CUL4A在小细胞肺癌发生发展中的作用及其调控机制

Lung cancer is one of the leading causes of cancer deaths in China. About 33% of people with small-cell lung cancer (SCLC) have limited-stage disease, but the majority of people have extensive-stage disease. It is important to develop novel approaches for prevention and treatment for SCLC. .The CUL4A ubiquitin ligase is a tetrameric complex in which the elongated CUL4A scaffold utilizes the adaptor proteins DDB1 and Rbx1/Roc1 to recruit the DCAF substrate receptors, which in turn bind specific substrates, and the E2 ubiquitin conjugating enzyme with activated ubiquitin, thereby bringing substrates and activated ubiquitin into close proximity to facilitate ubiquitin transfer. We recently demonstrated that the CUL4A ubiquitin ligase coordinately suppresses the nucleotide excision repair and G1/S DNA damage checkpoint pathways. This effect is achieved through CUL4A-mediated ubiquitination and degradation of DDB2 and XPC, the rate-limiting factors of nucleotide excision repair, and p21, the effector of the DNA damage checkpoint. We further demonstrated that CUL4A knockout mice are hyper-resistant to DMBA-induced skin tumors.In a high density SNP analysis, CUL4A gene was found amplified in 67% of small cell lung cancer cases. We have detected elevated CUL4A expression in small cell lung cancer specimens. Our recent study also indicated that the CUL4 E3 ligase targets topoisomerase I for ubiquitin-dependent degradation, thereby evading the cytotoxic effects of TOP1-directed anti-cancer drug camptothecin..We propose to investigate dysregulation of CUL4A expression in human SCLC specimens. We will evaluate whether lung cancer cells are addicted to high levels of CUL4A, and the relative contributions of known CUL4A substrates, and assess the efficacy of CUL4A inactivation in sensitizing SCLC to TOP1-directed chemotherapy. Results from these studies will provide insight into the potential clinical value of CUL4A dysregulation as a new biomarker for the susceptibility of this lung cancer type. This study also set the stage for developing pharmacological CUL4A inhibitors in prevention and/or personalized therapy against this human malignancy, for which effective targeted therapy is urgently needed.

小细胞肺癌（SCLC）占肺癌总数的10-20%，早期易发生转移，约67%的SCLC发现时即诊断为进展期肺癌，且癌细胞生长速度快，易发生化疗耐药，治疗效果不佳。因此探索在SCLC发生发展过程中发挥关键作用的分子，对于提高SCLC的防治效果具有重要意义。课题组在前期工作中发现肿瘤细胞中CUL4A表达升高，并且与肿瘤发生、细胞生长、凋亡及药物敏感性相关。本项目在此基础上，深入探讨CUL4A在SCLC发生发展中的作用。首先，分析SCLC中CUL4A泛素连接酶及其底物的表达，及其与患者临床指标、DNA损伤及修复情况的关系，评价CUL4A在SCLC发生当中的作用及机制；其次，观察以蛋白敲除联合shRNA方法下调CUL4A，评价对SCLC细胞生长、凋亡及药物敏感性的影响及其作用途径。总之，通过本项目研究，评价CUL4A在SCLC发生、发展中的作用及机制，为研发靶向CUL4A 的个体化防治策略奠定基础。

泛素化作为一类重要的体内蛋白质翻译后修饰，能够导致靶蛋白的降解。泛素连接酶（E3, Ubiquitin ligase）是泛素化过程中的关键酶之一，负责介导经泛素激活酶（E1, Ubiquitin-activiting enzyme）活化的泛素从泛素结合酶（E2, Ubiquitin-conjugating enzyme）转移至底物蛋白的过程。不同的E3连接酶能够识别不同的底物蛋白，从而决定了泛素化修饰的底物特异性。CUL4A和CUL4B是E3家族的重要成员，它们参与在翻译后水平精确调节细胞周期、DNA修复以及细胞凋亡等过程中关键蛋白的降解及活性，参与多种恶性肿瘤的发生、发展过程。.课题组收集天津医科大学肿瘤医院2000年1月-2011年6月期间手术的352例肺癌患者的肿瘤组织标本，以及62例肺癌患者的术后非肿瘤上皮组织标本,应用免疫组织化学染色（IHC）方法检测肺癌组织和非肿瘤上皮组织的CUL4A和CUL4B表达情况，分析CUL4A和CUL4B的表达与患者的临床病理学资料以及预后之间的关系。并通过稳定沉默CUL4A、CUL4B的肺癌细胞株模型的建立，探讨肺癌组织中高水平表达的CUL4A和CUL4B对肺癌细胞生长和凋亡的影响及其机制。.实验结果表明：肺癌组织中CUL4A和CUL4B的表达水平显著高于62例非肿瘤上皮组织（P<0.001）。CUL4A/CUL4B高表达组中，其DNA含量显著高于低表达组（P<0.001）。③CUL4A高表达组患者吸烟人数显著高于CUL4A低表达组。CUL4A和CUL4B表达与患者临床分期、肿瘤体积、远处转移和淋巴结转移，以及总生存（Overall Survival, OS）和无病生存（Disease-free Survival, DFS）显著相关。.CUL4A在肺癌细胞生长中的作用及机制：稳定沉默肺鳞癌细胞系NCI-H520和小细胞肺癌细胞系NCI-H446的CUL4A后，出现细胞周期S期缩短，G0/G1期阻滞现象，与此同时XPC和p21蛋白表达量升高；在沉默CUL4A的基础上沉默p21后，上述细胞周期变化得到逆转；并且在肺癌组织中CUL4A和XPC及p21的表达呈负相关。提示CUL4A可能通过p21调节肺癌细胞系的细胞周期。.通过小鼠模型验证CUL4A/B对肺癌生长的调节作用.

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