PAK5/beta-catenin信号通路在肝癌细胞耐药中的作用及机制研究

p21-activated kinases 5 (PAK5) is a serine/threonine kinase and has recently attracted considerable attention as that it is found closely related with carcinogenesis and tumor progression. Our previous study showed that PAK5 could promote the anti-apoptosis properties and enhance the resistance to cisplatin of hepatocelluar carcinoma cells, but the exact regulatory mechanism is unknown. Our preliminary data showed that PAK5 could promote the expression of ABCB1 and the phosphorylation of beta-catenin. On the basis of these findings, we propose the hypothesis that PAK5 phosphorylates beta-catenin and promotes its nuclear localization. The beta-catenin in nuclear together with TCF can bind to the promoter of ABCB1 and activate the transcription of ABCB1, ultimately induce the drug resistance of hepatocelluar carcinoma cells. Thus, by using hepatocelluar carcinoma specimens, cultured hepatocelluar carcinoma cells and nude mice, we intend to study the mechanism underlying the PAK5 mediated drug resistance to hepatocelluar carcinoma at molecular, cellular and animal levels. Our expected results will not only elucidate the drug resistance mechanism and discover a new signal pathway account for of drug resistance to hepatocelluar carcinoma, but also provide valuable idea for finding potential targets for molecular therapy of hepatocelluar carcinoma.

PAK5是一类丝氨酸/苏氨酸蛋白激酶，近年来的研究发现PAK5与肿瘤的发生发展密切相关。我们前期研究结果显示PAK5能增强肝癌细胞的抗凋亡功能并且产生对顺铂的抗性，但具体作用机制及其调控模式尚不清楚。我们初步研究发现PAK5可以促进耐药基因ABCB1的表达和beta-catenin的磷酸化，据此提出科学假说：PAK5通过磷酸化其底物beta-catenin，进而促进其核转位，入核的beta-catenin与TCF形成转录复合物进一步结合到ABCB1启动子上激活其转录从而引起肝癌细胞对药物的耐受。. 本项目拟以肝癌标本、培养的细胞及裸鼠为研究对象，从分子、细胞与整体水平研究PAK5介导肝癌细胞耐药的机制。预期研究结果将阐明肝癌耐药的机制，揭示PAK5-beta-catenin通路是调控肝癌细胞耐药的新的信号通路；为肝癌的分子靶向治疗提供理论依据和治疗靶点。

PAK5是一类丝氨酸/苏氨酸蛋白激酶，近年来的研究发现PAK5与肿瘤的发生发展密切相关。但是，PAK5是否与HCC的耐药性有关并未有研究报到，因此在本课题中我们探索了PAK5是否能够介导HCC的多重耐药性（MDR）。首先我们使用免疫组化来分析MDR的关键调节蛋白ABCB1和PAK5的表达，并且构建了具有多重耐药性并表现出癌症干细胞特征的HCC细胞株。之后我们使用蛋白质印迹，染色质免疫沉淀以及共聚焦成像研究PAK5是否促进耐药HCC细胞系中β-catenin的磷酸化和核转位。最后我们利用小鼠移植模型来验证PAK5和ABCB1在体内肿瘤生长中的作用。结果表明，与邻近的正常组织相比，HCC组织中PAK5和ABCB1的表达均显著较高。我们证明PAK5可以促进耐药HCC细胞株中β-catenin的磷酸化和核转位，进而可以增强ABCB1的转录活性。并且验证了在体内沉默PAK5和ABCB1的表达能够抑制肿瘤的生长。此外，本研究表明在耐药HCC细胞株中PAK5启动子甲基化降低，这可能导致了PAK5的异常表达。总之，PAK5介导的β-catenin的磷酸化和核转位促进了ABCB1的表达并促进了HCC的耐药特性。因此，这些数据表明PAK5是肝癌耐药的主要调节蛋白。我们的研究结果初步阐明了肝癌耐药的机制，揭示了调控肝癌细胞耐药的新的信号通路；为肝癌的分子靶向治疗提供理论依据和治疗靶点。