FBXO8通过泛素化降解CDK4调控结直肠癌增殖能力机制研究

In our previous study, we found that FBXO8 was down-regulated in colorectal cancer, which suppressed the proliferation and invasion of colorectal cancer. FBXO8 can serve as an independent prognostic factor of colorectal cancer patients. Till now, there is little known about the molecular mechanism of FBXO8 in tumor progression. An important approach to illustrate the function of FBXO8 is to identify the substrate and the signaling pathways of it. Based on the preliminary experiments and literature review, we proposed that FBXO8 inhibits the proliferation of CRC by targeting CDK4 for ubiquitin degradation, and subsequently regulating the cell cycle and apoptosis. To validate the hypothesis, this study will explore at the levels of cell lines, tissue specimens, nude mice and transgenic mice, (1) CDK4 is a substrate of FBXO8 for ubiquitin degradation, (2) the role of CDK4 in FBXO8 induced the proliferation of CRC cells, (3) the ubiquitin degradation of CDK4 inhibit the proliferation of CRC by regulates the cell cycle and apoptosis. This study aims to provide new clues for illustrating the molecular mechanism of tumor proliferation, and provide new targets and strategy for the diagnosis and treatment of colorectal cancer.

前期工作中我们发现FBXO8在结直肠癌组织中表达下调，抑制结直肠癌细胞体外增殖和侵袭能力,可作为结直肠癌患者的一个独立预后因子。目前对FBXO8在肿瘤演进中的作用机制知之甚少，而鉴定FBXO8泛素化底物及其信号机制是阐述FBXO8功能的一个重要途径。根据预实验及文献检索结果，我们提出了FBXO8作用的一个新机制：即FBXO8通过泛素化降解CDK4，进而影响细胞周期和凋亡进程，最终抑制结直肠癌增殖能力。为证实这一假设，本研究拟从细胞株、组织标本、裸鼠及转基因小鼠水平，（1）明确CDK4是FBXO8下游的泛素化底物；（2）检测CDK4泛素化降解在FBXO8抑制结直肠癌增殖中的作用；（3）验证CDK4泛素化降解在结直肠癌细胞周期阻滞，诱导凋亡过程中的作用。旨在为肿瘤增殖研究提供新的思路，为肿瘤诊治提供新的靶点和策略。

FBX8是F-box蛋白家族的一个新成员，该家族是SCF（Skp1 / Cul1 / F-box蛋白）泛素连接酶复合物的靶向底物。 FBX8在肿瘤中表达缺失，并在肿瘤进展中充当抑制因子。我们发现在15种细胞周期控制蛋白中，细胞周期蛋白依赖性激酶4（CDK4）被选为FBX8的潜在底物，明显受到FBX8的负调控，FBX8与CDK4有直接结合域。 FBX8和CDK4的共定位主要在CRC细胞的细胞质中，蛋白酶体抑制剂MG132增强了这种作用。此外，FBX8将CDK4靶向以泛素依赖性方式降解。功能测定的结果表明，FBX8通过靶向CDK4降解而在体外和体内抑制CRC细胞增殖。我们还确认CDK4是FBX8基因敲除小鼠模型中FBX8的底物。结直肠癌组织中FBX8表达与CDK4负相关。因此，我们的研究结果从从细胞株、组织标本、裸鼠及转基因小鼠水平三个水平证明，在结直肠癌中，FBX8通过控制CDK4降解而影响肿瘤进程，即FBX8通过泛素化降解CDK4，引起肿瘤细胞G1-S期阻滞，同时诱导肿瘤细胞发生凋亡，最终抑制结直肠癌细胞的增殖能力，发挥抑癌作用。