The Role of Cul4A in Genome Stability and Cancer Development

Cul4A 在基因组稳定性和癌症发展中的作用

• 批准号: 7986716
• 负责人: LIANG YOU
• 金额: $ 31.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-09 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7986716
• 关键词: 13q34; 9p21; Bladder; Breast; CDKN2A gene; Cancer Etiology; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cells; Chromosomes; Clinical; Crocidolite Asbestos; DNA amplification; Developing Countries; Development; Diagnosis; Drug resistance; Esophageal; Europe; Event; Excision; Gene Amplification; Genes; Genetic Models; Genome Stability; Genomics; Goals; Head and neck structure; Human; Human Development; Incidence; Injection of therapeutic agent; Intervention; Knock-out; Lung; Malignant Neoplasms; Malignant Pleural Mesothelioma; Mediating; Mesothelioma; Modality; Modeling; Molecular; Molecular Genetics; Mus; Mutation; Oncogene Activation; Oncogenes; Oncogenic; Patients; Phenotype; Play; Pleural; Post-Translational Protein Processing; Primary carcinoma of the liver cells; Protein p53; Proteins; Proto-Oncogenes; Radiation therapy; Recombinant Cytokines; Resistance; Role; Small Cell Carcinoma; Staging; Stomach; TP53 gene; Testing; Therapeutic Agents; Tissues; Transgenic Mice; Tumor Cell Line; Tumor Suppressor Genes; Tumor Suppressor Proteins; United States; base; carcinogenesis; cell growth; chemotherapy; cullin 4A; drug discovery; functional loss; gain of function; in vivo; knock-down; mouse model; novel therapeutics; oncoprotein p21; overexpression; public health relevance; small hairpin RNA; therapeutic target; tumor; tumorigenesis

DESCRIPTION (provided by applicant): In human malignances, DNA amplification leading to oncogene activation represents one of the major forms of genomic alterations that plays a causative role in tumorigenesis. The amplified genes may be viewed as primary oncogenic targets in the development of human cancer. The gain-of-function effect of gene amplification makes them ideal therapeutic targets for cancer. Amplification of ch13q34, the region that Cullin 4A (Cul4A) resides in, is amplified in several human cancers, including breast and hepatocellular cancer. The amplification of this region has also been observed in a variety of human cancers including esophageal, gastric, head and neck, bladder, small cell and non-small cell cancers. Since such the amplification involves multiple genes, the identification of the functional target has been difficult. To date, the study on the potential oncogenic role of Cul4A has been limited. Our hypothesis is that Cul4A is the key cancer-causing oncogene in this amplified region. In our preliminary study, we have identified frequent Cul4A amplification and overexpression in malignant pleural mesothelioma cell lines and tumors. Knockdown of Cul4A by shRNA leads to increased p21 protein, and subsequently induces cell cycle arrest and inhibits mesothelioma cell growth. Forced expression of Cul4A decreases p21 protein and promotes cell growth. The molecular genetics of mesothelioma are relatively homogenous, e.g., homozygous deletion of 9p21 (contains the INK4a/ARF locus) was found in more than 70% of mesothelioma tumors[12], thus make mesothelioma an unique model to study the mechanisms of human carcinogenesis. Currently, there is no Cul4A transgenic mouse tumor model available. The following specific aims outline our detailed plan to prove our hypothesis. To determine the role of Cul4A amplification in mesothelioma, we plan to perform additional Cul4A knockdown and overexpression study using more mesothelioma cell lines (Specific Aim 1). To elucidate the potential mechanisms through which Cul4A is a proto-oncogene, we have the detailed plan on the important Cul4A-related cell cycle and genome stability analysis (Specific Aim 2). To evaluate the potential oncogenic role of Cul4A in vivo and mimic the human mesothelioma, we have generated a conditional Cul4A transgenic mouse model and we plan to cross this model with three conditional tumor suppressor knockout models (Specific Aim 3). The specific aims to test our hypothesis: In aim 1, Investigate the role of Cul4A in human mesothelioma cell lines and tissues. We plane to investigate and validate the role of Cul4A in mesothelioma cell lines with amplification in the 13q34 region by knocking-down Cul4A and investigate the effects of enhanced Cul4A expression in normal and mesothelioma cell lines. In addition, we plan to determine the role of Cul4A amplification in large number of mesothelioma tissues; In aim 2, the goal is to investigate the potential mechanisms through which Cul4A plays an oncogenic role. We plan to investigate the mechanisms through which Cul4A regulates cell proliferation and genome stability; In aim 3, we plan to elucidate the role of Cul4A in mesothelioma development using knockout and transgenic mouse models. We have generated a Cul4A transgenic mouse model. We are now ready to use the Cul4A transgenic mice to test our hypothesis that Cul4A is an oncogene. PUBLIC HEALTH RELEVANCE: Malignant pleural mesothelioma is a highly aggressive and challenging cancer arising primarily from the pleural lining of the lung. Approximately 3,000 patients are diagnosed with mesothelioma in the United States annually and the incidence of this tumor is predicted to increase over the next decade in both Europe and developing nations. Since mesothelioma usually presents at an advanced stage, a curative resection is rarely possible. Radiotherapy has failed to show clinical benefit as a single treatment modality, and the administration of chemotherapy is mostly restricted to the advanced stage with limited efficiency. Alternative strategies based on pleural injections of recombinant cytokines have similarly proven unsatisfactory. Most patients die within 2 years after diagnosis. Since current interventions offer only limited benefit and overall survival is low, there is an urgent need to develop new therapeutic agents based on a greater understanding of mesothelioma's underlying molecular mechanisms. Loss of cell cycle control is a hallmark of human cancer including mesothelioma. For instance, the most predominant genetic change in human malignant pleural mesothelioma (>70%) is the homozygous deletion of INK4a/ARF locus within the 9p21 chromosome region. Despite that p21 is one of the most important effectors of p53, p21 can also be regulated by many p53-independent mechanisms including posttranslational modifications. For the majority of mesotheliomas that lacks both p14ARF and p16INK4A, up-regulating or stabilizing p21 protein may serve as a novel therapeutic strategy. In addition, functional loss of p21 can mediate drug-resistance phenotype, and that may be one of the key reasons why mesothelioma is resistant to classical chemotherapy and radiation therapy. Mesothelioma provides a very unique and homogeneous genetic model for human cancer. The current mesothelioma mouse models focused only on the conditional knockout of tumor suppressor genes, e.g., INK4a/ARF. Cul4A amplification represents the activation of a distinct proto-oncogene class in mesothelioma. To date, there is no Cul4A transgenic mouse model. Therefore, we have just established a conditional Cul4A transgenic mouse model to mimic human mesothelioma by incorporating this oncogenic event. This model may be used to study the role that Cul4A plays in mesothelioma carcinogenesis, and may also be potentially useful for further drug discovery, e.g., to screen for molecules that inhibiting Cul4A-mediated p21 degradation.

描述（由申请人提供）：在人类恶性肿瘤中，导致癌基因激活的DNA扩增代表了在肿瘤发生中起因作用的主要基因组改变的主要形式。扩增的基因可能被视为人类癌症发展中的主要致癌靶标。基因扩增的功能效果使它们成为癌症的理想治疗靶标。 CH13Q34的扩增是Cullin 4a（Cul4a）所在的区域，包括在包括乳腺癌和肝细胞癌在内的几种人类癌症中。在各种人类癌症中也观察到了该区域的扩增，包括食管，胃，头颈，膀胱，小细胞和非小细胞癌。由于这种扩增涉及多个基因，因此很难识别功能靶标。迄今为止，关于CUL4A潜在的致癌作用的研究一直受到限制。 我们的假设是CUL4A是该扩增区域中致癌的关键致癌癌基因。在我们的初步研究中，我们已经确定了恶性胸膜间皮瘤细胞系和肿瘤中频繁的CUL4A扩增和过表达。 SHRNA敲低CUL4A会导致P21蛋白增加，并随后诱导细胞周期停滞并抑制间皮瘤细胞的生长。 CUL4A的强制表达降低了P21蛋白并促进细胞生长。间皮瘤的分子遗传学相对均匀，例如，在超过70％的间皮瘤肿瘤中发现了9p21的纯合缺失（包含Ink4a/arf基因座）[12]，从而使间皮瘤成为研究人类癌作用机制的独特模型。当前，没有CUL4A转基因小鼠肿瘤模型可用。以下具体目的概述了我们证明我们的假设的详细计划。为了确定CUL4A扩增在间皮瘤中的作用，我们计划使用更多间皮瘤细胞系进行其他CUL4A敲低和过表达研究（特定的AIM 1）。为了阐明CUL4A是原始癌基因的潜在机制，我们制定了有关重要CUL4A相关细胞周期和基因组稳定性分析的详细计划（特定目标2）。为了评估Cul4a在体内的潜在致癌作用，并模仿人间皮瘤，我们产生了一个条件的Cul4a转基因小鼠模型，我们计划将该模型与三个条件抑制肿瘤抑制器敲除模型（特定AIM 3）交叉。具体目的是检验我们的假设：在AIM 1中，研究CUL4A在人间皮瘤细胞系和组织中的作用。我们通过敲击降低的CUL4A并研究了正常和间皮瘤细胞系中CUL4A表达增强的效果，从而在13q34区域进行了调查和验证CUL4A在间皮瘤细胞系中具有扩增和扩增的作用。此外，我们计划确定Cul4a扩增在大量间皮瘤组织中的作用。在AIM 2中，目标是研究CUL4A发挥致癌作用的潜在机制。我们计划研究CUL4A调节细胞增殖和基因组稳定性的机制。在AIM 3中，我们计划使用敲除和转基因小鼠模型阐明CUL4A在间皮瘤发育中的作用。我们已经生成了CUL4A转基因小鼠模型。现在，我们准备使用CUL4A转基因小鼠来测试我们的假设CUL4A是癌基因。 公共卫生相关性：恶性胸膜间皮瘤是一种高度侵略性且具有挑战性的癌症，主要由肺胸膜衬里引起。大约3,000名患者每年在美国被诊断出患有间皮瘤，预计该肿瘤的发生率在未来十年和发展中国家的未来十年中会增加。由于间皮瘤通常在晚期阶段出现，因此很少可以治愈切除术。放射疗法未能显示出作为单一治疗方式的临床益处，化学疗法的给药大部分限制为高级阶段，效率有限。基于重组细胞因子的胸膜胸膜注射的替代策略类似地证明了不令人满意的。大多数患者在诊断后2年内死亡。由于目前的干预措施仅提供有限的收益，总体生存率较低，因此迫切需要基于对间皮瘤的潜在分子机制的更深入的了解，开发了新的治疗剂。 细胞周期控制的丧失是包括间皮瘤在内的人类癌症的标志。例如，人类恶性胸膜间皮瘤（> 70％）中最主要的遗传变化是9p21染色体区域内Ink4a/ARF基因座的纯合缺失。尽管p21是p53最重要的效应子之一，但p21也可以受到许多独立于p53的机制（包括翻译后修饰）的调节。对于缺乏P14ARF和P16INK4A的大多数间皮瘤，上调或稳定的P21蛋白可能是一种新型的治疗策略。此外，p21的功能丧失可以介导药物抗药性表型，这可能是间皮瘤对经典化学疗法和放射治疗具有抵抗力的关键原因之一。 间皮瘤为人类癌症提供了非常独特和均匀的遗传模型。当前的间皮瘤小鼠模型仅集中在肿瘤抑制基因的条件敲除（例如Ink4a/arf）上。 CUL4A扩增代表间皮瘤中独特的原始癌细胞类别的激活。迄今为止，没有CUL4A转基因小鼠模型。因此，我们刚刚通过纳入了这种致癌事件来建立一个条件CUL4A转基因小鼠模型，以模仿人间皮瘤。该模型可用于研究CUL4A在女tophelioma癌中的作用，并且对于进一步的药物发现，例如筛选抑制CUL4A介导的p21降解的分子也可能有用。