利用基因敲除小鼠研究CUL4A在胃癌发生发展中的作用及其分子调控机制

Ubiquitin modification of cellular proteins plays a vital role in maintaining the balance between normal growth and uncontrolled proliferation. Dysregulated ubiquitination process has been shown to contribute to oncogenesis. The cullin family of ubiquitin ligases, comprised of CUL1, 2, 3, 4A, 4B, 5, and 7, represents the largest class of RING-type E3 ligases. Among these, CUL4A ubiquitin ligase has been drawing a lot of attentions due to its recurrent correlations to various tumors. The CUL4A gene was initially found to be amplified or overexpressed in primary breast cancer, which provided the first evidence that CUL4A is involved in carcinogenesis. Recently, CUL4A amplification has also been reported in several other malignancies. However, the expression status of CUL4A in gastric cancer and its roles in gastric carcinogenesis still remain unclear now..Amplification or overexpression of CUL4A in multiple tumor types leads to the hypothesis that CUL4A plays a role in promoting oncogenesis. Our previous work supports this scenario, as Cul4a knockout mice have shown to be markedly resistant to UV-induced skin carcinogenesis compared to wild-type mice. We have demonstrated that CUL4A ubiquitin ligase promotes UV-induced skin carcinogenesis by coordinately suppresses the nucleotide excision repair and G1/S DNA damage checkpoint pathways. Notably, our recent results show that CUL4A is essential in maintaining the growth and proliferation of gastric cancer cells, although it is dispensable in normal cells. This renders CUL4A an ideal molecule for the “targeting therapy” of gastric cancer..In this study, we propose to determine CUL4A expression in human gastric cancer specimens and evaluate the association between CUL4A expression and clinico-pathological factors in gastric cancer patients. We will further investigate the roles of CUL4A in gastric carcinogenesis as well as in maintaining the growth of gastric cancer by Cul4a knockout mouse. To elucidate the underlying molecular mechanisms of above processes, multiple approaches including proteomics, will be employed to identify the novel CUL4A substrates in addition to evaluate the contributions of the known ones..Results from this study will provide insight into the potential clinical value of CUL4A as a new biomarker for the pathologic evaluation and prognosis of gastric cancer patients. Importantly, with increased understanding of CUL4A’s roles in oncogenesis, strategies of CUL4A directed therapeutics, including personalized therapy against gastric cancer, are expected to be explored and developed in the near future.

蛋白泛素化的异常与肿瘤的发生、发展密切相关，其中CUL4A泛素连接酶与肿瘤的关系近来备受关注。目前，在多种肿瘤中均发现CUL4A的过表达现象，但其在胃癌中的表达情况及作用机制尚未见报道。申请人前期工作发现，CUL4A可以通过影响DNA损伤修复能力参与肿瘤的发生（Mol. Cell）；而且，CUL4A虽然对维持正常细胞的生理活动并非必需，但其对胃癌细胞的生长及凋亡却至关重要，这一特性使CUL4A成为胃癌靶向性治疗的理想靶点。本项目首先通过分析临床胃癌样本中CUL4A的表达情况，确立其与胃癌患者临床、病理指标的相关性；在此基础上，利用基因敲除小鼠，深入探讨CUL4A在胃癌发生、发展中的重要作用，并通过定量蛋白质谱（SILAC）等方法鉴定新的CUL4A底物蛋白，从而进一步阐明CUL4A在上述过程中的分子调控机制。本项目将为胃癌靶向性药物的研发及胃癌个体化治疗的突破开辟新的视野和思路。

胃癌是我国第二大常见肿瘤，具有恶性度高、进展快，易发生化疗耐药，预后较差等特点。因此探寻导致胃癌化疗耐药的关键分子，对于改善胃癌治疗具有重要意义。蛋白泛素化的异常与肿瘤的发生、发展密切相关，其中CUL4A泛素连接酶与肿瘤的关系近来备受关注。研究表明，CUL4泛素连接酶的底物涉及信号传导、细胞周期调控、基因组稳定等诸多重要生物学过程，然而其在化疗耐药中的具体作用及分子调控机制，目前尚无报道。本项目首先利用临床组织样本，分析了胃癌组织中CUL4A的表达情况与胃癌患者化疗疗效、生存预后的相关性。随后通过细胞学实验，评估了CUL4A对胃癌细胞增殖、细胞周期及顺铂耐药性的影响。最后进一步利用生化及分子生物学等方法探索CUL4A对胃癌细胞顺铂耐药性的分子调控机制。我们的胃癌组织样本多因素分析结果表明，CUL4A是独立于肿瘤TNM分期的预后危险因素，并且与患者的化疗治疗效果密切相关，可以作为指导胃癌患者临床治疗的重要指标。另外，我们在胃癌细胞中鉴定出一个新的CUL4A泛素连接酶底物及其受体蛋白，首次发现CUL4A泛素连接酶在DNA双链损伤修复中发挥重要作用，从而在分子水平揭示了CUL4A在胃癌细胞中对铂类化疗药物耐药性的调控作用机制。本项目为胃癌化疗增敏药物的研发提供了新的思路，为扭转目前胃癌治疗手段匮乏的局面、实现胃癌的精准治疗奠定了理论基础。