BRAP调控DDR在抑制奥沙利铂诱导肝癌细胞老化及逆转化疗耐药中的作用及机制研究

Oxaliplatin plays an important role in the comprehensive treatment of hepatocellular carcinoma. Recent evidences have demonstrated that oxaliplatin as a double-edged sword toward tumor cells, promoting tumor cell apoptosis while protecting tumor cells from death by inducing cell senescence. Therefore, the control of tumor cell senescence vs. apoptosis will likely be a new approach for hepatocellular carcinoma therapy. As a multifunctional protein, BRAP was initially identified as a BRCA1 associated binding protein. As part of the DNA damage response (DDR) network, BRCA1 was activated to facilitate DNA repair and cell cycle control. Our previous study has confirmed that the level of DNA damage and the number of senescence cells in the BRAP-/- MEF cells were increased. Furthermore, the level of express BRAP was down-regulated in oxaliplatin-resistant hepatocellular carcinoma cells, and the proportion of cell senescence in oxaliplatin-resistant cells was higher than in the parental cells. Based on these findings, we intend to comprehensively analyze the correlation between BRAP expression and cell senescence in hepatocellular carcinoma tissues and the clinical prognosis of patients. To further define the effect of the dose-time effect of oxaliplatin on the induction of cell senescence and apoptosis，and the role of such changes in the drug resistance of hepatocellular carcinoma cells. To explore the role and molecular mechanism of DDR modulated by BRAP in oxaliplatin-induced cell senescence and apoptosis. The success of this study will further deepen the understanding of the role of cell senescence on drug resistance, and provide some theoretical basis to individual chemotherapy strategies for hepatocellular carcinoma patients in the future.

奥沙利铂在肝癌的综合治疗中起到重要作用。奥沙利铂引发的细胞DNA损伤不仅促进细胞凋亡，还可显著诱导细胞老化发生从而保护肿瘤细胞逃避杀伤。如何调控肝癌细胞的老化与凋亡将成为逆转耐药的新策略。BRAP作为一个多功能蛋白，最先被发现是因它能与BRCA1信号肽结合，BRCA1对于DNA损伤有许多潜在的应答性，是DDR通路中的关键蛋白。前期研究证实，在BRAP-/-小鼠的MEF细胞内DNA损伤水平增高、细胞老化增强；并在对奥沙利铂耐药的肝癌细胞中BRAP表达下降，老化细胞比例增高。在此基础上，本项目拟全面分析肝癌组织中BRAP表达与细胞老化及患者临床预后的相关性；进一步明确奥沙利铂诱导肿瘤细胞老化和凋亡的量效关系和时程变化规律，及此种改变在肝癌细胞耐药中的作用；深入探索BRAP调控DDR致肿瘤细胞老化-凋亡相互转化的作用与分子机制。该研究结果将为针对细胞老化为靶点的逆转肿瘤耐药策略提供新的理论依据。

BRAP作为BRCA1结合蛋白在人类泛癌种中呈高表达状态，然而其在肝癌发生发展及耐药中的具体作用尚不清楚。我们首先通过对TCGA数据分析发现肝癌组织中BRAP基因表达水平明显高于正常肝组织，且随着TNM分期和微血管浸润程度升高，BRAP表达水平显著升高；BRAP高表达肝癌患者预后显著差于低表达组。此外，在BRAP高表达的肝癌组织中肿瘤突变负荷（TMB）值显着高于BRAP低表达组，其中，高表达组中TP53基因的突变率显着增加。BRAP的表达与肝癌组织内CD8＋T细胞浸润呈负相关性，与免疫抑制检查点PD-1和CTLA4等的表达显著呈正相关性，这提示BRAP高表达可能促使肝癌的抑制免疫微环境形成。我们进一步在对临床肝癌组织样本的检测中发现癌组织内BRAP蛋白的表达显著高于非癌对照组织；其在TNM病理分期高的癌组织中表达高于低分期的癌组织。此外，在肝癌细胞中的BRAP被敲减时，肝癌细胞的恶性增殖能力呈显著性降低改变。在此基础基础上，本项目又全面分析肝癌和结肠癌组织中BRAP表达与细胞老化的相关性；进一步明确奥沙利铂诱导的DDR在肿瘤细胞老化的量效关系和时程变化规律，及此种改变在肿瘤细胞耐药中的作用。并利用BRAP基因敲除小鼠模型深入探索BRAP调控DDR致肿瘤细胞老化的作用与分子机制，我们在BRAP-/-小鼠的MEF细胞内证实细胞的衰老伴随着持续的DNA双链断裂 (DSB) 损伤和BRCA1表达的增高以及组蛋白H2A单泛素化和多泛素化 (H2Aub)水平增加，升高的H2Aub不仅会启动细胞蛋白酶体介导的H2A蛋白水解，而且还会增加细胞内整个蛋白酶体的负荷，最终引起细胞衰老的发生。本课题组另有研究发现CAF作为胰腺癌微环境中最主要的成分在肿瘤恶性进展中发挥重要作用。由于CAF的异质性导致非特异性的抗CAF治疗并不理想。研究发现BRAP在促癌型CAF中呈高表达，相反在抑癌型CAF中呈低表达；在PSC中过表达BRAP可诱导其向促癌型CAF分化进而增强肿瘤抑制型免疫微环境的形成。以上研究结果将为针对BRAP为靶点抗肿瘤治疗及逆转肿瘤耐药策略提供新的理论依据。