p53依赖的放射诱导BRCA1出核增强PARP抑制剂敏感性研究

Poly (ADP-Ribose) Polymerase (PARP) inhibitor has shown to exhibit high selectivity in eliminating BRCA1 or BRCA2 mutated tumor cells while maintaining minimal toxicity in normal tissues. Preliminary data has demonstrated that the subcellular location of BRCA1 affects the DNA repair efficiency and sensitivity to PARP inhibitor. There is also evidence that radiation treatment can induce BRCA1 nuclear export, reducing the DNA repair efficiency and sensitize PARP inhibitor. Since BRCA1 nuclear export induced by radiation is dependent on the interaction of p53 and BRCA1, this project aims to study the mechanism and time pattern of p53 and BRCA1 interaction, and to locate domains associated with the p53 and BRCA1 binding interaction. In addition, the study also targets the interaction capacity of p53 common mutations with BRCA1 and their impact on radiation induced BRCA1 nuclear export and PARP inhibitor sensitivity. Lastly, the project verifies that the mutations occurred in the p53 and BRCA1 binding domain would have significant effect on radiation induced BRCA1 nuclear export and PARP inhibitor sensitivity. The results would be used to predict whether the p53 mutation pattern in breast cancer tumor tissue would affect radiation induced BRCA1 nuclear export and PARP inhibitor sensitivity.

PARP抑制剂高度选择性杀伤携带BRCA1或BRCA2突变的DNA修复功能缺陷肿瘤细胞，对正常组织毒性很小。本人前期研究表明BRCA1在细胞内的定位影响DNA修复功能及PARP抑制剂敏感性；放射可诱导野生型BRCA1出核，使细胞的DNA修复能力减低，从而增强PARP抑制剂敏感性。因放射诱导的BRCA1出核依赖于p53与BRCA1蛋白的正确结合，本项目拟研究放射后p53与BRCA1蛋白结合的机制和时间规律，p53与BRCA1蛋白结合的结构域。探讨乳腺癌常见p53突变型与BRCA1蛋白的结合能力，及其对放射诱导BRCA1出核和PARP抑制剂增敏作用的影响。验证p53与BRCA1蛋白结合结构域范围内的p53突变将会影响放射诱导的BRCA1出核和PARP抑制剂增敏作用。此研究结果将可预测乳腺癌患者肿瘤组织中的p53突变类型是否影响放射诱导的BRCA1出核和PARP抑制剂增敏作用。

我们的前期研究表明BRCA1出核是BRCA1功能完好的散发性乳腺癌细胞成为PARP抑制剂高度敏感细胞的必需机制。放射处理可诱导BRCA1出核并导致同源重组修复缺陷，增强乳腺癌细胞对PARP抑制剂的敏感性。在本课题中我们进一步证明放射诱导BRCA1出核及PARP抑制剂的联合致死作用依赖于野生型p53功能，转染p53SiRNA敲低MCF7细胞p53表达下降，或者在MCF7细胞稳定表达HPV-E6导致p53不表达，均可减少放射诱导的BRCA1出核作用，导致放射与PARP抑制剂的联合致死作用减弱。携带p53突变型的乳腺癌细胞系比携带p53野生型的乳腺癌细胞系的放射与PARP抑制剂的联合致死作用弱。放射与PARP抑制剂的联合致死作用依赖于p53与BRCA1蛋白的正确结合，与p53的转录功能无关。我们发现放射后2小时，胞核BRCA1和BARD1表达增加，放射后6小时开始，p53与BRCA1结合增加，胞核BRCA1和BARD1表达减少，胞浆表达增加，放射后24小时胞浆BRCA1达到高峰。我们的结果表明p53和BRCA1的结合促进BARD1与BRCA1的分离并暴露BRCA1的NES结构域，从而促进了BRCA1出核。我们发现调节放射诱导BRCA1出核相关的BRCA1与p53结合的位点为p53中部251-300aa之间。肿瘤组织中携带p53中部251-300aa之间的突变将影响放射处理诱导BRCA1出核导致的同源重组修复缺陷,不能增强PARP抑制剂敏感性。小分子化合物APR-246能够逆转p53突变，恢复p53突变型转录活性，明显增强携带突变型p53乳腺癌细胞放射与PARP抑制剂的联合致死作用。