VEGF MRNA STABILIZATION MECHANISMS IN TUMOR ANGIOGENESIS

血管生成与肿瘤进展之间的关系已成为抗癌新疗法发展的焦点。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的表达和释放，如血管内皮生长因子和碱性成纤维细胞生长因子。在不同类型的肿瘤细胞中，低氧、癌基因和细胞因子的激活可显著诱导血管内皮生长因子的表达。控制血管内皮生长因子表达的细胞内机制包括转录和稳定两方面。我们实验室和其他实验室的研究已经确定，在体外低氧诱导和体内肿瘤血管生成模型中，需要VEGF3‘-非翻译区(3’-UTR)才能获得适当和最大程度的表达。我们在人血管内皮生长因子3‘-非编码区中定义了一个新的125碱基的信使核糖核酸元件，它指导肿瘤细胞的低氧依赖的信使核糖核酸的稳定，我们称之为低氧稳定区(HSR)。此外，我们还鉴定了与HSR结合的五种蛋白质中的两种，其中一种是hnRNP L，它在低氧处理后表达增加，并与mR-NA结合。反义寡核苷酸对hnRNP L与HSR元件结合的功能干扰显著抑制缺氧诱导的VEGFmRNA和蛋白表达，并选择性地将mRNA半衰期降低到对照水平。另外一个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，它已经通过RNA亲和层析得到纯化，进一步的鉴定正在进行中。目前，参与缺氧时血管内皮细胞生长因子基因稳定的具体信号通路尚不清楚。一些证据表明，激活的蛋白激酶C途径，特别是PKC Zeta，有助于血管内皮生长因子mRNA的稳定。我们将继续鉴定与低氧介导的mRNA稳定有关的蛋白质，并确定它们之间的潜在相互作用。此外，我们还将确定VEGFHSR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。本研究的具体目的是：1)鉴定和鉴定人肿瘤细胞中的mRNA结合蛋白及其在稳定血管内皮生长因子中的作用；2)确定蛋白激酶C亚型在稳定血管内皮生长因子中的作用、这一作用所需的序列元件和涉及的RNA结合蛋白；3)了解缺氧和PKC Zeta激活对肿瘤血管生成、生长和转移的稳定作用。这些研究将极大地扩展我们对低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。

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.