FBXL7通过泛素化降解PFKFB4调控非小细胞肺癌糖代谢的功能及机制研究

代谢失调是肿瘤的重要标志，其机制尚未完全阐明。项目初步验证E3连接酶FBXL7在非小细胞肺癌表达下调，高表达FBXL7抑制癌细胞的增值、迁移及糖代谢；串联亲和纯化-质谱法鉴定糖酵解限速酶PFKFB4是FBXL7潜在底物，FBXL7与PFKFB4相互作用并介导其降解；FBXL7在低氧环境中下调，ChIP数据库显示FBXL7启动子有EZH2结合，并且敲低EZH2上调FBXL7。我们推测：在非小细胞肺癌中，FBXL7受EZH2转录抑制而表达下调，致使PFKFB4表达升高，促进非小细胞肺癌糖代谢重编及恶性进展。拟利用co-IP、泛素化分析等实验阐明FBXL7降解PFKFB4的分子机制；细胞与移植瘤模型中明确EZH2/FBXL7/PFKFB4通路对非小细胞肺癌糖代谢及恶性进展的影响；检测FBXL7表达在非小细胞肺癌中与EZH2、PFKFB4的相关性及临床意义，为非小细胞肺癌诊断与治疗提供新的策略。

Metabolic disorder is a hallmark of cancer, and its mechanism remains to be demonstrated. Our preliminary data of this study showed that the expression of E3 ligase FBXL7 was downregulated in non-small cell lung cancer specimens compared to normal lung tissues. Functionally, ectopic FBXL7 suppressed cancer cell proliferation, migration and glucose metabolism. Mechanistically, we identified PFKFB4, a crucial kinase of glycolysis pathway identified by Tandem affinity purification(TAP/MS), as a potential substrate of FBXL7, and interacted with and was degraded by FBXL7. In addition, FBXL7 was downregulated in non-small cell lung cancer cells in response to hypoxia. In ChIP database, we identified there exists a binding site of EZH2 transcriptional suppressor in FBLX7 promoter, and silence of EZH2 increases FBXL7 mRNA level in non-small cell lung cancer. Based on the results and other related reports, we make the hypothesis: EZH2 is overexpressed in non-small cell lung cancer, which transcriptionally represses FBXL7 expression, resulting in the downregulation of FBXL7. The reduced FBXL7 is unable to efficiently degrade PFKFB4 and leads to the upregulation of PFKFB4 in non-small cell lung cancer cells. Increased PFKFB4 improves the reprogramming of glucose metabolism in non-small cell lung cancer cells and hence the malignant progression in non-small cell lung cancer. To validate our hypothesis, Co-IP, ubiquitination assay, etc. will be used to identify the molecular mechanisms of FBXL7 reducing PFKFB4 protein level through ubiquitination-mediated degradation. We will confirm the roles of EZH2/FBXL7/PFKFB4 pathway in repressing the glucose metabolism, proliferation and migration of non-small cell lung cancer cells in vitro and in vivo assay. We will also reveal the clinical importance of the expression of FBXL7 and its correlations with EZH2 and PFKFB4 in non-small cell lung cancer specimens, which might provide a new strategy for diagnosis and therapy of non-small cell lung cancer.