RNA Binding Proteins in Cancer

描述（由申请人提供）：该项目的广泛目标是了解RNA结合蛋白RBM 3在癌细胞中mRNA稳定性和翻译的转录后水平调节基因表达的机制。我们已经确定，蛋白质的过表达导致正常细胞经历转化表型，导致细胞在免疫受损小鼠中形成肿瘤。另一方面，RBM 3表达的敲低导致由于有丝分裂灾难的细胞死亡。RBM 3与HuR和hnRNP A1相互作用，并与AU-RNA序列相互作用，以增强mRNA稳定性和富含AU的转录物如考克斯-2、VEGF和IL-8的翻译。此外，RBM 3过表达在Notch依赖性机制中增加雷帕霉素蛋白的哺乳动物靶标的活化。我们还鉴定了在RBM 3过表达时受调节的microRNA，包括下调抑制其自身表达的microRNA。我们的研究还表明，RBM 3在翻译后水平的磷酸化，泛素化和SUMO化的调节。根据这些观察，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RBM 3调节基因表达的机制。在这里，我们将识别与RBM 3相互作用的RNA序列。此外，我们将确定RBM 3介导的考克斯-2 mRNA稳定性和翻译所需的考克斯-2 3 'UTR中的测序。我们还将确定HuR和hnRNP-A1在此过程中的作用。在目标2中，我们将通过诱变确定RBM 3中经历磷酸化、泛素化和SUMO化的残基。我们还将确定突变体对mRNA稳定性和翻译的影响。在目标3中，我们提出确定RBM 3诱导肿瘤发生的机制。我们将使用异种移植癌症模型来确定mTOR和Notch通路在RBM 3介导的肿瘤发生中的作用。此外，microRNA在肿瘤发生中的作用也将被确定。这些实验的完成应该让我们更好地了解RNA结合蛋白RBM 3在正常上皮细胞中的功能，以及在肿瘤细胞中观察到的RBM 3表达的变化是否是肿瘤行为的原因。公共卫生相关性：癌症是美国的主要死亡原因。了解正常细胞如何发展为癌症将有助于我们开发针对这种可怕疾病的新疗法。我们已经鉴定了一种蛋白质RBM 3，其在癌细胞中的表达增加。过度表达RBM 3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RBM 3表达调节的机制，以及RBM 3如何诱导肿瘤发生。我们希望这项工作将导致对肿瘤发生过程的更好理解，随后将导致新的方法来阻止或减缓肿瘤发生。

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.