VEGF MRNA STABILIZATION MECHANISMS IN TUMOR ANGIOGENESIS

肿瘤血管生成中的 VEGF mRNA 稳定机制

• 批准号: 6633115
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633115
• 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新抗癌疗法发展的焦点。 肿瘤依赖性血管生成的一个关键要素是血管生成细胞因子（例如VEGF和BFGF）的表达和释放。 通过缺氧以及癌基因和细胞因子激活，在不同的肿瘤细胞类型中大大诱导了VEGF表达。 控制VEGF表达的细胞内机制包括mRNA转录和稳定。我们实验室和其他人的先前研究已经确定，需要在体外​​和体内缺氧和肿瘤血管生成模型中获得VEGF 3'-非翻译区（3'-UTR），以获得适当的和最大的VEGF表达。 我们已经在人VEGF3'-UTR中定义了一种新型的125个基本mRNA元素，该元素指导肿瘤细胞中缺氧依赖性mRNA稳定性，我们称之为缺氧稳定性区域（HSR）。此外，我们已经确定了五种与HSR结合的五种蛋白质中的两种，其中一种HNRNP L显示出与低氧治疗的表达增加和mRNA结合。 HNRNP L与HSR元件与反义寡核苷酸的结合显着抑制VEGF mRNA和缺氧诱导的蛋白质表达，并选择性地将mRNA半衰期降低至对照水平。 另一种RNA结合蛋白是在低氧细胞提取物中检测到的最突出的HSR结合蛋白，已通过RNA接近度色谱法纯化，进一步表征正在进行中。目前尚不清楚导致缺氧VEGF mRNA稳定的特定信号通路。 几条证据表明，活化的蛋白激酶C途径，尤其是PKC Zeta有助于VEGF mRNA稳定。 我们将继续鉴定参与缺氧介导的mRNA稳定的蛋白质，并定义其彼此的潜在相互作用。 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。 该建议的具体目的是：1）识别和表征mRNA结合蛋白及其在人类肿瘤细胞中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，所需的序列元件所需的序列元素对于这种作用和涉及的RNA结合蛋白，以及3）了解VEGF mRNA稳定在缺氧和PKC Zeta激活中对肿瘤血管生成，生长和转移的作用。 这些研究将大大扩展我们对缺氧介导的基因表达的理解，这可能会导致新型抗血管生成/抗癌靶标的发展。