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.
