通过BRCA1非依赖的DNA同源重组修复中关键分子改善三阴性乳腺癌治疗敏感性的研究

Triple negative breast cancer (TNBC) is commonly occurs in young women with high malignancy and poor prognosis. In contrast to breast cancer with ER+, PR+ and/or HER2+, TNBC often shows breast cancer suppressor gene 1 (BRCA1) deficiency and is resistant to endocrine therapy or anti-HER2 targeted therapy. Thus far, there is lack of effective chemotherapy or targeted therapy for TNBC. As BRCA1 plays a critical role in regulation of homologous recombination (HR)-mediated DNA repair pathway, recent studies reveal that BRCA1-mutant cells can be treated through a 'synthetic lethal' approach with PARP1 inhibitors (PARPi) that inhibit PARP1 mediated single-strand break repair (SSBR)/base excision repair (BER). Although there is a high response rate of BRCA1-mutant cells to treatment with PARPi in experimental systems, a substantial fraction of TNBC is still resistant to these agents during clinic trials. To better understand the mechanism(s) by which TNBC is resistant to PARPi, in this proposed study we will establish an in vitro HR/DNA repair system to examine BRCA1-deficient cells that acquire resistance to PARPi. We will examine if BRCA1-deficient cells acquire resistance to PARPi via 53BP1 deficient BRCA1-independent pathway to partially correct their defect in HR-mediated DNA repair. We will examine 53BP1 gene mutation and/or 53BP1 RNA and protein abnormal expression in TNBC samples and reveal their clinic significance. We will identify new molecular target(s) in which inhibition of its function results in BRCA1/53BP1-deficient TNBC cells sensitive to PARPi again. These studies will illustrate the potential mechanism(s) underlying TNBC resistant to PARPi via 53BP1 deficient BRCA1-independent HR-mediated DNA repair, and provide a new strategy for eradicating TNBC cells. Thus, our studies will help to improve current breast cancer treatment and have significant impact on breast cancer research.

三阴性乳腺癌（TNBC）好发于年轻妇女，恶性度高、治疗手段有限、预后差，多数存在BRCA1的缺失或产物异常。PARP1抑制剂是目前较有前景的靶向药物，但治疗效果仍不理想。其原因可能是TNBC细胞对PARP1抑制剂和BRCA1缺失造成的协同致死效应产生耐受。本项目将建立体外DNA同源重组修复检测系统，明确PARP1抑制剂对BRCA1缺失53BP1正常的TNBC细胞的致死效应，以及BRCA1、53BP1共同缺失的TNBC细胞对PARP1抑制剂的耐受性；通过检测53BP1在TNBC中的基因表达与蛋白功能的异常，明确其临床意义；筛选鉴定使BRCA1和53BP1共同缺失的TNBC细胞对PARP1抑制剂再次敏感的新分子靶点并研究其潜在的分子机制。本研究将为临床治疗TNBC提供理论基础与解决方案，并为发掘有效的干预靶点提供新思路，最终有望提高TNBC的治疗效果，改善其不良预后。

PARP抑制剂在BRCA1/2突变的乳腺癌和卵巢癌中显示出良好的疗效，这种高敏感性的原因是由于“协同致死”效应。BRCA1/BRCA2是参与细胞DNA损伤后同源重组修复（HR）的关键分子，而PARP-1则是介导DNA单链损伤后碱基切除修复过程的重要分子，二者具有协同作用。53BP1缺失是BRCA1突变细胞对PARP抑制剂的耐药的机制之一。53BP1缺失可逆转BRCA1缺失细胞中的部分HR缺损。ATM也是DNA双链损伤应答过程中的重要调节分子。细胞内出现DNA损伤之后，ATM迅速自磷酸化（重要标志位其丝氨酸1981位点磷酸化）,并磷酸化一系列下游底物，包括细胞周期检测点激酶Chk2、DNA修复相关分子BRCA1和转录因子p53等。ATM异常见于多种实体肿瘤和造血系统肿瘤。由于ATM与BRCA1一样，也参与PARP抑制剂导致的DNA损伤的修复过程，且可能主要是通过同源重组的方式。我们的研究使用PARP抑制剂Olaparib和ATM抑制剂Ku-55933处理三阴性及非三阴性乳腺癌细胞。结果表明ATM抑制剂能够增强PARP抑制剂在三阴性与非三阴性乳腺癌细胞中的细胞毒作用，表现在生长抑制、克隆形成能力的抑制和促进凋亡上；二者的协同作用在三阴性乳腺癌细胞系中似乎更加明显。此外，Olaparib和Ku-55933还能联合诱导γ-H2AX发生核聚集，提示联合用药增加了对DNA的损伤，特别是增加了双链损伤。利用慢病毒构建的稳定敲降53BP1细胞系中，Olaparib和Ku-55933以及二者联合使用对细胞的增殖抑制作用及对细胞克隆形成的抑制作用均下降，提示53BP1表达降低能够逆转ATM抑制剂与PARP抑制剂联合处理细胞的细胞毒作用。.以上实验结果说明PARP抑制剂Olaparib和ATM抑制剂Ku-55933对三阴性乳腺癌和非三阴性乳腺癌细胞系具有一定的杀伤效果，并且二者联合使用能够对乳腺癌产生协同致死效应，明显地提高杀伤细胞的作用。同时，我们还发现三阴乳腺癌细胞系对Olaparib和Ku-55933以及二者的联合使用的反应更为敏感。此外，本研究首次发现了53BP1功能缺陷能够导致乳腺癌细胞对Olaparib和Ku-55933以及二者的联合使用产生一定程度的耐药现象。这些结果将为临床治疗乳腺癌提供一定的理论指导。