14-3-3η在肝癌索拉非尼抵抗中的作用机制及三氧化二砷对其靶向干预研究

Intensively study the mechanism of the sorafenib resistance and looking for the precise intervention mode to improve the overall outcome of hepatocellular carcinoma (HCC) is one of the major problems to be solved urgently. Based on our previous studies and the researches worldwide, we suggest a new hypothesis: the HIF-1α/14-3-3η positive feedback loop was activated by hypoxia to induce the sorafenib resistance in hepatocellular carcinoma and vascular endothelial cells, meanwhile, the exosomes of 14-3-3η were released to induce the interaction between the two cells and cascade amplificated the resistance effect. The conformation of PARK2 was changed when the 14-3-3η binding to the site of Y391 in PARK2-E3 ligase was blocked by the target binding of arsenic trioxide (ATO). The ubiquitin was then presented to HIF-1α & 14-3-3η to induce the ubiquitination degraded subsequently and the resistance effect was reverse eventually. This project intends to perform the work in the cell model, animal model, as well as in the population. The expected results can not only reveal the key process of sorafenib resistance induced by the interaction of HCC and the microenvironment, but also can provide theoretical evidence that 14-3-3η as new intervention target during the sorafenib therapy and ATO as a potential targeted drug to reverse the sorafenib resistance.

深入研究索拉非尼抵抗机制，探索精准干预方式，提升肝癌总体疗效是目前临床亟待解决的重大问题之一。我们在聚焦国内外相关研究现状及发展趋势基础上，结合预实验证据，提出科学假说：缺氧微环境激活HIF-1α/14-3-3η正反馈环路，在诱导肝癌和血管内皮细胞产生索拉非尼抵抗同时，释放外泌体14-3-3η诱导二者交互作用，级联放大抵抗效应。三氧化二砷通过与14-3-3η靶向结合，阻断其与PARK2-E3连接酶Y391结合，改变PARK2构象，促成其将泛素呈递至14-3-3η和HIF-1α，导致此二者泛素化降解，最终逆转抵抗效应。本项目拟在细胞、动物和人群水平系统验证科学假说。预期成果不仅能够揭示肝癌与微环境交互作用进而产生索拉非尼抵抗的部分实质和关键过程，亦可为是否能将14-3-3η作为新的索拉非尼治疗联合干预靶标，以及将三氧化二砷作为潜在的14-3-3η靶向药物进而逆转抵抗提供理论和实践支撑。

深入研究Sorafenib抵抗的分子机制，探索精准干预方式逆转抵抗，提升肝癌总体疗效成为临床亟待解决的重大问题之一。本项目在细胞和整体动物水平从反向和正向验证我们提出的科学假说：缺氧微环境激活HIF-1α/14-3-3η正反馈环路，在诱导肝癌和血管内皮细胞产生索拉非尼抵抗同时，释放外泌体14-3-3η诱导二者交互作用，级联放大抵抗效应。三氧化二砷通过与14-3-3η靶向结合，诱导14-3-3η泛素化降解，最终逆转抵抗效应。主要研究结果如下：.[1].构建了肝癌sorafenib耐药的HepG2和HuH7细胞株，发现14-3-3η通过增强细胞的CSCs样特性，诱导sorafenib抵抗。.[2].细胞水平验证了HIF-1α/14-3-3η正反馈环路。.[3].细胞水平验证了14-3-3η能够通过诱导血管内皮细胞的间质表型，继而上调TAFs样特性，导致内皮细胞对sorafenib产生抵抗。.[4].细胞和动物水平验证了外泌体14-3-3η介导肝癌和内皮细胞的Cross-Talk。.[5].细胞和动物水平验证了ATO能够通过与14-3-3η靶向结合，诱导14-3-3η的泛素化降解，逆转sorafenib抵抗。.[6].拓展研究揭示了若干新的肿瘤免疫微环境分子与肝癌耐药复发的关系。.项目研究成果不仅揭示了肝癌细胞与微环境交互作用，进而产生Sorafenib抵抗的部分实质和关键过程，亦为将14-3-3η作为新的肝癌Sorafenib治疗联合干预靶标，以及将ATO作为潜在的14-3-3η靶向药物进而逆转Sorafenib抵抗提供理论和实践依据。