细胞自噬作用角色的转变在肝癌索拉非尼耐药机制中的研究

The resistance to sorafenib by hepatocellular carcinoma (HCC) greatly affects its clinical efficacy, but the mechanisms remain unclear. The autophagy pathway crosstalks with many key cellular signaling pathways involved in cancer cells, and contributes to cell proliferation, apoptosis and tumor angiogenesis, thus has become a hot spot in cancer research field. We have recently found for the first time that autophagy switches from a cytoprotective function to a death-promoting role and turns to be the type II programmed cell death in sorafenib-resistant HCC cells, accompanied with the downregulated mTORC1, the key protein in autophagy pathway, and increased levels of phosphorylated Akt at ser473 site. It has been reported that mTORC1 activates phosphorylation of Akt through regulating mTORC2. Therefore, we hypothesized that inhibition of mTORC1 and regulation mTORC2 could lead to the activation of Akt and function switch of autophagy by sorafenib, contributating to the molecular mechanism of acquired resistance to sorafenib. The present project aims to further investigate the autophagic molecular signaling pathway, its crosstalk with the PI3K/AKT/mTOR pathway and the feedback regulatory mechanism at cellular levels by applying specific inhibitors, small interfering RNA(SiRNA) and expression vectors targeting the key factors of the pathways. The present project will further clarify the molecular mechanism of resistance to sorafenib in HCC, and help seeking novel therapeutic targets for overcoming the acquired resistance.

肝癌对索拉非尼耐药是影响疗效的严峻问题，而其机制尚未阐明。自噬与多种信号通路交互作用（crosstalk）,影响肿瘤增殖、凋亡和新生血管生成，是近年癌症研究的热点。我们前期首次发现索拉菲尼耐药中自噬功能转变现象，即由保护机制转变为Ⅱ型程序性死亡方式。随着自噬关键靶点mTORC1被抑制，上游Akt ser473磷酸化增强。mTORC1可反馈调控mTORC2，引起Akt Ser-473磷酸化。因此，我们认为索拉菲尼经抑制mTORC1，反馈调控mTORC2，进而激活Akt，转变自噬功能是导致耐药的关键。为验证该假说，本课题依托我们前期建立的耐药研究平台，在细胞和整体水平，以特异抑制剂、SiRNA、表达载体交叉干预，在基因和蛋白层面，对自噬角色转变中起关键作用的PI3K/AKT/mTOR和自噬通路反馈调控机制进行研究。本研究将从新的视角揭示索拉菲尼获得性耐药机理，为逆转肝癌耐药提供新思路和新靶点。

索拉非尼是目前唯一一线治疗药物，但耐药问题较为突出，我们前期研究发现肝癌对索拉非尼耐药过程中PI3K/AKT通路激活同时，其下游自噬通路受到抑制，且自噬从保护性机制变成了自噬性死亡。因此在本项目资助下，我们进一步探讨PI3K/AKT通路激活及其介导的自噬通路在逆转肝癌对索拉非尼耐药中的作用。取得成果如下。1）三氧化二砷（ATO）可协同索拉非尼抑制增殖和诱导肝癌细胞凋亡。其机理为ATO抑制索拉非尼激活AKT同时，激活索拉非尼不可激活内源性凋亡通路。2）肝癌对索拉非尼耐药过程中耐药细胞出现上皮间质转化及多药耐药现象。索拉非尼持续作用下激活PI3K/Akt通路经调控EMT介导肝癌对索拉非尼多药耐药。3）肝癌对索拉非尼耐药过程中PI3K/AKT通路激活与miR-21相关。索拉非尼持续作用经上调miR-21，抑制PTEN，激活p-Akt，抑制自噬性死亡而介导肝癌对索拉非尼耐药。4）双重抑制AKT和c-Met作为二线治疗可协同逆转肝癌对索拉非尼耐药。肝癌对索拉非尼耐药过程中c-met和Akt通路被激活。抑制c-met可抑制Akt激活，但抑制Akt可反馈激活c-met，因此，双重抑制AKT和c-Met可阻断环路协同逆转肝癌耐药。5）Bufalin经依赖于IRE1的内质网应激通路抑制Akt，可抑制索拉非尼对Akt的激活作用，协同索拉非尼促进凋亡，逆转耐药。6）生物信息学分析二线药物Regorafenib与Sorafenib作用靶点差异，筛选逆转耐药新靶点。另外，课题组尚受到其他课题资助，研究过程中人员分配及实验试剂的使用存在交叉，因此发表的论文上也注明了国家自然基金资助和项目编号。截止目前在本项目资助下，课题组共发表论文7篇。培养博、硕士研究生6名。