利用DNA修复报告小鼠模型研究肝细胞性肝癌发生发展过程中DNA双链断裂修复能力变化及其调控机制

DNA double strand breaks (DSBs) are the most detrimental type of DNA damage. Two repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), are responsible for fixing the broken ends. If DSBs are not repaired or improperly repaired, they may lead to tumorigenesis. Ironically, previous research has also indicated that tumor cells can take advantage of DNA DSB repair machineries to overcome replication stresses, making these pathways good targets for cancer therapy. However, most of research on tumorigenesis associated DNA DSB repair, and developing therapeutic methods by targeting DNA DSB repair, was performed using in vitro cultured cells. Whether DNA DSB repair changes during tumorigenesis in vivo has not been assessed due to a lack of an appropriate tool. Liver cancer is one of the deadliest cancers among all types of tumors and it is listed as the second leading cause of cancer mortality in the world. The methods of inducing hepatocellular carcinoma (HCC), the major type of liver cancer, in mice have been well established, rendering it an ideal model for studying the change of physiological parameters during tumorigenesis in vivo. Here using our newly developed knock-in mouse models for in vivo analysis of HR and NHEJ efficiency and fidelity we propose to accomplish the following goals: aim 1. to optimize the conditions for analyzing the efficiency of both pathways and NHEJ fidelity in mouse livers using our reporter mice; aim 2. to analyze the change of DSB repair efficiency and fidelity during HCC tumorigenesis; aim 3. to elucidate the regulatory mechanisms of tumorigenesis associated change of DSB repair using mouse models, clinical samples and data mining methods; aim 4. to study whether it is possible to prevent liver tumorigenesis by promoting DNA DSB repair efficiency and stabilizing genomes; aim 5. to study whether it is possible to treat HCC by targeting two DNA DSB repair pathways with small molecules.

DNA双链断裂在所有DNA损伤中最为严重，这种损伤可被两条独立的通路——同源重组（HR）以及非同源末端连接（NHEJ）修复，而这种损伤的发生与修复的平衡与肿瘤发生、维持以及治疗关系紧密。肝癌恶性程度高，预后较差，是导致癌症相关死亡的第二大肿瘤。但是由于缺少合适的小鼠研究工具，在肝癌发生发展过程中，HR和NHEJ的效率及精确性的变化以及相应调控机制尚未被深入研究。申请人计划利用本课题组可用于体内检测HR和NHEJ的报告小鼠模型，结合化合物诱导小鼠肝癌发生的方法，研究肝癌发生过程中HR和NHEJ效率及精确性的变化。基于该现象的研究，申请人计划与临床合作获得大量的肝癌和癌旁标本，并结合TCGA数据库中相关数据，阐述肝癌发生过程中以及发生后HR和NHEJ的变化的调控机制。基于机制研究，申请人将探索是否可通过提高DNA修复能力预防肝癌，以及通过靶向HR和NHEJ治疗肝癌，为临床应用奠定理论基础。

DNA双链断裂修复是双刃剑，高效精确的DNA双链断裂损伤修复既可以防止正常细胞恶性转化，又可以被肿瘤细胞所利用促进其生存能力提升，因此理解肿瘤和正常组织的DNA双链断裂修复的不同可为发展针对DNA修复通路的治疗肿瘤的方法提供理论依据。但是，领域内缺少研究体内DNA双链断裂损伤修复两条通路-同源重组（HR）和非同源末端连接（NHEJ）-的动物模型。因此，本项目中，项目负责人建立了新的研究HR修复通路的报告小鼠模型，并结合已经建立的NHEJ小鼠模型，采用尾静脉高压注射的方法向肝脏中导入了诱导双链断裂发生的I-SceI表达载体，实现了在肝脏中检测HR和NHEJ的研究目标。项目负责人利用化学诱导肝癌发生的方法诱导了这些报告小鼠模型肝癌的发生，基于此，结合上述检测DNA修复的方法，项目负责人发现，相比较正常肝脏组织，肝细胞癌中HR和NHEJ修复均显著上调。结合小鼠和临床组织的进一步机制研究发现，肝癌组织中PARP1和DNA-PKcs表达显著提升。而通过抑制PARP1酶活活性，项目负责人发现肝癌组织中的HR修复效率显著下降。深入的机制研究发现抑制PARP1酶活可抑制ALC1招募至双链断裂损伤位点，从而导致损伤位点核小体密度维持在较高水平，阻止HR修复通路中RPA2以及RAD51的招募，进而抑制了HR修复。项目负责人进一步的临床前研究发现，通过同时抑制PARP1和DNA-PKcs的酶活，可协同抑制小鼠的原位肝癌组织和病人的PDX组织的增殖。这些研究为后续临床治疗肝癌提供了新思路。