以同源重组为靶点的胆囊癌个性化化疗研究

Homologous recombination(HR) plays an important role in drug resistance in cancer chemotherapy. Based on our prophase research, it was found suppression of autophagy by chloroquine(CQ) sensitizes 5-fluorouracil-mediated cell death in GBC cells, 5-fluorouracil (5-FU) treatment resulted in a general increase of the apoptotic rate and G0/G1 arrest of gallbladder carcinoma(GBC) cells, and the effect was potentiated by CQ pre-treatment. Preliminary experiment shows the autophagy inhibition by CQ diminishes HR, resulting in the net accumulation of mutations and chromosomal aberrations that cause genomic instability..On the other hand, when both the discovery and prevalence screens were combined, the overall mutation prevalence in GBC for breast cancer susceptibility gene 1(BRCA1) associated protein-1(BAP1) was 6%, indicating BRCA1-BAP1 complex might loss function on account of inactivating mutations in GBC, leads to a deficiency in HR and resistance to chemotherapy. Our data of the immunohistochemical staining with BAP1 also supports this conclusion. While it has demonstrated that HR-deficient cancer cells harboring BRCA1/2 mutation, are hypersensitive to poly(ADP-ribose) polymerase(PARP) inhibition (e.g. olaparib) based on the theory of synthetic lethality, a novel targeting HR personalized chemotherapy of human gallbladder carcinoma might be feasible..Here we develop a green fluorescent protein(GFP)-based reporter system to quantitatively detect HR efficiency and will directly measure HR proficiency and test the sensitization to chemotherapeutics after treatment with autophagy inhibitor or PARP inhibitor according to the HR efficiency in this study. We attempt to establish the liver metastasis model of GBC in nude mice, take the strategy of using autophagy inhibitor or PARP inhibitor to increase the clinical efficacy of 5-FU. It is believed that understanding the molecular mechanism underlying the chemotherapeutic resistance of GBC may provoke the translational studies that ultimately will provide the clinical evidence for treating GBC with a novel chemotherapy.

同源重组（HR）在肿瘤化疗抵抗中发挥关键作用。前期研究发现自噬抑制剂氯喹（CQ）能提高氟尿嘧啶（5-FU）对胆囊癌细胞的化疗效果，增加G0/G1期细胞数，预实验提示CQ可能通过下调HR途径，增加DNA错配修复发生率，与5-FU发挥协同作用。乳腺癌1号基因（BRCA1）的结合蛋白-BRCA1相关蛋白1（BAP1）在胆囊癌病人中较高的突变率及免疫组化结果提示部分病人中BRCA1-BAP1复合体可能存在功能缺失性突变导致HR缺陷，无法通过CQ下调HR的化疗增敏手段治疗，需要寻找新的靶点。基于合成致死理论，上述HR缺陷细胞可用聚腺苷酸二磷酸核糖转移酶抑制剂（PARPi）药物治疗，实现以HR为靶点的胆囊癌个性化化疗。.我们的系统可定量研究HR过程，本课题针对HR正常或缺陷胆囊癌细胞分别以CQ或PARPi尝试提高5-FU化疗效果，利用胆囊癌异体原位移植肝转移裸鼠模型评估其疗效，建立胆囊癌新型化疗技术

目前对胆囊癌的防治研究仍处于起步阶段，临床治疗多采用手术为主的综合治疗，中晚期患者常失去手术机会。胆囊癌对许多传统的化疗药物不敏感，化疗效果受病人个体差异影响较大，但术后化疗仍有改善预后的意义，且可能是很多病人唯一能得到的治疗。本项目中FAM（氟尿嘧啶+阿霉素+丝裂霉素）药物组合是现有治疗包括胆囊癌在内消化道肿瘤的常用化疗方案之一，然与其他胃肠道肿瘤相比，FAM对胆囊癌的化疗敏感性欠佳。本课题基于个体化化疗策略，利用已有的同源重组（HR）相关指标评估胆囊癌细胞内HR是否存在缺陷，依据HR正常或HR缺陷分别以抑制自噬和抑制聚腺苷酸二磷酸核糖转移酶（PARP）处理，在细胞和动物水平证实上述处理可有效提高FAM化疗效果，建立了胆囊癌新型化疗技术。.主要研究成果包括：1）构建稳定表达HR报告系统的胆囊癌细胞模型，选取在HR修复过程中起关键作用的基因并验证评估；2）抑制自噬显著增强FAM对HR正常的胆囊癌细胞的促凋亡和细胞周期的阻滞作用；3）PARP抑制剂Olaparib显著增强FAM对HR缺陷的胆囊癌细胞的杀伤作用；4）动物实验验证胆囊癌个体化FAM化疗策略的有效性。