FBXO43通过调节CDKIs 蛋白稳定性促进肿瘤细胞增殖的作用机制研究

F-box proteins are subunit recruiting modules of SCF (SKP1-Cullin 1-F-box protein) E3 ligase complexes, and determine the substrate specificity of the complexes. FBXO43 is a member of FBXO sub-family of F-box proteins. While it is reported that FBXO43 is involved in the regulation of meiosis and embryonic development, little is known about the biological function of the protein, especially its role during human malignancies. Our preliminary results indicated both FBXO43 gene and protein levels are significantly highly expressed in the clinical samples of liver cancer, as compared with normal tissues. Also, knockdown of FBXO43 caused significant growth inhibition in human hepatocellular carcinoma cells. These data suggest FBXO43 may play a tumorigenic role during cancer development. We also observed the expression of p27 and p21 proteins, both of which belong to CDKIs family, were simultaneously increased upon FBXO43 depletion. As some SCF complexes have been reported to be capable of modulating CDKIs protein stability, we speculated that p27 and/or p21 may be the substrate proteins regulated by the SCF-FBXO43 complex as an E3 ubiquitin ligase. In this project, we aim to thoroughly study the effect of FBXO43 on the proliferation and survival of human liver cancer cells by altering the protein level of FBXO43. Mechanistically, the role of FBXO43 in regulating p27 and/or p21 protein stability through ubiquitin degradation will be systematically examined. This novel project will provide first evidence to demonstrate the function of FBXO43 in human malignancies, which may serve as a potential biomarker as well as therapeutic target for human hepatocellular carcinoma.

SCF复合物是环指类E3泛素连接酶家族，其关键组分F-box蛋白负责底物蛋白的特异性识别和招募。FBXO43是F-box蛋白家族成员，目前报道的其功能主要局限于生殖发育，而该蛋白在肿瘤发生发展中的作用和可能机制尚无任何报道。我们前期结果提示，FBXO43在肝癌临床样本中显著高表达。敲低FBXO43后，人肝癌细胞出现显著生长抑制，伴随p27、p21的表达升高。结合SCF复合物与CDKIs关系的研究进展，我们推测以p27和p21代表的CDKIs可能是SCF-FBXO43复合物作为E3泛素连接酶调节的底物蛋白。本项目拟以人肝癌细胞为主要研究对象，系统研究FBXO43在促进肿瘤细胞增殖中的确切作用，并重点从泛素化降解方面明确FBXO43调节可能底物蛋白p27和/或p21稳定性的分子机制。本项目属于国内外首次对FBXO43蛋白在肿瘤中的作用和机制进行系统深入研究，有望为难治性肝癌提供新型诊治靶标。

SCF复合物是环指类E3泛素连接酶家族，F-box蛋白作为其中的关键组分，负责底物蛋白的特异性识别和招募，决定了该类复合物的作用特异性。BXO43 是F-box蛋白家族亚类FBXO成员，目前报道的其功能主要局限于生殖发育，该蛋白在肿瘤发生发展中的作用和可能机制目前报道较少。本项目研究发现，TCGA数据库和肝癌临床样本分析发现FBXO43在肝癌组织中的表达显著高于正常组织，且高表达肝癌患者预后较差。功能分析发现敲低FBXO43后能抑制肝癌细胞的增殖和克隆形成能力、促进细胞衰老、诱发细胞G2/M期阻滞，伴随p27、p21的表达升高。此外敲低FBXO43后能抑制肝癌细胞体内成瘤能力。细胞周期同步之后发现FBXO43在细胞周期中的蛋白水平变化与SKP2一致，与SKP的底物蛋白p27相反。通过内源性和外源性co-IP实验发现FBXO43能与SKP2相结合，与p27不结合。敲低FBXO43后的肝癌细胞内SKP2蛋白水平降低，但其转录水平未呈现显著变化。敲低FBXO43能够缩短SKP2降解半衰期，且MG132逆转了敲低FBXO43导致的SKP2减少。泛素化实验发现FBXO43能抑制SKP2的自泛素化，且FBXO43的N端结构域和SKP2的丝氨酸72位点对于FBXO43发挥抑制SKP2自泛素化功能至关重要。综上，我们得出结论：FBXO43在肝癌的形成和发展中起到促进作用，通过影响SKP2的自泛素化来调控SKP2的稳定性，促进肿瘤细胞周期进展，从而发挥其促癌功能。本项目属于国内外首次对FBXO43蛋白在肿瘤中的作用机制进行系统深入的研究，有助于全面理解FBXO43在肿瘤中的作用特点，有望为难治性肝癌提供新型生物标志和潜在干预靶点。