KSR1与Raf结合调控ERK1/2信号诱导肝癌索拉非尼耐药机制研究

Sorafenib has become the first-line drugs for advanced hepatocellular carcinoma due to blockage of Raf signal, but its objective response rate is only 41.6%, which is probably related to resistance. Our previous series of studies focused on the role of sorafenib in HCC and mechanisms of drug resistance. We found that HCC cells with high level of Cryab often expressed sorafenib resistance (Hepatology, 2013). Recently, we found that KSR1 expression in HCC tissues and cell lines with sorafenib resistance was significantly higher than that of non-resistance group using iTRAQ technology followed by laser capture microdissection,and p-KSR1 nuclear translocation using immunohistochemistry or immunocytochemistry. Existing theory about the role of KSR1 in Raf signal can not explain the reasons for resistance, and the role and mechanism of nuclear translocation of p-KSR1 is not clear.Therefore, we suppose that KSR1 mediate sorafenib resistance in HCC through Raf activation or neuclear translocation after phosphoration. Our aims are to detect expression of KSR1 and p-KSR1 in HCC tissues and analyze the relationship among their expression, progression of HCC and sorafenib resistance. After modification of KSR1 expression in HCC cells we investigate the biological function of HCC cells and the relationship between KSR1 expression and drug sorafenib. Combination co-immunoprecipitation and mass spectrometry, KEGG analysis and pull-down technology, we screen and verify its interacting protein. We construct different domains of plasmid of KSR1 and detect its functional domain. In vivo and in vitro experimental study are use to reveal the role of nuclear translocation of p-KSR1 and its mechanism. Above all, This study will further disclose the role and mechanism of KSR1 in HCC cells from new view of different subcellular location and provide a new target for HCC patients with sorafenib resistance.

索拉非尼因可阻断Raf信号等已成为晚期肝癌治疗一线药物，但其客观反应率仅41.6%，学者认为此与耐药有关。我们前期研究证实高Cryab表达肝癌细胞对其耐药(Hepatology,2013)；最近用激光显微切割后iTRAQ检测等发现KSR1在索拉非尼耐药肝癌组织与细胞系中表达显著高于非耐药组，且免疫组化/荧光显示p-KSR1核表达更为明显，这与p-KSR1通过影响胞浆Raf信号获得功能相饽。为此我们提出假说：KSR1通过Raf或磷酸化后核易位介导肝癌索拉非尼耐药。故我们通过检测肝癌组织KSR1与p-KSR1表达，明确其与索拉非尼耐药关系；调节肝癌KSR1表达，用免疫共沉淀结合质谱筛选、KEGG分析、pull-down及构建KSR1不同结构域质粒等探讨KSR1诱导索拉非尼耐药机制；体内验证。本研究将从KSR1不同亚细胞定位这个新视点揭示肝癌索拉非尼耐药机制，为索拉非尼耐药患者提供新治疗靶点。

索拉非尼是晚期肝癌治疗经典一线药物，耐药是限制其临床应用的瓶颈。我们采用激光显微切割结合iTRAQ检测发现KSR1在索拉非尼耐药肝癌组织表达显著高于索拉菲尼敏感组，免疫组化/荧光显示p-KSR1定位于肝癌细胞的细胞核，高表达KSR1和p-KSR1肝癌患者预后明显差于低表达者；上调KSR1表达促进肝癌细胞生长、侵袭、转移和索拉菲尼耐药，下调KSR1表达抑制肝癌细胞生长、侵袭和转移；免疫共沉淀结合质谱研究显示KSR1与DDX5形成复合物；干扰DDX5表达显著降低肝癌细胞克隆形成能力和迁徙，促进肝癌细胞凋亡，逆转KSR1诱导的索拉菲尼耐药；DDX5在肝癌组织中表达明显高于癌旁，高表达DDX5肝癌患者预后较差；生存分析显示共高表达DDX5、p-KSR1的肝癌患者预后最差。我们的研究揭示了KSR1在肝癌索拉非尼耐药中的作用及机制，KSR1可作为索拉菲尼耐药肝癌患者预测预后的参数和新的治疗靶点。