AMPKα调控DNA双链断裂修复的机制及其在卵巢癌中的功能研究

DNA double-strand breaks repair pathways in mammalian cells are closely related to the occurrence and treatment of cancer. The increased genomic instability and somatic cell mutations caused by DNA double-strand break defect is one of the important causes of cancer. It is essential to dissect the key steps and screen novel regulators of DNA double-strand breaks repair process in understanding the mechanisms of cancer development. AMP-dependent protein kinase AMPKα is the sensor of cellular energy state and core regulator of metabolic pathways, but its involvment in DNA damage response has rarely been reported. Using laser micro-irradiation technique, we induced DNA double-strand breaks in specific regions of nucleus, and subsequently observed rapid recruitment of AMPKα (including AMPKα1 and AMPKα2) to damage sites. Inhibitor treatment assays revealed the recruitment was regulated by poly-ADP-ribosylation (PAR) modification. Moreover, AMPKα knockdown leaded to the persistence of DNA damage marker γH2AX in X-Ray treated cells, which indicated the decreased repair efficiency due to AMPKα deficiency. Based on the above results, in this project, we will focus on the function of AMPKα in double-strand breaks repair, identify its downstream target, elaborate the molecular mechanisms of its function and then explore the pathological significance of this function in ovarian cancer. This project will complement mechanisms of DNA double-strand breaks repair and provide new ideas for understanding the treatment of ovarian cancer.

DNA双链断裂修复通路与癌症发生及治疗密切相关，其缺陷引起的基因组不稳定性增加及体细胞突变是癌症发生的重要原因之一。解析DNA双链断裂修复过程的核心步骤及挖掘该过程的关键调控因子是理解癌症发生机制的重要途径。AMP依赖的蛋白激酶AMPKα是细胞能量状态的感受器及代谢途径的核心调节因子，但其在DNA损伤应答中的作用还少有报道。应用激光微辐射技术在核内定点诱发DNA双链断裂后，我们发现蛋白AMPKα（AMPKα1和AMPKα2亚基）被快速招募至断裂位点，且招募过程受多聚ADP-核糖化修饰（PAR）调控。AMPKα敲低导致X线造成的DNA损伤持续存在，表明其导致细胞修复速率下降。由此，本项目将重点研究AMPKα在双链断裂修复中的作用，鉴定其下游蛋白，解析其发挥功能的分子机制，进而以卵巢癌为模型探讨该功能的病理意义。研究结果将为DNA损伤修复机制提供新内容并为卵巢癌的治疗提供新思路。

DNA双链断裂是细胞内最为致命的损伤类型，负责修复该类损伤的通路包括同源重组（HR）和非同源末端连接(NHEJ)，其中同源重组修复是维持基因组稳定性和保真性的重要机制，该通路的缺陷与癌症发生及化疗耐药密切相关。卵巢癌是女性生殖器官常见的恶性肿瘤，遗传学与癌症基因组图谱研究表明，约50%的卵巢癌细胞中存在同源重组修复通路基因的遗传性或表观遗传改变。因此，鉴定调控通路中的关键蛋白并以此为靶点设计理想的化合物与铂类药物联合用药对卵巢癌的治疗有重要意义。本研究鉴定到了一个新的DNA双链断裂修复的调控因子AMPKα，我们发现AMPKα的两个亚型AMPKα1与AMPKα2都可被招募至DNA双链断裂位点，且PARP1介导的蛋白PAR化是AMPKα招募所必需的。AMPKα1可与PARP1互作，且该互作在诱导DNA双链断裂后增强。在卵巢癌细胞SKOV3及HO-8910中敲除AMPKα1或抑制AMPK激酶活性引起细胞对博莱霉素的敏感性增加；彗星实验发现AMPKα1敲除后细胞有更长的彗星尾巴；并且DNA断裂的标志物H2AX，53BP1及BRCA1蛋白聚集点显著降低，这些都说明AMPKα1调控了细胞的DNA双链修复能力。进一步的功能性实验发现AMPKα1敲低显著影响同源重组修复效率，且重新表达全长AMPKα1可挽救该缺陷。从分子机制上，我们发现AMPKα1敲除显著降低了DNA损伤后ATM的活化，且与多种DNA损伤修复蛋白互作，如RAD50, BRCA1, FANCD2,UNR5等等。AMPKα1在阿霉素耐药的癌细胞中显著上调表达，说明AMPKα1可能通过调控DNA双链断裂修复增强了肿瘤耐药性。本研究发现细胞内的能量感受器AMPKα参与DNA双链断裂的新功能，为AMPKα的激活剂作为卵巢癌治疗靶点的提供了新的思路和可能性。