit of the NuRD complex, providing a molecular basis for the interplay of histone demethylation and deacetylation in chromatin remodeling. By enlisting LSD1, the NuRD complex expands its chromatin remodeling capacity to include ATPase, histone deacetylase, and histone demethylase. Our experiments indicate that LSD1 regulates the metastatic potential of breast cancer, supporting the pursuit of LSD1 as a target for cancer therapy.Key words: LSD1, MTA2, NuRD complex, TGFβ1, Breast Cancer Metastasis。