探索曲妥珠单抗对cyclin D3的调控及其在乳腺癌细胞中的耐药机制

发布时间：2017-12-31 15:31

本文关键词：探索曲妥珠单抗对cyclin D3的调控及其在乳腺癌细胞中的耐药机制　出处：《浙江大学》2017年博士论文　论文类型：学位论文

更多相关文章： 乳腺癌 HER2 曲妥珠单抗 cyclin D3 泛素 ERK

【摘要】：背景:乳腺癌是目前女性发病率最高的恶性肿瘤,其中20%-30%患者伴有HER2基因的异常扩增。曲妥珠单抗的问世显著改善了 HER2过表达的患者的生存期。然而其使用过程中耐药的发生也是目前治疗中面临的重大难题。研究其耐药机制及克服耐药的方式具有重要的科学价值。目的:通过构建曲妥珠单抗耐药细胞模型,探索曲妥珠单抗的作用机制及可能的耐药机制。检测曲妥珠单抗处理后引起细胞周期改变的蛋白分子,阐明曲妥珠单抗对其调控的方式,明确该分子在曲妥珠单抗靶向治疗中的作用及与耐药产生的关系。寻找敏感和耐药细胞中信号通路的差异,探索治疗曲妥珠单抗耐药的新策略。方法:首先我们构建曲妥珠单抗耐药的HER2阳性乳腺癌细胞模型,通过MTS、凋亡实验、细胞周期实验验证其对曲妥珠单抗敏感性的改变。通过western blot方法寻找曲妥珠单抗处理后周期蛋白的变化,通过RT-qPCR及蛋白降解相关抑制剂寻找周蛋白变化的原因。利用TCGA数据库分析cyclinD3mRNA水平与预后的关系。最后我们通过敏感和耐药细胞中信号通路的差异进行分析,利用MTS及克隆形成实验寻找曲妥珠单抗耐受后治疗的新策略。结果:通过细胞周期检测发现曲妥珠单抗可以在敏感细胞中通过下调cyclin D3蛋白表达而诱导G1期阻滞,而曲妥珠单抗不能下调耐药细胞中的cyclinD3蛋白表达。敏感和耐药细胞中cyclinD3 mRNA表达在未发生显著改变,且曲妥珠单抗处理后敏感细胞cyclinD3 mRNA不会发生改变,通过TCGA数据库发现cyclin D3 mRNA水平与HER2阳性乳腺癌患者预后相关性不明显。曲妥珠单抗通过泛素-蛋白酶体途径中的SCF复合体促进cyclin D3蛋白发生泛素化降解。cyclin D3蛋白可以和CUL1蛋白结合,CUL1的缺失可以拮抗曲妥珠单抗对cyclin D3蛋白引起的降解。MEK抑制剂、PKCδ抑制剂、GLS抑制剂、CDK4/CDK6抑制剂在曲妥珠单抗耐受细胞中更为敏感。曲妥珠单抗耐药细胞中可能存在ERK信号通路的异常。MEK抑制剂和CDK4/CDK6抑制剂也能很好的抑制曲妥珠单抗耐药细胞的增殖。MEK抑制剂可能通过mTOR信号通路调控cyclinD3的降解。结论:曲妥珠单抗通过泛素-蛋白酶体系统下调cyclin D3蛋白的表达而诱导G1期阻滞,而在耐药细胞中不会下调cyclin D3蛋白的表达。MEK抑制剂、PKCδ抑制剂、GLS抑制剂、CDK4/CDK6抑制剂可能作为曲妥珠单抗耐药治疗的新药物。创新点:1.阐明曲妥珠单抗通过泛素蛋白酶体途径中SCF复合体促进cyclin D3蛋白降解进而引起细胞G1期阻滞。2.发现曲妥珠单抗耐受细胞中cyclin D3蛋白不受曲妥珠单抗调控。3.Cyclin D3是一个潜在作为曲妥珠单抗治疗有效的蛋白标记物。4.发现MEK抑制剂、PKCδ抑制剂、GLS抑制剂、CDK4/CDK6抑制剂在曲妥珠单抗耐受细胞中更为敏感。可能作为曲妥珠单抗耐药后的新的治疗策略。
[Abstract]:Background: breast cancer is the most common malignant tumor in women. 20% to 30% of the patients were accompanied by abnormal amplification of the HER2 gene. The survival time of patients with overexpression of HER2. However, the occurrence of drug resistance in the course of its use is also a major problem in current treatment. It is of great scientific value to study the mechanism of drug resistance and the ways to overcome it. :. The drug resistant cell model was constructed by triptozumab. To explore the action mechanism and possible drug resistance mechanism of tratuzumab, to detect the protein molecules which caused cell cycle change after treatment with tritozumab, and to elucidate the regulation mode of trotozumab on it. To clarify the role of this molecule in the targeting therapy of tratuzoite and its relationship with drug resistance, and to find out the difference of signal pathway between sensitive and resistant cells. Methods: first of all, we constructed the HER2 positive breast cancer cell model of tratozumab resistance, through MTS, apoptosis experiment. Cell cycle assay was used to verify the change of the sensitivity to tratozumab. The changes of cyclin were detected by western blot method. To find out the cause of the changes of pericylein by RT-qPCR and inhibitor of protein degradation. To analyze the relationship between the level of cyclinD3mRNA and prognosis by TCGA database. Finally, we analyze the relationship between the level of cyclinD3mRNA and prognosis through sensitivity. The difference of signal pathway in drug-resistant cells was analyzed. MTS and clone formation assay were used to search for a new strategy for the treatment of tratozumab after tolerance. Results: by cell cycle detection, it was found that trotozumab could down-regulate cyclin in sensitive cells. The expression of D3 protein induced G1 arrest. The expression of cyclinD3 protein was not down-regulated by tritozumab, but the expression of cyclinD3 mRNA in sensitive and resistant cells did not change significantly. And the cyclinD3 mRNA of the sensitive cells was not changed after the treatment of trotozumab. Discovery of cyclin D3 through TCGA Database. The level of mRNA was not correlated with the prognosis of HER2 positive breast cancer patients. Tritozumab promoted cyclin through the SCF complex in the ubiquitin proteasome pathway. Cyclin D3 protein can bind to CUL1 protein. The absence of CUL1 could antagonize the degradation of cyclin D3 protein induced by tritozumab. CDK4/CDK6 inhibitors are more sensitive in tratuzumab resistant cells. There may be abnormal ERK signaling pathway in tratuzumab resistant cells. MEK inhibitors and CDK4/CDK6 inhibition. Mek inhibitor may regulate the degradation of cyclinD3 through mTOR signaling pathway. Tratozumab induced G1 phase arrest by down-regulating the expression of cyclin D3 protein in the ubiquitin proteasome system. However, the expression of cyclin D3 protein was not down-regulated in drug-resistant cells. CDK4/CDK6 inhibitor may be used as a new drug for drug resistance therapy of tratozumab. Innovation: 1. Clarify that tratozumab promotes cyclin through SCF complex in ubiquitin proteasome pathway. The degradation of D3 protein resulted in G1 phase arrest. 2. It was found that the cyclin D3 protein was not regulated by trotozumab in tratuzumab tolerant cells. 3. Cyclin. D3 is a potential protein marker for the treatment of trotozumab. 4. MEK inhibitor was found. PKC 未 inhibitor GLS inhibitor CDK4 / CDK6 inhibitor is more sensitive in tetozumab resistant cells, and may be a new therapeutic strategy for tratuzumab resistant cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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