激酶基因TTK通过调节AKT通路促进肝癌发生发展的分子机制研究

Liver cancer, hepatic cellular carcinoma (HCC), is one of the common cancers world-wide, and the mechanisms of hepatocellular oncogenesis are unclear. Convincing evidence of dysregulated expression of kinase genes has been reported to be involved in the development of HCC. In our previous study, we found that TTK protein kinase gene, which encodes a dual specificity protein kinase with the ability to phosphorylate tyrosine, serine and threonine, was significantly up-regulated in 90.79%(138/152) HCC specimens at mRNA level compared with non-HCCs using RT-PCR and Panomic platform (P<0.01). Notably, clinicopathological analyses demonstrated that elevated TTK expression correlated with large tumor size (P=0.006), advanced TNM-stage (P=0.020), more presence of PVTT (P=0.003), more distant metastasis (P=0.04), poorer disease-free survival rate (P=0.033) and overall survival (P=0.018). In addition, the overexpression of TTK can significantly promote cell growth，cell migration and colony formation of human hepatoma cells (SMMC-7721 and BEL-7404). The knockdown of TTK gene by RNAi can inhibit the cell growth, cell migration and colony formation of SMMC-7721 and QGY-7703 cells. Mechanismly, up-regulated TTK was found to induce phosphorylation disorders of AKT in hepatoma cells. All the current results in our lab suggest the up-regulation of TTK gene promotes cell proliferation and migration via activating AKT phosphorylation in HCC...The objects of this project is first to study the molecular mechanism of AKT phosphorylation by TTK overexpression using protein interaction analysis. The second part will focus on the signaling pathways provoked by TTK-AKT activation, which induced cell proliferation and migration in HCC in vivo, based on both xenograft model and orthotopic model. Thirdly, we will test whether the knock down of TTK can enhance the effects of Sorafinib in inhibiting nude mice tumorigenicity. ..In summary, the project’s aim is to identify the way in which TTK provoked the phosphorylation of AKT, the functional pathways TTK played in hepatocellular oncogenesis and the synergistic inhibitory effect of silencing TTK gene on Sorafinib as a molecular targeting agent in HCC, to reveal the molecular mechanisms involving into TTK-AKT activaion leading to hepatic cancer and lay the foundation for developing an novel strategy of liver cancer therapy in the future.

肝癌恶性程度高，发病机制不明；激酶基因的失调与肝癌发生发展密切相关。本课题组前期利用人激酶组表达谱芯片发现苏氨酸酪氨酸激酶（TTK）在肝癌组织中明显高表达，且表达水平与肝癌病人生存期负相关，与肿瘤大小、肝门静脉转移和远端转移正相关；进一步研究发现在肝癌细胞中过表达TTK能促进肝癌细胞增殖和迁移，并促进AKT激酶磷酸化。据此我们推测TTK通过激活AKT通路进而调控肝癌细胞增殖和迁移。本项目拟深入研究：1.通过蛋白间相互作用分析，探讨TTK高表达促进AKT磷酸化的作用途径；2. 采用裸鼠成瘤和原位肝转移模型，明确TTK-AKT活化影响肝癌成瘤和转移的下游信号通路；3. 抑制TTK表达与索拉非尼联用对小鼠成瘤的影响，寻求基于TTK-AKT信号通路增强肝癌分子治疗效果的可能性。以期阐明激酶基因TTK通过激活AKT信号通路促进肝癌发生发展的分子机制，为以TTK为靶点治疗肝癌提供理论依据。

本课题组前期利用人激酶组表达谱芯片发现苏氨酸酪氨酸激酶（TTK）在肝癌组织中明显高表达，且表达水平与肝癌病人生存期负相关，与肿瘤大小、肝门静脉转移和远端转移正相关；进一步研究发现在肝癌细胞中过表达TTK能促进肝癌细胞增殖和迁移，并促进AKT激酶磷酸化。据此我们推测TTK通过激活AKT通路进而调控肝癌细胞增殖和迁移，拟研究TTK激活AKT通路的相关机制及下游相关信号通路，以阐明激酶基因TTK通过激活AKT信号通路促进肝癌发生发展的分子机制。本课题利用免疫共沉淀和免疫荧光技术研究了TTK与AKT的作用形式，发现TTK并非通过直接的相互作用激活AKT；挖掘新的TTK相互作用蛋白，鉴定到DBN1、EPPK1作为新的TTK互作蛋白。利用磷酸化蛋白组学研究发现，肝癌细胞BEL-7404中TTK消减可导致263个蛋白磷酸化水平上调，162个蛋白磷酸化水平下调。通过蛋白差异KEGG富集明确了TTK在肝癌细胞中通过激活AKT- AKT1S1- mTORC1- EIF4EBP1(autophagy) /EIF4B (protein synthesis)通路调节肝癌细胞生物学特性，参与肝癌发生发展的分子基础。此外，以斑马鱼为模型，在体内水平分析了TTK基因过表达对发育和肿瘤相关通路的影响，佐证TTK通过PI3K-AKT通路激活mTORC1复合物，并提示TTK作为多靶点激酶的其它可能作用途径。本研究在原有基础上进一步阐明TTK在肝癌中的作用机制，为以TTK为靶点的肝癌治疗研究奠定实验基础。