新BRCA2调控因子RINT1在DNA同源重组修复及乳腺癌发生中的功能和分子机制研究

Defective DNA homologous combination repair (HR) predisposed to the breast cancer. However, neither the breast cancer tumorigenesis nor the drug resistance of the PARP inhibitor therapy could be comprehensively accounted for the HR deficiency dysregulated by the germline and acquired mutations within discovered susceptible genes. It indicates that some unknown factor and involved mechanism function as regulator imposing on HR signaling pathway. Based on our previous published studies（Nature Comms, 2016; Cell Reports, 2015） we have identified a novel BRCA2 regulator, RINT1. Our preliminary data suggest that RINT1 is involved in DNA damage response and is important for the proper progression of HR signaling pathway. Thus, concerning the published germline mutations of the RINT1, we propose the further study as below to dissect the functional role of RINT1 in DNA damage response, HR signaling regulation and breast cancer tumorigenesis..1. Study the role of RINT1 in response to DNA damage. .2. Study the regulation of BRCA2 by RINT1 and the subsequent outcome in HR repair signaling. .3. Through cellular and xenograft assay, study the function of the RINT1 germline mutation in the DNA homologous repair signaling and its role in the drug response with PARP inhibitor chemotherapy which targeting the defective HR pathway. .Our research would expand our knowledge of the role of RINT1 in DNA damage response, HR signaling and breast cancer tumorigenesis which will contribute to the resolution of the drug resistance of PARP inhibitor chemotherapy as well.

DNA同源重组修复缺陷与乳腺癌发生显著相关，但已知的易感基因突变不能完全解释其肿瘤发生及靶向药物耐药机理，提示有重要分子和调控机制待发掘。我们基于自身前期研究（Nature Comms, 2016; Cell Reports, 2015），鉴定了同源重组修复关键因子BRCA2的新调控蛋白RINT1，初步研究表明其参与DNA损伤应答和同源重组修复。结合乳腺癌病例中生殖系突变报道，本研究拟：1.在细胞水平确证RINT1在DNA损伤应答中的功能定位及调控机制；2.鉴定RINT1/BRCA2互作区段，构建乳腺癌致瘤性RINT1突变的稳定细胞株，通过突变-遗传回补分析解析在细胞水平该复合物调控同源重组修复的分子机制；3.建立基于病理性突变的荷瘤裸鼠模型，评估荷瘤对PARP inhibitor药物的响应，确证乳腺癌病理性RINT1突变与同源重组修复之间功能联系, 完善乳腺癌发生机理为其诊疗提供理论参考

我们根据本项目计划任务书完成了所有研究计划内容，实现了各项研究目标。我们对DNA同源重组修复相关蛋白RINT1在同源重组修复中的分子调控机制及其在乳腺癌肿瘤的发生发展中的功能作用机制开展了研究。. 我们鉴定了同源重组修复关键因子BRCA2的新结合蛋白RINT1。RINT1是一个BRCA2的直接结合蛋白。DNA损伤发生后，RINT1在DNA损伤灶发生募集。RINT1在DNA损伤后增强与BRCA2结合进而调控同源重组修复的进行。RINT1在同源重组修复中的功能受到蛋白激酶ATM/ATR的调控。DNA损伤发生后，RINT1的第521位苏氨酸残基（T521）发生磷酸化促使RINT1在DNA损伤位点处募集，磷酸化的RINT1与BRCA2的相互作用显著增强，进而调控同源重组修复的正常进行。我们的研究还显示，PALB2对于RINT1在DNA损伤位点的募集至关重要。BRCA1-PALB2-BRCA2轴直接调控细胞同源重组修复，因而，我们的研究完成了对RINT1在DNA损伤修复级联通路中的功能定位，即：RINT1是一个DNA损伤修复相关蛋白，参与DNA损伤应答，并通过与BRCA2的相互作用实现对同源重组修复的调控。. 采用基因组编辑技术和基于遗传拯救的实验策略，我们在细胞水平和荷瘤裸鼠模型水平解析了RINT1在乳腺癌发生发展中的致病分子机理。我们发现RINT1在乳腺癌中的生殖系细胞突变导致DNA损伤后细胞的同源重组修复效率显著降低。在荷瘤裸鼠中，病理突变体导致了乳腺癌肿瘤细胞成瘤能力显著增强并且对parp inhibitor敏感度显著提高。这些结果表明，RINT1调控同源重组修复的功能对于乳腺癌的发生发展至关重要，其病理性突变促进了乳腺恶性肿瘤的发生和发展。. 我们的研究是对DNA损伤同源重组修复通路调控机制的新发现，有助于为乳腺癌新型诊断标志物的发现、新型靶向药物的研发以及个性化医疗提供了重要参考。