Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, is often overexpressed in cancers. In many in vivo models, inhibition of VBGF function arrests tumor growth. While hypoxia has long been recognized to be a potent inducer of VEGF, VEGF can also be expressed in normoxia. Recent evidence indicates that angiogenesis can develop in tumor masses before they have grown to a size large enough to contain hypoxic regions, suggesting that angiogenic factors may be expressed by these tumors under normoxic conditions. In contrast to the induction of VEGF rnRNA under hypoxia whose mechanism is known to involve the hypoxia-inducible factor-1 (HIF-1), the upregulation of VEGF in normoxia is much less well understood. The overall aim of this grant is to study mechanisms of VEGF upregulation in normoxia by alterations commonly found in cancers: specifically, epidermal growth factor receptor (EGFR) activation and mutations in Ras and PTEN. VEGF mRNA levels and promoter activity in U87 human glioblastoma cells are increased by EGFR stimulation. Introduction of wild type PTEN into U87 cells, in which PTEN is inactivated, decreases VEGF mRNA levels and promoter activity.
Specific Aim 1 will focus on defining the elements in this pathway, which appears to be PI(3) kinase dependent but independent of HIF- 1. H-ras transformation of Rat 1 fibroblasts leads to a six-fold increase in VEGF mRNA expression in normoxia. Furthermore, the level of HIF-1alpha protein is increased in normoxic Rat1-ras cells, an unexpected finding given that HIF-1alpha has traditionally been thought to only be induce under hypoxic conditions. The focus of Specific Aim 2 is to determine whether this increase in HIF- 1 alpha causes the increase in VEGF expression under normoxia.
Aim 2 will also examine the signaling pathways that link Ras activation, HIF-1alpha and VEGF expression. These experiments will lead to a better understanding of HIF-1 alpha regulation, mechanisms of VEGF overexpression in cancers, and the effects of EGFR activation and Ras and PTEN mutations on gene expression.
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