索拉非尼促进ATG4B磷酸化的分子机制及其在HCC细胞耐药中的意义

保护性自噬是导致肝细胞癌（HCC）细胞对索拉非尼耐药的重要原因，相关机制亟待深入阐明。本课题组在成功建立自噬关键酶ATG4B活性检测新方法的基础上，发现ATG4B的活性升高是引起HCC细胞保护性自噬增强的重要环节，而ULK1对ATG4B的磷酸化修饰可能是导致ATG4B活性升高的关键所在，并据此提出“索拉非尼可能通过调控MTOR与AMPK途径而激活ULK1，进而促进ATG4B的磷酸化，从而使HCC细胞保护性自噬增强并耐药”这一研究假设。本项目拟首先分析MTOR和AMPK在ULK1激活中的协调机制，然后重点解析ULK1促进ATG4B磷酸化的分子机制，揭示ATG4B分子中受磷酸化修饰的位点；随后在细胞系及扩大的HCC临床样本中探讨p-ATG4B水平与索拉非尼耐受的关系，最后在体内外研究阻断ATG4B磷酸化对HCC细胞保护性自噬与索拉非尼耐受的影响，以期为探寻增敏索拉非尼的可能策略提供科学依据。

Protective autophagy plays an important role in the process of hepatocellular carcinoma (HCC) cells resisting to sorafenib, but the associated mechanisms are not well known. Previously, we developed a new system to detect the activity of autophagy-related 4B (ATG4B). Furthermore, our studies show that enhanced ATG4B activity mediated by Unc-51 like kinase-1 (ULK1) may contribute to the protective autophagy genesis and sorafenib resistance in HCC cells. Based on these data, we hypotheses that sorafenib may activates ULK1 through MTOR (mechanistic target of rapamycin) and AMPK (AMP-activated protein kinase) pathways, and ULK1 sequentially phosphorylates and activates ATG4B directly which initiates protective autophagy and drug resistance in HCC cells. In this project, we will focus on several key questions as follow: ① the mechanisms of MTOR- and AMPK-mediated ULK1 activtion; ② the molecular mechanisms of ULK1-mediated ATG4B phosphorylation; ③ the phosphorylation sites in ATG4B which were regulated by ULK1; ④ the relation of the level of phospho-ATG4B and sorafenib resistance in HCC tumor tissues and HCC cells; ⑤ the effect on protective autophagy and sorafenib resistance in HCC after blocking ATG4B phosphorylation in vitro and in vivo. The answers of these questions will provide scientific evidence for designing and developing new strategy to sensitize sorafenib in HCC.