1、ZEB1通过诱导ER-α启动子甲基化调控乳腺癌抗雌激素治疗耐药的机理研究 2、新型VEGFR2小分子抑制剂YLL54

发布时间：2018-08-14 13:19

【摘要】：乳腺癌是一种雌激素依赖性肿瘤。雌激素通过与雌激素受体结合,激活特异的下游信号通路表达,刺激乳腺癌细胞的增殖,介导了乳腺癌的发生和发展。抗雌激素治疗作为乳腺癌重要的辅助治疗手段,可以明显改善患者预后。然而并不是所有乳腺癌都能采用抗雌激素治疗。对抗雌激素治疗药物的先天性耐药和获得性耐药,都可引起抗雌激素抵抗,导致治疗的失败。因此,明确抗雌激素治疗抵抗的分子机制,将极大的促进乳腺癌抗雌激素治疗的发展,提高乳腺癌患者的治疗效果,改善预后。目前已知雌激素受体ER-α表达下调或功能异常,是抗雌激素治疗耐药的主要原因。而ER-α转录表达的异常与表观遗传调节密切相关。但是ER-α表达下调的具体机制尚不完全清楚。本研究中,我们发现转录因子ZEB1可以通过招募甲基转移酶DNMT3B和去乙酰化酶HDAC1到ER-α基因启动子,促进其发生高甲基化,从而诱导乳腺癌细胞中ER-α表达下调,导致抗雌激素治疗耐药。首先,我们通过在线数据库预测分析发现,在ER-α基因启动子区存在一个大小为267 bp的Cp G岛。利用DNMT3B和HDAC1抗体行染色质免疫共沉淀实验Ch IP证明这个Cp G岛是一个潜在的差异性甲基化调控区DMR。而在我们的前期研究中发现,ZEB1与乳腺癌组织中ER-α的表达呈负相关。并且我们同样通过数据库分析发现这个Cp G岛内包含有两个ZEB1的潜在转录结合元件E2box序列。于是,我们检测了乳腺癌细胞系MDA-MB-231/SUM-159和MCF-7/ZR75-1中ER-α基因启动子甲基化程度,并且通过过表达及沉默ZEB1,从功能上证明了ZEB1对ER-α基因启动子的甲基化调控作用。接下来,我们通过体外实验中使用抗雌激素治疗的代表性药物他莫西芬(tamoxifen)和氟维司群(fulvestrant)处理乳腺癌细胞,证明了ZEB1过表达可以下调乳腺癌细胞ER-α的表达,并且降低乳腺癌细胞对抗雌激素药物的敏感性;相反地,沉默ZEB1的表达可以部分恢复ER-α的表达,并且提高乳腺癌细胞对抗雌激素药物的敏感性。机制研究证明,ZEB1可以同时与甲基转移酶DNMT3B和去乙酰化酶HDAC1在ER-α启动子上形成蛋白复合物,进而诱导DNA发生高甲基化,抑制ER-α的转录,沉默其表达。更重要的是,通过RNA干扰敲低ZEB1表达后可引起ER-α启动子的去甲基化,进而部分恢复ER-α的表达,增加乳腺癌对抗雌激素治疗的敏感性。最后,经过对乳腺癌临床标本的分析,我们发现ZEB1和ER-α的蛋白表达评分呈负相关。在乳腺癌ER-α阴性病例中,ZEB1高表达,并且与ER-α启动子高甲基化呈正相关,而在ER-α阳性性病例中,结果与之相反。在小鼠移植瘤模型中,我们进一步证明了下调ZEB1可以部分恢复ER-α的表达,并提高乳腺癌对抗雌激素治疗的敏感性。综上所述,我们的研究证明了ZEB1是介导乳腺癌抗雌激素耐药的关键因子。ZEB1及其下游信号通路可能作为乳腺癌治疗的新靶点。靶向ZEB1,并联合应用特异的表观调控抑制剂,有望成为克服乳腺癌抗雌激素治疗耐药的新策略。侵袭和转移是恶性肿瘤患者死亡的主要原因,而肿瘤血管生成是其侵袭和转移的重要因素。在肿瘤血管生成过程中,血管内皮生长因子VEGF信号通路发挥了关键的调控作用。靶向血管内皮生长因子受体VEGFR2的小分子抑制剂,作为有效的抗血管新生药物,已经广泛应用于恶性肿瘤的临床治疗。其中代表性药物为治疗晚期肾癌的小分子药物索拉非尼(sorafenib)和舒尼替尼(sunitinib)。虽然临床前研究和初期的临床实验得到了理想结果,但是随着临床的逐渐应用,发现大多数这类药物都存在一些未知的副作用,并且其疗效也需要进一步验证。因此,开发新型而低毒的VEGFR2小分子抑制剂仍然是当前研究的热点。我们在本课题中,首先以sorafenib为药物主体结构,采用计算机辅助设计,生物化学合成的方法,得到25个新型的靶向VEGFR2的小分子抑制剂。接着,以sorafenib为阳性对照,利用转基因斑马鱼(Fli-1:EGFP)模型,对这些化合物的抗血管生成作用进行筛选,得到了一种高效且低毒的VEGFR2小分子抑制剂YLL545。接下来,我们同样以sorafenib为阳性对照,用不同浓度的YLL545处理人脐静脉血管内皮细胞HUVECs。通过CCK8细胞活力实验检测发现YLL545抑制HUVECs细胞增殖的IC50为5.844μM。而进一步用Ed U细胞增殖实验检测发现,YLL545抑制HUVECs的增殖是通过减少细胞S期DNA合成导致的。此外,划痕实验和transwell实验提示YLL545可以抑制HUVECs细胞的迁移和侵袭。重要的是,成管实验和Matrigel胶塞实验证明,与sorafenib相比,YLL545能够更有效地抑制HUVECs细胞的成管能力及其在小鼠体内的血管生成作用。进而,我们需要明确YLL545抑制血管生成的分子机制。通过WB检测发现,YLL545可以明显抑制由VEGF诱导的VEGFR2磷酸化,及其下游信号调节因子的蛋白磷酸化水平,包括细胞外调节蛋白激酶ERK、信号转导子与转录激活子STAT3和雷帕霉素靶蛋白m TOR。此外,利用RT2 Profiler PCR Array,我们还发现,YLL545可以通过VEGFR2非依赖途径抑制其他血管生成相关基因的表达来影响血管生成,其中包括:FN1、TEK、ENG、THBS1和ITGAV。由于乳腺癌细胞同样表达VEGFR2、m TOR、STAT3和ERK,提示YLL545具有潜在的靶向肿瘤细胞的作用。为此,我们选择了乳腺癌细胞MDA-MB-231进行研究。通过CCK8、Ed U和平板克隆形成实验均证明了2.5μM YLL545就能明显抑制肿瘤细胞增殖,并且Annexin V和PI染色证明了YLL545还能促进MDA-MB-231细胞的凋亡。而YLL545抑制正常乳腺上皮细胞和肝上皮细胞增殖的IC50分别是35.83和33.40μM,表明YLL545对正常组织细胞的毒性甚小,其特异性抑瘤作用与细胞毒无关。最后,我们在小鼠移植瘤模型中验证了YLL545抑制血管生成的体内作用。结果显示,口服YLL545 50 mg/kg/d即可抑制乳腺癌移植瘤的血管生成,进而抑制肿瘤生长,抑制率达50%。并且与对照组相比,YLL545实验组小鼠并未出现药物毒副反应。综上所述,作为一种新型、安全、高效的血管生成小分子抑制剂,YLL545有望将来应用于肿瘤,特别是乳腺癌的治疗。
[Abstract]:Breast cancer is an estrogen-dependent tumor. Estrogen stimulates the proliferation of breast cancer cells by binding to estrogen receptors and activating specific downstream signaling pathways. Antiestrogen therapy, as an important adjuvant therapy for breast cancer, can significantly improve the prognosis of patients. All breast cancer can be treated with anti-estrogen therapy. Congenital and acquired resistance to anti-estrogen drugs can lead to anti-estrogen resistance and lead to treatment failure. Therefore, to clarify the molecular mechanism of anti-estrogen resistance will greatly promote the development of anti-estrogen therapy for breast cancer and improve the treatment of breast cancer patients. The abnormal expression of ER-alpha is closely related to epigenetic regulation. However, the specific mechanism of the down-regulation of ER-alpha expression is still unclear. In this study, we found that the transcription factor ZEB1 can pass through. Recruitment of promoters of methyltransferase DNMT3B and deacetylase HDAC1 to ER-alpha promoters promotes hypermethylation and induces down-regulation of ER-alpha expression in breast cancer cells, leading to resistance to estrogen therapy. First, we found a 267 BP CpG island in the promoter region of ER-alpha gene by online database prediction analysis. Chromatin immunoprecipitation assay with antibodies to DNMT3B and HDAC1 demonstrated that the Cp G island was a potential differentially methylated regulatory region DMR. In our previous study, ZEB1 was found to be negatively correlated with the expression of ER-alpha in breast cancer tissues. Thus, we examined the methylation of ER-alpha promoter in breast cancer cell lines MDA-MB-231/SUM-159 and MCF-7/ZR75-1, and functionally demonstrated the methylation regulation of ER-alpha promoter by ZEB1 overexpression and silencing. Treatment of breast cancer cells with tamoxifen and fulvestrant, a representative antiestrogen drug, demonstrated that ZEB1 overexpression could down-regulate the expression of ER-alpha in breast cancer cells and reduce the sensitivity of breast cancer cells to antiestrogen drugs; on the contrary, silencing ZEB1 expression could partially restore the expression of ER-alpha. Mechanisms have shown that ZEB1 can form protein complexes with methyltransferase DNMT3B and deacetylase HDAC1 on ER-a promoter simultaneously, which induces DNA hypermethylation, inhibits ER-a transcription and silences ER-a expression. More importantly, ZE is knocked down by RNA interference. After the expression of B1, the demethylation of ER-alpha promoter was induced, and the expression of ER-alpha was partly restored, which increased the sensitivity of breast cancer to anti-estrogen therapy. Promoter hypermethylation was positively correlated with ER-alpha in ER-alpha-positive cases, but the opposite was true in ER-alpha-positive cases. In a mouse transplanted tumor model, we further demonstrated that down-regulation of ZEB1 partially restored the expression of ER-alpha and increased the sensitivity of breast cancer to anti-estrogen therapy. ZeB1 and its downstream signaling pathways may be new targets for breast cancer treatment. Targeting ZEB1 and combining with specific inhibitors of epigenetic regulation may be a new strategy to overcome the resistance of breast cancer to estrogen therapy. Vascular endothelial growth factor (VEGF) signaling pathway plays a key role in tumor angiogenesis. As an effective anti-angiogenesis drug, small molecule inhibitor targeting vascular endothelial growth factor receptor (VEGF R2) has been widely used in the clinical treatment of malignant tumors. Sorafenib and sunitinib are small molecule drugs for the treatment of advanced renal cell carcinoma. Although ideal results have been obtained in preclinical studies and initial clinical trials, with the gradual application of these drugs in clinic, some unknown side effects have been found in most of them, and their efficacy needs to be further verified. Therefore, the development of novel and low toxic small molecule inhibitors of VEGFR2 is still a hot research topic. In this project, we first used sorafenib as the main structure of the drug, using computer-aided design, biochemical synthesis methods, to obtain 25 novel small molecule inhibitors targeting VEGFR2. Then, sorafenib as a positive control, the use of. After screening the anti-angiogenesis effects of these compounds in transgenic zebrafish (Fli-1:EGFP) model, a high-efficiency and low-toxicity small molecule inhibitor of VEGFR2, YLL545, was obtained. Next, we treated HUVECs of human umbilical vein endothelial cells with different concentrations of YLL545 and sorafenib as positive control. The IC50 of YLL545 inhibited the proliferation of HUVECs was 5.844 mu M. Further studies using Ed U cell proliferation assay showed that YLL545 inhibited the proliferation of HUVECs by reducing S-phase DNA synthesis. In addition, scratch and Transwell experiments suggested that YLL545 could inhibit the migration and invasion of HUVECs cells. In addition, we need to clarify the molecular mechanism of YLL545 inhibiting angiogenesis. WB assay showed that YLL545 significantly inhibited the phosphorylation of VEGFR2 induced by VEGF, and YLL545 significantly inhibited the angiogenesis of HUVECs. Protein phosphorylation levels of downstream signal regulators, including extracellular regulated protein kinase ERK, signal transducer and activator of transcription STAT3 and rapamycin target protein M TOR, were measured. In addition, using RT2 Profiler PCR Array, we also found that YLL545 could inhibit the expression of other angiogenesis-related genes through the non-dependent pathway of VEGFR2. Because breast cancer cells also express VEGFR2, m TOR, STAT3 and ERK, YLL545 has the potential to target tumor cells. Therefore, we chose breast cancer cell MDA-MB-231 for our study. CCK8, Ed U and platelet cloning experiments have all proved that 2.5 mu YLL545 is effective. YLL545 could also promote the apoptosis of MDA-MB-231 cells. The IC50 of YLL545 inhibited the proliferation of normal mammary epithelial cells and hepatic epithelial cells were 35.83 and 33.40 mu M, respectively, indicating that YLL545 had little toxicity to normal tissue cells, and its specific inhibitory effect on tumor was not related to cytotoxicity. Finally, we validated the inhibitory effect of YLL545 on angiogenesis in vivo in mice transplanted tumor model. The results showed that oral administration of YLL545 50 mg/kg/d could inhibit angiogenesis of breast cancer transplanted tumor, and then inhibit tumor growth, the inhibitory rate was 50%. Moreover, compared with the control group, YLL545 experimental group mice did not show any toxic and side effects. YLL545, as a novel, safe and effective angiogenesis inhibitor, is expected to be used in the treatment of cancer, especially breast cancer.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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