FGFR3/TET2/p-TET2轴异常激活对肝癌生物学特性影响及调控机制

In our previous study, FGFR3Δ7-9 was identified with high frequency in HCC. FGFR3Δ7-9 can apparently promote the proliferation, migration, and metastases of HCC cells both in vitro and in vivo. Co-immumoprecipation and SPR assay showed the binding affinity of FGFR3Δ7-9 to FGFs was significantly higher than that of wild type FGFR3 (P<0.01). FGFR3Δ7-9 can be self-activated by self-dimerization and autophosphorylation even in the absence of ligand (FGFs). We further confirmed FGFR3Δ7-9 could trigger the activation of TET2 and the phosphorylation of TET2..Evidenced by the construction of site-directed mutation of TET2, TET2 (Y1902) was confirmed as a tyrosine phosphorylation site. TET2 (Y1902) could be specifically mediated by FGFR3Δ7-9. In this study, based on paired cellular models and animalmodel, we will focus on the function and regulation of TET2(Y1902F) and TET2 in HCC. All those researches will be very helpfu1 to understand the relationship between FGFR3/TET2/p-TET2 and hepatocarcinogenesis. This study will provide not only newer diagnostic tools, prognostic markers, and therapeutic modu1ation for HCC, but also newer viewpoint in HCC research.

本课题组发现肝癌存在FGFR3Δ7-9(外显子7-9缺失)剪接变异体，并证实FGFR3Δ7-9可以独立于配体，形成二聚体受体自激活，通过多个下游信号通路促进肝癌细胞的增殖和转移。预实验首次发现FGFR3Δ7-9能够介导TET2蛋白磷酸化修饰(p-TET2)，TET2第1902位络氨酸是FGFR3Δ7-9特异性结合位点和磷酸化重要功能位点。进一步发现1902位络氨酸突变体TET2 (Y1902F)和野生型TET2调控肝癌生物学特性完全相反。据此本课题首先明确FGFR3Δ7-9与TET2 (Y1902F)和TET2间的互作关系，继而深度挖掘FGFR3Δ7-9对TET2(Y1902F)蛋白及p-TET2的调控机制和功能性意义，从而明确FGFR3Δ7-9/TET2/p-TET2轴对肝癌生物学行为的作用机制，初步阐明肝癌发生发展的分子生物学机制，为临床治疗肝癌提供供新思路和理论依据。

本课题组前期研究发现了肝癌中存在一种新的FGFR3剪接变异体—FGFR3∆7-9，其能显著促进肝细胞癌（HCC）的恶性进展，但其具体机制仍有待进一步研究。通过免疫共沉淀联合质谱检测发现FGFR3∆7-9能特异性地结合TET2。而TET2在肝癌中显著下调，且TET2的表达量与肝癌不良预后呈负相关。FGFR3∆7-9能显着缩短TET2的半衰期，促进TET2降解，且添加FGF1后这种现象更显著。进一步研究表明FGFR3Δ7-9能直接磷酸化TET2的Y1902位点，从而导致TET2的泛素化和蛋白酶体介导的降解。去磷酸突变TET2（Y1902F）的过表达显着降低了FGFR3Δ7-9在体外和体内的促癌潜力。此外，FGFR3Δ7-9通过TET2-PTEN-AKT途径显着增强HCC细胞增殖。具体而言，TET2通过直接与PTEN启动子结合并增加5-hmC水平来抵消FGFR3Δ7-9诱导的p-AKT水平升高。因此，通过磷酸化和降解TET2，FGFR3降低PTEN表达并促进AKT激活以刺激HCC增殖。FGFR3Δ7-9与TET2之间的相互作用揭示了致癌性FGFR3Δ7-9在肝癌中的重要功能。FGFR3Δ7-9通过TET2的磷酸化和泛素化降解促进HCC进展。靶向FGFR3Δ7-9/TET2-PTEN-AKT信号轴可能有助于开发新的HCC治疗策略。本研究不仅能够为肝癌的分子标志物和治疗靶点的研发提供实验依据，而且可望在肝癌的研究方面开拓新的学术生长点，具有重要的理论和现实意义。