The reactive oxygen-generating enzyme Nox1 transforms NIH 3T3 cells, rendering them highly tumorigenic and, as shown herein, also increases tumorigenicity of DU-145 prostate epithelial cells. Although Nox1 modestly stimulates cell division in both fibroblasts and epithelial cells, an increased mitogenic rate alone did not account fully for the marked tumorigenicity. Herein, we show that Nox1 is a potent trigger of the angiogenic switch, increasing the vascularity of tumors and inducing molecular markers of angiogenesis. Vascular endothelial growth factor (VEGF) mRNA becomes markedly up-regulated by Nox1 both in cultured cells and in tumors, and VEGF receptors (VEGFR1 and VEGFR2) are highly induced in vascular cells in Nox1-expressing tumors. Matrix metalloproteinase activity, another marker of the angiogenic switch, also is induced by Nox1. Nox1 induction of VEGF is eliminated by coexpression of catalase, indicating that hydrogen peroxide signals part of the switch to the angiogenic phenotype.

Oncogenes and tumor suppressor genes are implicated in the regulation of the angiogenic switch. Much of the data accumulated to date uses NIH 3T3 cells, which are deficient in the tumor suppressor gene p16, as models for these studies. We have used a novel system, derived by sequential introduction of a temperature-sensitive SV40 large T antigen and oncogenic H-ras, to study the angiogenic switch. The results from our studies differ from those using NIH3T3 cells, but have been confirmed by multiple other groups. The data from all of these studies suggest that there is synergy between inactivation of the p53 tumor suppressor gene and activation of the phosphoinositol-3-kinase pathway (PI-3-K), as well as synergy between inactivation of the p16 tumor suppressor gene and activation of the MAP kinase pathway. These findings suggest that there are predictable behaviors of tumors that may be assessed by the status of p53 or p16 in a biopsy, and that these predictable changes in signal transduction may be useful both prognostically and in the design of rationally based drug therapy of benign and malignant tumors.