VEGF MRNA STABILIZATION MECHANISMS IN TUMOR ANGIOGENESIS

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中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。 VEGF的表达在不同的肿瘤细胞类型中被缺氧以及癌基因和细胞因子激活极大地诱导。 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，VEGF 3 '-非翻译区（3'-UTR）是获得体外缺氧诱导的VEGF适当和最大表达所必需的，并且在体内肿瘤血管生成模型中。 我们已经在人VEGF 3 '-UTR中定义了一个新的125个碱基的mRNA元件，其指导肿瘤细胞中的低氧依赖性mRNA稳定，我们将其称为低氧稳定区（HSR）。此外，我们已经确定了两个五种蛋白结合的HSR，其中之一，hnRNP L，显示增加的表达和mRNA结合与缺氧处理。 用反义寡核苷酸对hnRNP L与HSR元件的结合进行功能性干扰显著抑制由缺氧诱导的VEGF mRNA和蛋白质表达，并选择性地将mRNA半衰期降低至对照水平。 一个额外的RNA结合蛋白，这是在缺氧细胞提取物中检测到的最突出的HSR结合蛋白，已被纯化的RNA亲和层析和进一步的表征正在进行中。目前还不清楚有助于低氧VEGF mRNA稳定的特定信号传导途径。 一些证据表明，活化的蛋白激酶C途径，特别是PKC ζ，有助于VEGF mRNA的稳定。 我们将继续鉴定参与低氧介导的mRNA稳定化的蛋白质，并确定它们之间的潜在相互作用。 此外，我们将确定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。 这项建议的具体目标是：1）鉴定和表征mRNA结合蛋白及其在人肿瘤细胞中VEGF mRNA稳定化中的功能，2）确定蛋白激酶C同种型在VEGF mRNA稳定化中的作用，该作用所需的序列元件和所涉及的RNA结合蛋白，3）了解缺氧条件下VEGF mRNA的稳定和PKC zeta的激活在肿瘤血管生成、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，这将潜在地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.