VEGF MRNA STABILIZATION MECHANISMS IN TUMOR ANGIOGENESIS

肿瘤血管生成中的 VEGF mRNA 稳定机制

• 批准号: 6097465
• 负责人: Kevin P. Claffey
• 金额: $ 27.32万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6097465
• 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.

血管生成和癌症进展之间的联系已成为新抗癌疗法开发的焦点。 肿瘤依赖性血管生成的关键要素之一是血管生成细胞因子（例如 VEGF 和 bFGF）的表达和释放。 缺氧以及癌基因和细胞因子激活在不同肿瘤细胞类型中极大地诱导 VEGF 表达。 控制 VEGF 表达的细胞内机制包括 mRNA 转录和稳定。我们实验室和其他实验室之前的研究已经确定，需要 VEGF 3'-非翻译区 (3'-UTR) 才能获得体外缺氧和体内肿瘤血管生成模型中诱导的 VEGF 适当且最大的表达。 我们在人 VEGF3'-UTR 中定义了一个新的 125 个碱基 mRNA 元件，它指导肿瘤细胞中缺氧依赖性 mRNA 的稳定性，我们将其称为缺氧稳定区 (HSR)。此外，我们还鉴定了与 HSR 结合的五种蛋白质中的两种，其中一种 hnRNP L 在缺氧处理下表现出表达和 mRNA 结合增加。 用反义寡核苷酸对 hnRNP L 与 HSR 元件的结合进行功能性干扰，可显着抑制缺氧诱导的 VEGF mRNA 和蛋白表达，并选择性地将 mRNA 半衰期缩短至对照水平。 另一种 RNA 结合蛋白是缺氧细胞提取物中检测到的最重要的 HSR 结合蛋白，已通过 RNA 亲和层析纯化，进一步表征正在进行中。目前尚不清楚有助于缺氧 VEGF mRNA 稳定的具体信号通路。 多项证据表明，激活的蛋白激酶 C 通路，特别是 PKC zeta，有助于 VEGF mRNA 的稳定。 我们将继续鉴定参与缺氧介导的 mRNA 稳定的蛋白质，并确定它们之间潜在的相互作用。 此外，我们将定义 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。 该提案的具体目标是：1) 鉴定和表征 mRNA 结合蛋白及其在人肿瘤细胞中 VEGF mRNA 稳定中的功能，2) 定义蛋白激酶 C 同工型在 VEGF mRNA 稳定中的作用，以及所需的序列元件了解这种效应和所涉及的 RNA 结合蛋白，以及 3) 了解缺氧时 VEGF mRNA 稳定和 PKC zeta 激活对肿瘤血管生成、生长和转移的作用。 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，这将有可能导致新型抗血管生成/抗癌靶点的开发。