蛋白乙酰化修饰引起的磷酸烯醇丙酮酸羧激酶同工酶转换在肝癌靶向治疗中的机制研究

索拉非尼是晚期肝癌一线靶向药物，但因癌细胞耐药使其应用受限。我们前期发现，耐药细胞线粒体代谢活跃，替代碳源利用显著增强，发生了代谢重塑。对敏感和耐药细胞的蛋白质组、磷酸化和乙酰化修饰蛋白质组等多组学分析提示，耐药细胞除磷酸化异常外，还有广泛的乙酰化改变并富集于糖代谢相关通路，其中鉴定到磷酸烯醇丙酮酸羧激酶（PCKs）的新位点乙酰化增加。由此推测索拉非尼通过磷酸化->乙酰化的蛋白翻译后修饰调控网络，促进PCKs胞浆和线粒体同工酶类型转换，导致代谢重塑，使细胞获得生存优势并产生耐药。本研究拟1）发现、验证和完善索拉非尼耐药的蛋白磷酸化-乙酰化-代谢重编程调控网络；2）揭示PCKs乙酰化新位点的乙酰化和去乙酰化功能和机制，及其促进PCKs同工酶转换的分子机理；3）体内实验评价PCKs在肝癌发生发展中的作用，及其抑制剂与索拉非尼联用的治疗效果。本研究将为靶向药物的耐药机制和治疗干预研究提供新思路。

As a kind of multi-targeted tyrosine kinase inhibitor, sorafenib is the only first-line targeted agent for advanced liver cancer that enters China's national medical insurance catalog since October 2017. However, its disease control rate is less than 10%, and resistance is an outstanding issue. Recently, it had been found that sorafenib induced central carbon metabolism remodeling. However, how multi-kinase inhibitor affects metabolism remains unclear. Our preliminary findings showed that sorafenib resistance led to an increase in mitochondrial metabolism and utilization of alternative carbon sources. Subsequently, proteomics, phosphor-proteomics and acetyl-proteomics of sensitive and sorafenib-resistant cells were analyzed. Besides abnormal protein phosphorylation, there were extensively altered acetylation in sorafenib-resistant cells, which were enriched in carbohydrate metabolism-related pathways. Among them, two new acetylation sites were identified in phosphoenolpyruvate carboxykinases (PCKs) and their acetylation levels were increased in sorafenib-resistant cells. Therefore, we speculated that sorafenib leads to metabolic remodeling through the post-translational modification regulatory network of phosphorylation and acetylation. And the isoenzyme transition of PCKs plays the critical role. The metabolic reprogramming endows sorafenib-resistant cells with survival advantage by promoting cell proliferation and survival in nutrient-deficient environment, that results in a drug-resistant phenotype. To address the hypothesis, we propose to 1) optimize our protein phosphorylation-acetylation-metabolic reprogramming interactive network of sorafenib resistance via sorafenib-related quantitative proteomics, phosphor-proteomics and acetyl-proteomics analysis and subsequent experimental validation; 2) explore the acetylation and deacetylation mechanisms of our newly identified acetylation sites in PCKs, as well as the molecular mechanism by which acetylation promotes isoenzyme transition of PCKs; and 3) investigate the role of PCKs in hepatocarcinogenesis and the therapeutic effect of its inhibitor combined with sorafenib through in vivo experiments, including using PCK conditional knockout mice model and cell-derived tumor xenografts. The results will help to find the novel metabolic vulnerability sites and therapeutic intervention strategy for sorafenib-resistant liver cancer patients. This study provides a new perspective and direction for the mechanistic study and therapeutic interventions of resistance to targeted drugs.