Yap1-ROS信号轴维持肺鳞癌启动细胞放疗抵抗的机制研究

Lung squamous cell carcinoma (LSCC), lack of effective personal therapies, tends to relapse after radiotherapy. To improve the prognosis, the key is to explore the mechanism underlying the radioresistance of lung squamous tumor-initiating cells (TICs). We found that Yap1 mediated lung squamous TICs, thus hypothesizing that the intrinsic mechanism of maintaining the radioresistance of lung squamous TICs is to bind Yap1 to IAP (inhibitor of apoptosis protein), then transcript and activate Nrf2, and downregulate active oxygen species (ROS) to form Yap1/IAP-Nrf2-ROS signaling axis. Establishing mouse models of spontaneous LSCC and Patient Derived Xenograft (PDX) of LSCC, this project aims to 1) explore the role of signaling axis in regulating TICs radioresistance on molecular level by detecting the expression at signal nodes; 2) verify the role of signaling axis in maintaining TICs radioresistance on functional level through overexpression of Lentivirus vector, Crispr/Cas9 knockout and IAP-Nrf2 rescue test; 3) confirm the role of signaling axis from analysis of direct interaction by validating the binding sites of Yap1/IAP, and IAP-Nrf2 using co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP-seq) and electrophoretic mobility shift assay (EMSA); 4) test the clinicopathologic significance of this sinaling axis in the radiotherapeutic effect on LSCC and its prognosis based on clinical cases. The results will be of great significance to further the understanding of the essence and radioresistance of lung squamous TICs.

肺鳞癌缺乏个体化治疗策略，放疗后易残留及复发，阐明肺鳞癌启动细胞(TICs)放疗抵抗机制是改善患者预后的关键。我们发现Yap1介导肺鳞癌TICs放疗抵抗，推测Yap1结合IAP、转录激活Nrf2、下调ROS形成Yap1/IAP-Nrf2-ROS信号轴，是维持肺鳞癌TICs放疗抵抗的内在机制。本项目以自发性肺鳞癌小鼠模型和人源肺鳞癌移植小鼠模型为研究对象，首先，检测各信号节点表达水平，从分子水平验证信号轴调控TICs放疗抵抗；其次，慢病毒过表达、Cas9基因敲除及IAP-Nrf2回复实验，从功能水平验证信号轴维持TICs放疗抵抗；再次，CoIP、ChIP-seq、EMSA技术验证Yap1/IAP、IAP-Nrf2的结合位点，从直接互作分析深度证实信号轴的真实性；最后，从临床病例验证信号轴与肺鳞癌放疗疗效及预后的临床病理意义。研究结果对于加深认识肺鳞癌TICs本质和放疗抵抗机制具有重要意义。

阐明肺鳞癌启动细胞放疗抵抗的分子机制，对改善患者预后具有重要的科学意义。本研究发现：肺鳞癌（LUSC）组织中CD49fhighEpCAMlow与肺腺癌有显著不同的染色比例，且肺鳞癌CD49fhighEpCAMlow亚群高表达Yap1，并显著高表达干细胞转录因子，具有极强的成球能力、成瘤能力及放疗抵抗性，显示出干细胞特性；利用慢病毒载体转染稳定敲减Yap1基因后，可逆转TICs放疗抵抗；CellRox检测结果表明TICs内ROS低于NTCs，而Yap1敲减（Yap1-KD）后TICs内ROS显著升高，显示Yap1-ROS信号轴可调控TICs放疗抵抗。本课题构建了Yap1KI/Trp53KO转基因小鼠，通过病理鉴定获得了自发性肺癌小鼠模型。Yap1 CpG位点中cg19298498、cg22104427、cg25484790和cg05260877甲基化与LUSC患者的预后不良有关，Yap1高表达可能在调节巨噬细胞向M0、M1表型极化、减少CD8+T细胞、活化的CD4+T细胞、滤泡辅助T细胞、NK细胞和活化的树突细胞中发挥作用。公共数据库和自建免疫治疗的肺癌数据集分析，提示Yap1高表达与总生存期（OS）或无进展生存期（PFS）较短相关。本研究阐明了Yap1在肺鳞癌TICs和放疗抵抗调控的重要作用及分子机制。