The Role of Cul4A in Genome Stability and Cancer Development

描述(申请人提供)：在人类恶性肿瘤中，DNA扩增导致癌基因激活是基因组改变的主要形式之一，在肿瘤发生中起到致病作用。扩增的基因可能被视为人类癌症发展的主要致癌靶点。基因扩增的功能增强效应使其成为治疗癌症的理想靶点。Cullin 4A(Cul4A)所在的区域ch13q34的扩增在几种人类癌症中都被扩增，包括乳腺癌和肝细胞癌。该区域的扩增也在多种人类癌症中观察到，包括食道癌、胃癌、头颈部癌、膀胱癌、小细胞癌和非小细胞癌。由于这种扩增涉及多个基因，因此识别功能靶标一直很困难。到目前为止，对Cul4A的潜在致癌作用的研究还很有限。我们的假设是，Cul4A是这个扩增区域中关键的致癌基因。在我们的初步研究中，我们发现在恶性胸膜间皮瘤细胞系和肿瘤中存在频繁的Cul4A扩增和过表达。通过shRNA对Cul4A基因的敲除可导致p21蛋白表达增加，进而诱导细胞周期停滞，抑制间皮瘤细胞生长。强制表达的Cul4A降低了p21蛋白，促进了细胞生长。间皮瘤的分子遗传学具有相对同源性，例如，在超过70%的间皮瘤中发现9p21纯合缺失(包含INK4a/ARF基因座)[12]，从而使间皮瘤成为研究人类致癌机制的独特模型。目前，尚无转基因小鼠肿瘤模型可用。以下具体目标勾勒出我们详细的计划，以证明我们的假设。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基因敲除和过度表达研究(特定目标1)。为了阐明Cul4A是原癌基因的潜在机制，我们在与Cul4A相关的重要细胞周期和基因组稳定性分析(特定目标2)方面有了详细的计划。为了评估Cul4A在体内的潜在致癌作用并模拟人类间皮瘤，我们建立了一个条件性Cul4A转基因小鼠模型，并计划将该模型与三个条件性肿瘤抑制基因敲除模型(特异性目标3)交叉。具体目的是为了验证我们的假设：在目标1中，研究Cul4A在人间皮瘤细胞系和组织中的作用。我们计划通过下调间皮瘤细胞系中的13q34区扩增基因，来研究和验证Cul4A在间皮瘤细胞系中的作用，并研究在正常细胞系和间皮瘤细胞系中增强的Cul4A表达的作用。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调控细胞增殖和基因组稳定性的机制；在目标3中，我们计划通过基因敲除和转基因小鼠模型来阐明Cul4A在间皮瘤发展中的作用。我们已经建立了一种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关于Cul4A是癌基因的假设。

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.