【作者】 张立将；

【导师】 李永泉； 张晓霆；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 乳腺癌是最常见的恶性肿瘤之一,严重威胁女性的健康与生命,而近年来我国乳腺癌发病率快速上升,在全国多数大城市中乳腺癌已占据女性恶性肿瘤之首。以雌激素受体(ER)为靶点的纯抗雌激素内分泌治疗药物Fulvestrant(氟维司群)在乳腺癌临床控制中发挥着重要作用,然而随之而来的耐药性问题大大减弱了其治疗效果；同时,远端的侵袭转移更是导致乳腺癌患者死亡的最大诱因。内分泌治疗耐药和侵袭转移已成为乳腺癌临床治疗面临的两大难题。转录中介体亚基1(MED1)作为ER关键的共激活因子,在ER依赖的基因表达和雌激素依赖的乳腺癌生长过程中起到关键性作用,而且参与了另一类内分泌治疗药物Tamoxifen的耐药形成过程,与乳腺癌患者的不良预后有着高度相关性。针对上述乳腺癌治疗的两大难题,并鉴于已报道的MED1在乳腺癌中贡献作用,本论文对MED1在乳腺癌治疗时Fulvestrant耐药及侵袭转移中的作用进行了较系统的科学分析,并对其分子机理做了较深入的机制研究。1、成功建立了可稳定表达萤火虫荧光素酶和诱导型MED1shRNA的可诱导型MED1沉默乳腺癌细胞株(MCF7-tet-shMED1、BT474-tet-shMED1ZR75-1-tet-shMED1)。在确保doxycycline(Dox)诱导MED1表达确实明显下调的同时,验证分析了MED1对乳腺癌细胞生长增殖中的关键作用：MED1沉默明显抑制细胞生长增殖、贴壁克隆及非贴壁软琼脂克隆形成。这些诱导型细胞株的建立,为后续试验提供了可靠的实验材料保障。2、首次对MED1在乳腺癌内分泌治疗药物Fulvestrant耐药性中的贡献作用做了较为系统的科学分析及机制研究。体外、体内实验研究揭示,沉默MED1增强了Fulvestrant耐药乳腺癌细胞株BT474、ZR75-1和MCF7-F细胞(体外实验)以及BT474细胞裸鼠原位移植瘤(体内实验)对Fulvestrant药物处理的敏感性。机制研究初步阐明：沉默MED1联合Fulvestrant处理协同抑制了乳腺癌细胞的细胞周期进程及ERa靶基因表达；染色质免疫沉淀(ChIP)实验揭示,协同影响ERα、RNA聚合酶Ⅱ及转录共抑制因子HDAC1在启动子上的招募是MED1参与Fulvestrant抑制ER靶基因表达及耐药形成的主要分子机理；HER2与MED1的相互调控环也参与了Fulvestrant耐药机制的形成。这些结果提示,MED1对乳腺癌细胞体外、体内Fulvestrant耐药形成起着关键性作用。3、首次建立了MED1与乳腺癌细胞侵袭转移能力的机制性联系。通过体外、体内实验研究发现,MED1通过调控MMP9表达在ER阳性乳腺癌细胞侵袭转移进程中发挥着决定性的作用。沉默MED1能有效地抑制诱导型乳腺癌细胞体内外的侵袭转移以及MMP9的表达；外源性MMP9过表达质粒可以挽救重建这种被MED1沉默所抑制的细胞侵袭能力；而过表达MED1则可以通过上调MMP9来有效增强乳腺癌细胞的侵袭性。MED1调控MMP9转录的分子机制研究初步揭示：MED1通过调节启动子近端AP-1位点上AP-1、共抑制因子HDAC1和共激活因子CBP的招募,参与AP-1转录复合体的组装以及染色质构象重组过程,从而完成对MMP9的转录调控。总之,这些结果表明,MED1通过对MMP9的转录调控在乳腺癌侵袭转移进程中扮演着非常重要的角色。综上所述,本论文研究初步揭示了MED1在乳腺癌的Fulvestrant治疗耐药和侵袭转移中的贡献作用,并较深入地阐明了其可能涉及的分子机理。MED1可以作为一个很有前景的潜在的乳腺癌分子治疗靶点,通过靶向沉默MED1来克服乳腺癌临床内分泌治疗耐药、控制远端复发转移。更多还原

【Abstract】 Breast cancer is the most common malignancy and one of the leading causes of cancer-related mortality amongst female worldwide. The incidence of breast cancer in China, especially in most big cities, is dramatically increasing nowadays. Pure anti-estrogen fulvestrant has been shown to be a promising estrogen receptor (ER) antagonist for locally advanced and metastatic breast cancer. Unfortunately, a significant proportion of patients developed resistance to this type of endocrine therapy but the molecular mechanisms governing cellular responsiveness to this agent remain poorly understood. Meanwhile, metastasis is the major cause of death associate with breast cancer, and the molecular mechanisms of metastasis of breast cancer also remain unclear. MED1(Mediator subunit1), as a known ER coactivator, plays a crucial role in ER-dependent gene expression and estrogen-dependent breast cancer cell growth, and is associated with the resistance of tamoxifen, another class of endocrine therapy agent, and poor outcome of breast cancer patients. Here, this study investigated the role of MED1in the processes of fulvestrant resistance and metastasis of breast cancer.1. This study successfully constructed Tet-inducible MED1shRNA lentiviral system using pLKO-Tet-On vector and generated breast cancer cell sublines that stably express firefly luciferase and Tet-inducible MED1shRNA (MCF7-tet-shMED1, BT474-tet-shMED1and ZR75-1-tet-shMEDl). The expressions of MED1in all these Tet-inducible cells were significantly down-regulated after Dox (doxycycline) treatment. Consistent with the previous reports, MED1knockdown induced by Dox strongly inhibited the cell proliferation, anchorage-dependent and anchorage-independent colony formation. In a word, these inducible cells are reliable and useful for further studies.2. This study first investigated the role of MEDl on fulvestrant resistance. It was revealed that knockdown of MED1sensitized fulvestrant resistance breast cancer cells (BT474, ZR75-1, MCF7-F) to fulvestrant treatment in vitro. MED1knockdown further promoted cell cycle arrest induced by fulvestrant. Using an orthotopic BT474xenograft mouse model, it was found that knockdown of MED1further significantly potentiated tumor growth inhibition by fulvestrant. Mechanistic studies indicated that combination of fulvestrant treatment and MED1knockdown was able to cooperatively inhibit the expression of ER target genes. Chromatin immunoprecipitation (ChIP) assays further supported a role for MED1in regulating the recruitment of RNA polymerase II, ER and transcriptional corepressor HDAC1on endogenous ER target gene promoter in the presence of fulvestrant. Furthermore, there is a regulator loop between HER2and MED1in controlling fulvestrant resistance of human breast cancer cells. These results demonstrate a role for MED1in mediating resistance to the pure anti-estrogen fulvestrant both in vitro and in vivo.3. This study first established the relationship between MED1and the aggressiveness of ER-positive breast cancer cells. Using Tet-inducible MED1knockdown cells, it was revealed that silencing MED1efficiently blocks the abilities of cell migration and invasion in vitro, as well as the metastasis of breast carcinoma in vivo using orthotopic or i.v. xenograft mice models. Meanwhile, the expression of matrix metalloproteinase9(MMP9) was down-regulated by silencing MED1. Exogenous restoration of MMP9can rescue cell migration and invasion decreased by MED1knockdown. Whereas, over-expression of MED1significantly enhances the migration and invasion of MCF7and BT474cells through increasing MMP9expression. Mechanistically, promoter reporter assays indicated that the proximal AP-1binding site is required for MED1-modulated transcriptional regulation of MMP9. ChIP assays further supported that MED1regulated the assembly of AP-1complex and chromatin remodeling through modulating the recruitment of AP-1, transcriptional corepressor HDAC1and coactivator CBP on the proximal AP-1binding site of endogenous MMP9promoter. This perhaps is the main mechanism for MED1activating MMP9transcription. Collectively, our results reveal that MED1plays a key role on invasion and metastasis of ER-positive breast carcinoma through transcriptional regulation of MMP9.Taken together, this study has demonstrated the key role of MED1on modulating fulvestrant resistance and metastasis of breast cancer. MED1therefore may offer advantageous therapeutic outcome by overcoming both fulvestrant resistance and metastasis of breast cancer. Targeted intervention of MEDl expression may provide a novel therapeutic strategy to inhibit breast cancer endocrine resistance and distant metastasis.更多还原