Regulation of cell growth by RNA binding proteins

描述(申请人提供)：肝脏肿瘤的发展涉及许多基因表达的改变。阐明正常细胞和癌细胞中调控基因表达的分子机制是发展肿瘤治疗的重要一步。蛋白质的翻译是基因表达中的关键事件之一。我的实验室研究了RNA CUG三联体重复序列结合蛋白CUGBP1的生物学活性，以及该蛋白在肝脏增殖和肝脏肿瘤中的作用。在肝脏中，CUGBP1调节几种蛋白质的翻译，包括C/EBP家族的成员C/EBP？，它是肝脏增殖的关键调节因子。CUGBP1与C/EBP？m RNA的5‘端结合，增加优势负性分子C/EBP？-LIP的翻译，从而抑制全长C/EBP蛋白的生物学活性，促进肝脏增殖。我们最近在肝脏中发现了一个新的CUGBP1靶点。我们获得的证据表明，CUGBP1结合到编码组蛋白脱乙酰基酶1 HDAC1的5‘端mRNA区域，并增加了HDAC1蛋白在肿瘤肝脏中的翻译。我们发现，CUGBP1的翻译活性主要受CUGBP1的磷酸化水平控制。在肿瘤肝脏和部分肝切除(PH)后的增殖肝脏中，CUGBP1被过度磷酸化，这种磷酸化增加了CUGBP1的翻译活性。我们发现CDK4是一种使CUGBP1磷酸化并增加其翻译活性的激酶。因此，这一应用的主要假设是CDK4介导的CUGBP1在肝脏中的激活导致C/EBP？LIP和HDAC1翻译增加，从而导致肿瘤的发生(见左图)。与这一假说相一致的是，CDK4的激活剂细胞周期蛋白D1和D3在人肝肿瘤细胞质提取液和小鼠肝部分切除后增殖的细胞质中表达增加。具体目的1研究增殖的肝脏增加细胞质中CDK4-D型细胞周期蛋白复合体的数量的机制。特异性AIMS 2和3决定了CUGBP1-C/EBP？-LIP和CUGBP1-HDAC1通路在肝脏增殖中的作用。我们将检验这样一种假设，即CUGBP1介导的HDAC1的上调抑制了肝脏特异性肿瘤抑制基因的启动子，如C/EBPA。在这项应用中，我们还将确定在某些条件下，如急性时相反应，肝脏阻断C/EBP？-LIP促生长活性的机制。了解C/EBPβ-LIP和HDAC1在肿瘤中升高的机制将有助于开发预防肝脏恶性转化的方法。公共卫生相关性：本项目研究RNA结合蛋白CUGBP1在肝肿瘤发生和肝部分切除(PH)后肝脏增殖中的作用。这项应用的目标是1)研究RNA结合蛋白在肝脏肿瘤发展中的作用；2)阐明增殖肝脏激活RNA结合蛋白的机制；3)确定增殖肝脏中RNA结合蛋白CUGBP1的下游靶点。阐明肝脏恶性转化的分子机制将为开发预防肿瘤的治疗方法提供依据。

DESCRIPTION (provided by applicant): Development of liver tumors involves alterations in expression of many genes. The elucidation of molecular mechanisms that regulate gene expression in normal cells and in cancer cells is an important step for the development of therapeutic treatments of the tumor. Translation of proteins from mRNAs is one of the critical events in the expression of the genes. My laboratory investigates biological activities of RNA CUG triplet repeat binding protein, CUGBP1, and the role of this protein in liver proliferation and in liver tumors. In the liver, CUGBP1 regulates translation of several proteins including a member of C/EBP family, C/EBP¿, which is a key regulator of liver proliferation. CUGBP1 binds to the 5' region of C/EBP¿ mRNA and increases translation of a dominant negative molecule C/EBP¿-LIP, which inhibits biological activities of full-length C/EBP proteins and promotes liver proliferation. We have recently identified a new target of CUGBP1 in the liver. We obtain evidence that CUGBP1 binds to the 5' region of mRNA coding for a histone deacetylase 1, HDAC1, and increases translation of HDAC1 protein in tumor liver. We found that the translational activity of CUGBP1 is controlled mainly on the level of phosphorylation of CUGBP1. In tumor livers and in livers proliferating after partial hepatectomy (PH), CUGBP1 is hyper-phosphorylated and this phosphorylation increases translational activities of CUGBP1. We have found that cdk4 is a kinase which phosphorylates CUGBP1 and increases its translational activity. Therefore, the major hypothesis of this application is that the cdk4-mediated activation of CUGBP1 in the liver leads to the increased translation of C/EBP¿-LIP and HDAC1 and to development of tumor (see diagram on the left). Consistent with this hypothesis, levels of cyclin D1 and cyclin D3, which are activators of cdk4, are increased in cytoplasmic extracts of human liver tumors and in cytoplasm of mouse liver proliferating after partial hepatectomy. Specific Aim 1 examines mechanisms by which proliferating livers increase amounts of cdk4-D-type cyclins complexes in cytoplasm. Specific Aims 2 and 3 determine the role CUGBP1-C/EBP¿-LIP and CUGBP1-HDAC1 pathways in liver proliferation. We will test the hypothesis that CUGBP1-mediated elevation of HDAC1 inhibits promoters of liver specific tumor suppressor genes such as C/EBPa. In this application, we will also determine mechanisms by which liver blocks growth promotion activities of C/EBP¿-LIP under certain conditions such as Acute Phase Response. The understanding of the mechanisms of the elevation of C/EBP¿-LIP and HDAC1 in tumor will help to develop approaches to prevent malignant transformations in the liver. Public Health Relevance: This project investigates the role of RNA binding protein CUGBP1 in development of liver tumors and in the liver proliferation after partial hepatectomy (PH). The goals of this application are 1) to examine the role of RNA binding proteins in the development of liver tumors; 2) elucidate mechanisms by which proliferating livers activate RNA binding proteins; and 3) determine down-stream targets of RNA binding protein CUGBP1 in proliferating livers. The elucidation of molecular mechanisms of malignant transformations in liver will provide basement for the development of therapeutic approaches to prevent tumors.