The Role of Cul4A in Genome Stability and Cancer Development

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致病作用的基因组改变的主要形式之一。扩增的基因可被视为人类癌症发展中的主要致癌靶点。基因扩增的功能获得效应使其成为癌症的理想治疗靶点。Cullin 4A（Cul4A）所在区域ch13q34的扩增在几种人类癌症中扩增，包括乳腺癌和肝细胞癌。该区域的扩增也已在多种人类癌症中观察到，包括食道癌、胃癌、头颈癌、膀胱癌、小细胞癌和非小细胞癌。由于这样的扩增涉及多个基因，因此难以鉴定功能性靶标。迄今为止，对Cul4A潜在致癌作用的研究有限。 我们的假设是Cul4A是该扩增区域中的关键致癌基因。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。通过shRNA敲低Cul4A导致p21蛋白增加，随后诱导细胞周期停滞并抑制间皮瘤细胞生长。Cul4A的强制表达降低p21蛋白并促进细胞生长。间皮瘤的分子遗传学是相对同质的，例如，在超过70%的间皮瘤肿瘤中发现了9p21（含有INK4a/ARF基因座）的纯合缺失[12]，从而使间皮瘤成为研究人类致癌机制的独特模型。目前，还没有Cul4A转基因小鼠肿瘤模型可用。以下具体目标概述了我们证明假设的详细计划。为了确定Cul4A扩增在间皮瘤中的作用，我们计划使用更多的间皮瘤细胞系进行额外的Cul4A敲低和过表达研究（特定目的1）。为了阐明Cul4A作为原癌基因的潜在机制，我们对重要的Cul4A相关细胞周期和基因组稳定性分析（具体目标2）进行了详细的计划。为了评估Cul4A在体内的潜在致癌作用并模拟人间皮瘤，我们已经产生了条件Cul4A转基因小鼠模型，我们计划将该模型与三种条件肿瘤抑制基因敲除模型（特异性目标3）杂交。具体目的是验证我们的假设：目的1，研究Cul4A在人间皮瘤细胞系和组织中的作用。我们计划通过敲低Cul4A来研究和验证Cul4A在13q34区域扩增的间皮瘤细胞系中的作用，并研究Cul4A表达增强在正常和间皮瘤细胞系中的作用。此外，我们计划确定Cul4A扩增在大量间皮瘤组织中的作用;在目标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;在目标3中，我们计划使用基因敲除和转基因小鼠模型阐明Cul4A在间皮瘤发展中的作用。我们建立了Cul4A转基因小鼠模型。我们现在准备使用Cul4A转基因小鼠来验证我们的假设，Cul4A是一个致癌基因。 公共卫生相关性：恶性胸膜间皮瘤是一种高度侵袭性和挑战性的癌症，主要发生于肺的胸膜衬里。在美国，每年约有3,000名患者被诊断患有间皮瘤，预计未来十年欧洲和发展中国家的间皮瘤发病率将增加。由于间皮瘤通常出现在一个先进的阶段，根治性切除术是很少可能的。放射治疗作为单一的治疗方式未能显示出临床益处，并且化疗的施用主要限于晚期，效率有限。基于重组细胞因子的胸膜注射的替代策略同样被证明是不令人满意的。大多数患者在诊断后2年内死亡。由于目前的干预措施只能提供有限的好处和总生存率低，有迫切需要开发新的治疗药物的基础上更好地了解间皮瘤的潜在分子机制。 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，人恶性胸膜间皮瘤中最主要的遗传变化（> 70%）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也可以通过许多非p53依赖的机制进行调节，包括翻译后修饰。对于大多数同时缺乏p14ARF和p16INK4A的间皮瘤，上调或稳定p21蛋白可作为一种新的治疗策略。此外，p21功能缺失可介导耐药表型，这可能是间皮瘤对经典化疗和放疗耐药的重要原因之一。 间皮瘤为人类癌症提供了一个非常独特和同质的遗传模型。目前的间皮瘤小鼠模型仅关注肿瘤抑制基因的条件性敲除，例如，INK4a/ARF。Cul4A扩增代表间皮瘤中一种独特的原癌基因类的激活。迄今为止，还没有Cul4A转基因小鼠模型。因此，我们刚刚建立了一个条件Cul4A转基因小鼠模型，模拟人类间皮瘤通过纳入这一致癌事件。该模型可用于研究Cul4A在间皮瘤癌变中的作用，并且也可能潜在地用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。

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.