Regulation of cell growth by RNA binding proteins

描述（由申请人提供）：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节基因表达的分子机制是肿瘤治疗发展的重要一步。mrna中蛋白质的翻译是基因表达的关键事件之一。我的实验室研究了RNA CUG三联体重复结合蛋白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调节多种蛋白质的翻译，包括C/EBP家族成员C/EBP¿，这是肝脏增殖的关键调节因子。CUGBP1结合到C/EBP¿mRNA的5'区，增加显性负分子C/EBP¿-LIP的翻译，从而抑制全长C/EBP蛋白的生物活性，促进肝脏增殖。我们最近在肝脏中发现了CUGBP1的新靶点。我们获得的证据表明，CUGBP1与编码组蛋白去乙酰化酶1 （HDAC1）的mRNA的5'区结合，并增加肿瘤肝脏中HDAC1蛋白的翻译。我们发现CUGBP1的翻译活性主要受CUGBP1磷酸化水平的控制。在肿瘤肝脏和部分肝切除术（PH）后增殖的肝脏中，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是一种磷酸化CUGBP1并增加其翻译活性的激酶。因此，该应用的主要假设是cdk4介导的CUGBP1在肝脏中的激活导致C/EBP¿-LIP和HDAC1的翻译增加并导致肿瘤的发生（见左图）。与这一假设相一致的是，在人肝肿瘤细胞质提取物和部分肝切除术后增殖的小鼠肝细胞质中，cdk4的激活剂cyclin D1和cyclin D3的水平升高。特异性目的1研究了增殖肝脏增加细胞质中cdk4- d型细胞周期蛋白复合物数量的机制。特异性目的2和3确定CUGBP1-C/EBP¿-LIP和CUGBP1-HDAC1途径在肝脏增殖中的作用。我们将验证cugbp1介导的HDAC1升高抑制肝脏特异性肿瘤抑制基因（如C/EBPa）启动子的假设。在本应用中，我们还将确定肝脏在某些条件下（如急性期反应）阻断C/EBP¿-LIP促进生长活性的机制。了解C/EBP¿-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：本项目探讨RNA结合蛋白CUGBP1在肝肿瘤发生和肝部分切除术（PH）后肝脏增殖中的作用。本应用程序的目的是1)检查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发展预防肿瘤的治疗方法提供基础。

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.