Cullin1-FBXL6泛素连接酶作为肝癌治疗靶点及其作用分子机制

SKP1-Cullin-1-F-box (SCF/CRL1) ubiquitin ligase complexes utilize the F-box family protein (consisting of 69 members) as substrate receptors. By using the Cancer Genome Atlas (TCGA), we have recently found that the F-box family member, FBXL6, is overexpressed in liver cancer tissue. Further, we found that the protein was highly expressed in metastatic liver cancer cell lines. Knockdown of FBXL6 by RNA interference significantly inhibited metastatic liver cancer cell proliferation and transwell migration in vitro, while ectopic expression of FBXL6 in liver cancer cells with low metastatic potential facilitated their proliferation and transwell migration. Importantly, these biological characteristics of FBXL6 were found to be dependent of SKP1-Cullin-1-FBXL6 ligase activity. Collectively, these results suggest that FBXL6 may serve as a potential molecular target for liver cancer therapy. In this study, we will explore this hypothesis by focusing on the work as follows: 1) To study the effect of FBXL6 expression on malignancy of liver cancer using cell line and animal tumor models; 2) To identify and characterize SKP1-Cullin-1-FBXL6 substrate(s) that contribute(s) to liver cancer growth and metastasis. Our work may hopefully define the role of SKP1-Cullin-1-FBXL6 ubiquitin ligase in liver cancer and contributes to develop an effective approach to treat this malignancy.

FBXL6蛋白是cullin-1-RING泛素连接酶（CRL1）的一个底物识别亚基。我们前期发现FBXL-6基因的mRNA表达水平在肝癌组织异常高表达；随后证实FBXL6蛋白表达下调能够抑制肝癌细胞增殖与迁移，而其过度表达则促进肝癌细胞增殖与迁移；进一步研究证实FBXL6蛋白促进肝癌恶性表型的生物学特性与CRL1-FBXL6复合物的连接酶活性密切相关。我们据此推测CRL1-FBXL6连接酶可以作为抗肝癌治疗的干预靶点。为了证实这一假说，本项目拟分别从细胞水平和动物模型层面评价FBXL6蛋白差异表达对肝癌细胞恶性表型的影响；通过质谱和生化技术鉴定肝癌相关CRL1-FBXL6泛素连接酶底物，并探索其在肝癌细胞恶性表型中的作用。本项目预期能够鉴定以CRL1-FBXL6泛素连接酶为靶点的抗肝癌治疗新策略并揭示其作用分子机制，为今后肝癌临床治疗提供科学依据。

原发性肝癌是我国最为常见的恶性肿瘤之一。因其恶性程度高、病程进展迅速，大部分患者初诊时已发展至疾病中晚期，最终因肿瘤转移而加速患者死亡。因此，控制肿瘤转移是提高临床疗效的关键措施。但迄今仍无安全、特异和有效的抗肿瘤转移治疗方法。本研究发现CRL1-FBXL6泛素连接酶能够通过促进肿瘤细胞STK16丝氨酸/苏氨酸激酶泛素化修饰及活性，后者对其下游FGFR1受体进行磷酸化作用，从而激活FGFR1及相关信号通路，最终促进肝癌细胞发生肝内或远端转移。因此，通过靶向CRL1-FBXL6 -STK16信号轴将能够有效抑制肝癌细胞转移。针对上述靶点，本研究设计了STK16激酶的泛素化抑制性多肽；动物试验发现该小分子多肽在肿瘤细胞内表达能够有效抑制肿瘤细胞远端转移，具有一定的临床应用前景。