RNA Binding Proteins in Cancer

描述(由申请人提供)：该项目的主要目标是了解RNA结合蛋白RBM3在转录后水平上调节癌细胞中mRNA稳定性和翻译的基因表达的机制。我们已经证实，该蛋白的过度表达会导致正常细胞经历转化的表型，从而导致免疫受损小鼠的细胞形成肿瘤。另一方面，RBM3的表达下调会导致细胞因有丝分裂灾难而死亡。RBM3与HUR和hnRNP A1以及AU-RNA序列相互作用，以增强mRNA的稳定性和富含AU的转录物，如COX-2，VEGF和IL-8。此外，RBM3的过表达以一种Notch依赖的机制增加了哺乳动物雷帕霉素蛋白靶标的激活。我们还发现了调控RBM3过度表达的microRNAs，包括下调一个抑制其自身表达的microRNAs。我们的研究还表明，RBM3在翻译后的磷酸化、泛素化和SUMO化水平上受到调控。基于这些观察，我们提出了三个具体目标。在目标1中，我们将确定RNA结合蛋白RBM3调节基因表达的机制。在这里，我们将识别与RBM3相互作用的RNA序列。此外，我们还将确定RBM3介导的COX-2mRNA的稳定性和翻译所需的COX-2 3‘非编码区的序列。我们还将确定HUR和hnRNP-A1在这一过程中的作用。在目标2中，我们将通过突变来确定RBM3中经过磷酸化、泛素化和SUMO化的残基。我们还将确定突变体对信使核糖核酸稳定性和翻译的影响。在目标3中，我们建议确定RBM3诱导肿瘤发生的机制。我们将使用异种移植癌模型来确定mTOR和Notch通路在RBM3介导的肿瘤发生中的作用。此外，还将确定microRNAs在肿瘤发生中的作用。这些实验的完成应该会让我们更好地了解RNA结合蛋白RBM3在正常上皮细胞中的功能，以及在肿瘤细胞中观察到的RBM3表达的变化是否与肿瘤行为有关。与公共健康相关：癌症是美国主要的死亡原因。了解正常细胞如何发展为癌症将有助于我们为这种可怕的疾病开发新的治疗方法。我们已经确定了一种蛋白质，RBM3，它在癌细胞中的表达增加。过量表达RBM3蛋白会导致正常细胞转化为癌细胞。我们目前的建议涉及确定RBM3表达受调控的机制，以及RBM3如何诱导肿瘤发生。我们期望这项工作将导致对肿瘤发生过程的更好的理解，这将随后导致阻止或减缓肿瘤发生的新方法。

DESCRIPTION (provided by applicant): The broad goal of the project is to understand the mechanisms by which RNA binding protein RBM3 regulates gene expression at the posttranscriptional level of mRNA stability and translation in cancer cells. We have identified that overexpression of the protein causes a normal cell to undergo a transformed phenotype resulting in the cells forming tumors in immunocompromized mice. On the other hand, knockdown of RBM3 expression results in cell death due to mitotic catastrophe. RBM3 interacts with HuR and hnRNP A1, and with AU-RNA sequences to enhance mRNA stability and translation of AU-rich transcripts such as COX-2, VEGF and IL-8. In addition, RBM3 overexpression increases the activation of the mammalian target of rapamycin protein in a Notch dependent mechanism. We have also identified microRNAs regulated upon RBM3 overexpression, including downregulating one that inhibits its own expression. Our studies also suggest that RBM3 is regulated at the posttranslational levels of phosphorylation, ubiquitination and SUMOylation. Based on these observations, we propose three specific aims. In Aim 1, we will determine the mechanism by which RNA binding protein RBM3 regulates gene expression. Here, we will identify the RNA sequences that interact with RBM3. In addition, we will determine the sequenced in COX-2 3'UTR that are required for RBM3-mediated COX-2 mRNA stability and translation. We will also identify the role of HuR and hnRNP-A1 in the process. In Aim 2, we will determine the residues in RBM3 that undergo phosphorylation, ubiquitination and SUMOylation by mutagenesis. We will also determine the effect of the mutants on mRNA stability and translation. In Aim 3, we propose to determine the mechanisms by which RBM3 induces tumorigenesis. We will use an xenograft cancer model to determine the role of mTOR and Notch pathway in RBM3 mediated tumorigenesis. Also, the role of microRNAs in the tumorigenesis will be determined. Completion of these experiments should give us a better understanding of how the RNA binding protein RBM3 functions in normal epithelial cells, and whether changes in the RBM3 expression that is observed in tumor cells is responsible for tumor behavior. PUBLIC HEALTH RELEVANCE: Cancer is the leading cause of death in the United States. Understanding how the normal cell progresses to a cancer will aid in our developing novel therapies for this dreaded disease. We have identified a protein, RBM3 whose expression is increased in cancer cells. Overexpressing RBM3 protein causes a normal cell to become transformed into a cancer cell. Our current proposal deals with identifying mechanisms by which RBM3 expression is regulated, and also how RBM3 induces tumorigenesis. We expect that the work will lead to a better understanding of the tumorigenesis process which should subsequently lead to novel methods to stop or slow down tumorigenesis.