NOD2-RIPK2途径通过调节细胞自噬而促进化学物诱导的肺癌发生

Chronic inflammation was essential to tumor initiation and progression. NOD2, a pattern recognition receptor, played complicated roles in inducing innate and adaptive immunity and was also involved in tumorigenesis. In this proposal, we hypothesize that NOD2 activation by chemical carcinogens will produce a tumor-promoting microenvironment in lung through receptor-interacting protein kinase 2 (RIPK2) pathway, and then augment tumor formation by inhibiting autophagy. Firstly, two mouse lung cancer models induced by urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT), respectively, will be prepared to show the dynamic expressions of mRNAs/proteins of NOD-like receptors. Then, NOD2-knockout and RIPK2-knockout mice will be used to investigate the impact of NOD2-RIPK2 pathway on chemically induced lung carcinogenesis. Cancer associated signaling pathways and biomarkers will be detected in the lung tissues and carcinomas of both wild-type, NOD2-KO and RIPK2-KO mice. The regulation of autophagy by NOD2-RIPK2 cascade will be investigated in HEK293 or lung carcinoma cell lines. At last, the target autophagy proteins of NOD2 signaling will be found and the physical interaction between target and NOD2 signal molecules will be discovered. To our knowledge this is the first time the hypothesis “NOD-like receptors activation augment chemically induced lung tumor formation by inhibiting autophagy” has been proposed. We hope this study will be helpful for new drugs design and treatment of lung cancer.

慢性炎症在肿瘤发生进展中起重要作用。NOD2是一种模式识别受体，可诱导固有和获得性免疫反应，参与了多种肿瘤发病。本研究在前期基础上提出假说：在化学物诱导的肺癌发生过程中存在NOD2的活化，NOD2通过RIPK2-NF-kB途径促进慢性炎症，抑制肺细胞自噬，最终促进肿瘤形成。本研究首先建立乌拉坦和MCA-BHT诱导的两种小鼠肺癌模型，观察自噬标志基因和NOD样受体的动态变化；随后在NOD2和RIPK2敲除小鼠上观察该通路敲除对肺癌发生的影响；在组织水平探讨NOD2通路敲除对小鼠肺癌发生中细胞生长、代谢、自噬的影响；随后建立NOD2-RIPK2过表达/敲减的细胞模型，观察该通路对自噬的影响，筛选其调节的自噬靶基因；最后在细胞模型上探讨NOD2-RIPK2通路与上述自噬靶基因的蛋白相互作用，分析NOD2对细胞自噬的调节机制。本研究的完成，将对炎症和肿瘤的关系提出新见解，为防治肺癌提供新靶点。

本课题研究了NOD2-RIPK2信号途径在肺癌发生发展中的作用。取得的主要成果有：发现乌拉坦诱导的小鼠肺癌模型中存在NOD2受体及下游炎性因子高表达；发现RIPK2基因敲除可显著抑制乌拉坦诱导的小鼠肺癌；发现NOD2-RIPK2基因敲除有利于小鼠肺癌皮下移植瘤生长；建立了过表达/基因敲除NOD2-RIPK2的肺癌细胞株；发现过表达NOD2-RIPK2可促进肺癌细胞生长、迁移，而基因敲除NOD2-RIPK2可抑制肺癌细胞生长、迁移；发现RIPK2可抑制细胞自噬；采用转录组学、蛋白质组学和非靶代谢组学分析了NOD2-RIPK2过表达/基因敲除对肺癌细胞分子变化的整体影响；初步确定IL6-JAK-STAT3和CXCL8为NOD2-RIPK2调节肺癌发生发展的靶点；在22个候选基因中，筛选出TNFRSF10C、PRKCDBP和EPAS1三个基因，发现其启动子甲基化在肺癌组织显著上调；对NOD-RIPK2信号在TCGA数据集进行了泛癌分析和免疫浸润分析。