Oncogenes and tumor angiogenesis: Differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts

A possible link between oncogenes and tumor angiogenesis has been implicate d by the finding that expression of various oncogenes, particularly mutant ms, can lead to a marked induction of a potent paracrine stimulator of angi ogenesis, vascular endothelial growth factor (VEGF), We sought to determine how oncogenic ras induction of VEGF is mediated at the molecular level and whether the mechanisms involved differ fundamentally between transformed e pithelial cells and fibroblasts. Our results suggest that in a subline (cal led RAS-3) of immortalized nontumorigenic rat intestinal epithelial cells ( IEC-18) that acquired a tumorigenic phenotype upon transfection of mutant r as, up-regulation of VEGF occurs in the absence of an autocrine growth fact or circuit. The expression of VEGF mRNA and protein by RAS-3 cells was stro ngly suppressed in the presence of LY294002, an inhibitor of phosphatidylin ositol 3'-kinase, but remained largely unaffected in the same cells treated with an inhibitor (PD98059) of mitogen-activated protein/extracellular sig nal-regulated kinase kinase 1 (MKK/MEK-1). This is consistent with the obse rvation that overexpression of a constitutively activated mutant of MEK-1 ( Delta N3/S222D) in the parental IEC-18 cells did not result in up-regulatio n of VEGF production. The impact of mutant ras on VEGF expression was also significantly amplified at high cell density, conditions under which RAS-3 cells became less sensitive to LY294002-induced VEGF downregulation. In marked contrast to cells of epithelial origin, ras-transformed murine fi broblasts (3T3RAS) up-regulated VEGF in a manner that was strongly inhibita ble by MEK-1 blockade (i.e. treatment with PD98059), whereas these cells we re relatively unaffected by treatment with the phosphatidylinositol 3'-kina se inhibitor LY294002. In addition, VEGF was upregulated by 2-3-fold in NIH 3T3 cells overexpressing mutant MEK-1, Collectively, the data suggest that the stimulatory effect of mutant ms on VEGF expression is executed in a non autocrine and cell type-dependent manner and that it can be significantly e xacerbated by physiological/environmental influences such as high cell dens ity.