Aurora-A磷酸化及降解BRCA2促进染色体不稳定性和卵巢癌发生的机理研究

Chromosomal instability (CIN) is the major driver in development of malignancy. Both Aurora-A and BRCA2 regulate cell mitosis, but their abnormal expression promotes cell chromosomal aneuploidy or polyploidy, which causes chromosomal instability and tumorigenesis, but the underlying mechanism is unclear. Our previous study revealed that Aurora-A binds to BRCA2 and inhibits the expression and localization of BRCA2 at midbody during cytokinesis, which induces abnormal cytokinesis and increases cell multinuclearity, resulting in chromosomal polyploidy and ovarian tumorigenesis. Thus, we hypothesize that Aurora-A may regulate the expression of BRCA2 through phosphorylation and ubiquitination-mediated degradation at the end of cell mitosis to control chromosomal instability and tumorigenesis. This project is designed to use ovarian cancer as a model, at the different levels in molecules, cells, animals, and human tissues, to investigate the effective sites and mechanism of BRCA2 phosporylation and ubiquitnation-mediated degradation driven by Aurora-A, from which to reveal the regulatory mechanism of chromosomal instability and ovarian cancer formation and development. The fulfillment of this project may provide theoretical evidences and clinical applications for effective diagnosis and targeted therapy of Aurora-A-associated ovarian cancer.

染色体不稳定性（CIN）是恶性肿瘤发生的主要原因。Aurora-A 和BRCA2调节细胞有丝分裂，其异常表达促进细胞染色体非整倍性或多倍性，诱导染色体不稳定性和肿瘤发生，但其作用机制十分不清。本课题组在前期研究中发现过量的Aurora-A 与BRCA2结合并抑制BRCA2在胞质分裂中间体的表达和定位，诱导细胞胞质分裂异常和细胞多核性增加，导致染色体多倍性/非整倍性和肿瘤形成。我们推测Aurora-A很可能在细胞有丝分裂末期，通过磷酸化和促泛素化降解作用调节BRCA2 蛋白表达，因而调控染色体不稳定性和肿瘤发生。本课题拟以卵巢癌为模型，从分子、细胞、动物和人体组织水平分别研究Aurora-A磷酸化和促泛素化降解BRCA2的作用位点和机制，并揭示其对细胞染色体不稳定性和卵巢癌发生、发展的调节规律。本课题的实施有望为Aurora-A相关卵巢癌的有效诊断和靶向治疗提供一定的理论依据和临床应用基础。

染色体不稳定性（CIN）是恶性肿瘤发生的主要原因。Aurora-A 和BRCA2调节细胞有丝分裂，其异常表达促进细胞染色体非整倍性或多倍性，诱导染色体不稳定性和肿瘤发生，但其作用机制十分不清。本课题组在前期研究中发现过量的Aurora-A 与BRCA2结合并抑制BRCA2在胞质分裂中间体的表达和定位，诱导细胞胞质分裂异常和细胞多核性增加，导致染色体多倍性/非整倍性和肿瘤形成。在拿到本项目资助后，我们立即将BRCA2全长cDNA分为16个片段进行大肠杆菌的诱导表达，并利用原核细胞中表达和纯化的Aurora A进行了大量的体外磷酸化检测，发现BRCA2氨基端第一个片段有Thr/ser磷酸化现象，并用商业够买的Aurora A激酶进行了ELISA和Western blot测定，最后将BRCA2-1与Aurora A反应的产物进行磷酸化质谱分析，发现Thr10有强烈的磷酸化，而这个磷酸化位点目前没有任何文献报道，该磷酸化位点的突变体已经构建，正在检测其磷酸化位点的功能（包括BRCA2泛素化降解）及其对细胞基因组不稳定性、卵巢癌发生、发展以及化疗抵抗的。本课题的研究结果有望为Aurora-A相关卵巢癌的有效诊断和靶向治疗提供一定的理论依据和临床应用基础。