VEGF MRNA STABILIZATION MECHANISMS IN TUMOR ANGIOGENESIS

肿瘤血管生成中的 VEGF mRNA 稳定机制

• 批准号: 6721470
• 负责人: Kevin P. Claffey
• 金额: $ 25.07万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-05 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6721470
• 关键词: RNA binding protein; angiogenesis; athymic mouse; biological signal transduction; carcinogenesis; chimeric proteins; enzyme activity; gene expression; genetically modified animals; growth factor receptors; hypoxia; luciferin monooxygenase; messenger RNA; metastasis; molecular cloning; neoplastic growth; northern blottings; nucleic acid sequence; phorbols; posttranscriptional RNA processing; protein kinase C; transfection; vascular endothelial growth factors; vascular endothelium

The connection between angiogenesis and cancer progression has become a focal point in the development of new anti-cancer therapies. One key element in tumor-dependent angiogenesis is the expression and release of angiogenic cytokines such as VEGF and bFGF. VEGF expression is greatly induced in different tumor cell types by hypoxia as well as oncogene and cytokine activation. Intracellular mechanisms which control VEGF expression include both mRNA transcription and stabilization. Previous investigations from our lab and others have determined that the VEGF 3'-untranslated region (3'-UTR) is required to obtain appropriate and maximal expression of VEGF induced by hypoxia in vitro and in tumor angiogenesis models in vivo. We have defined a novel 125 base mRNA element in the human VEGF3'-UTR which directs hypoxia-dependent mRNA stabilization in tumor cells, which we have termed Hypoxia Stability Region (HSR). In addition, we have identified two of five proteins which bind to the HSR, one of which, hnRNP L, shows increased expression and mRNA binding with hypoxic treatment. Functional interference of the binding of hnRNP L to the HSR element with an antisense oligonucleotide significantly represses VEGF mRNA and protein expression induced by hypoxia, and selectively reduced mRNA half-life to control levels. An additional RNA-binding protein which is the most prominent HSR-binding protein detected in hypoxic cell extracts, has been purified by RNA-affinity chromatography and further characterization is in progress. Specific signaling pathways which contribute to hypoxic VEGF mRNA stabilization is currently unclear. Several lines of evidence suggest that activated protein kinase C pathways, and in particular, PKC zeta, contributes to VEGF mRNA stabilization. We will continue to identify proteins involved in hypoxia-mediated mRNA stabilization and define their potential interactions with each other. In addition, we will define the role of the VEGF HSR and its RNA-binding proteins in tumor angiogenesis, growth and metastasis in vivo. The specific aims of this proposal are to: 1) identify and characterize mRNA binding proteins and their function in VEGF mRNA stabilization in human tumor cells, 2) define the role of protein kinase C isoforms in the stabilization of VEGF mRNA, the sequence elements required for this effect and the RNA-binding proteins involved, and 3) understand the role of VEGF mRNA stabilization with hypoxia and PKC zeta activation to tumor angiogenesis, growth and metastasis. These investigations will greatly expand our understanding of hypoxia-mediated gene expression which will potentially lead to the development of novel anti-angiogenic/anti-cancer targets.

血管生成与癌症进展之间的联系已成为开发新的抗癌疗法的焦点。肿瘤依赖性血管生成的一个关键因素是血管生成细胞因子如VEGF和bFGF的表达和释放。在不同类型的肿瘤细胞中，缺氧以及癌基因和细胞因子的激活均可显著诱导VEGF的表达。细胞内控制VEGF表达的机制包括mRNA转录和稳定。我们实验室和其他研究人员先前的研究已经确定，在体外缺氧和体内肿瘤血管生成模型中，VEGF 3'-非翻译区（3'-UTR）需要获得适当和最大的VEGF表达。我们在人VEGF3'-UTR中定义了一个新的125碱基mRNA元件，该元件指导肿瘤细胞缺氧依赖的mRNA稳定，我们将其称为缺氧稳定区（HSR）。此外，我们已经确定了与HSR结合的5个蛋白中的2个，其中一个hnRNP L在缺氧处理下表达和mRNA结合增加。用反义寡核苷酸干扰hnRNP L与HSR元件结合的功能，可显著抑制缺氧诱导的VEGF mRNA和蛋白表达，选择性地降低mRNA半衰期至控制水平。另一个rna结合蛋白是在缺氧细胞提取物中检测到的最突出的hsr结合蛋白，已被rna亲和层析纯化，进一步的表征正在进行中。促进缺氧VEGF mRNA稳定的特定信号通路目前尚不清楚。一些证据表明，活化的蛋白激酶C通路，特别是PKC zeta，有助于VEGF mRNA的稳定。我们将继续确定参与缺氧介导的mRNA稳定的蛋白质，并确定它们之间潜在的相互作用。此外，我们将明确VEGF HSR及其rna结合蛋白在体内肿瘤血管生成、生长和转移中的作用。本课题的具体目的是：1)鉴定和表征mRNA结合蛋白及其在人肿瘤细胞中稳定VEGF mRNA的功能；2)确定蛋白激酶C同工型在稳定VEGF mRNA中的作用，此作用所需的序列元件和所涉及的rna结合蛋白；3)了解缺氧和PKC zeta活化稳定VEGF mRNA在肿瘤血管生成、生长和转移中的作用。这些研究将极大地扩展我们对缺氧介导的基因表达的理解，这将有可能导致新的抗血管生成/抗癌靶点的开发。