Oncogenes as inducers of tumor angiogenesis.

Dominantly acting transforming oncogenes are generally considered to contribute to tumor development and progression by their direct effects on tumor cell proliferation and differentiation. However, the growth of solid tumors beyond 1-2 mm in diameter requires the induction and maintenance of a tumor blood vessel supply, which is attributed in large part to the production of angiogenesis promoting growth factors by tumor cells. The mechanisms which govern the expression of angiogenesis growth factors in tumor cells are largely unknown, but dominantly acting oncogenes may have a much greater impact than hitherto realized. An example of this is the induction of expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by mutant H- or K-ras oncogenes, as well as v-src and v-raf, in transformed fibroblasts or epithelial cells. Besides VEGF/VPF, mutant ras genes are known to upregulate the expression of a variety of other growth factors thought to have direct or indirect stimulating effects on angiogenesis, e.g. TGF-beta and TGF-alpha. This effect may be mediated through the ras-raf-MAP kinase signal transduction pathway, resulting in activation of transcription factors such as AP1, which can then bind to relevant sites in the promoter regions of genes encoding angiogenesis growth factors. In principle, similar events could take place after activation or overexpression of many other oncogenes, especially those which can mediate their function through ras-dependent signal transduction pathways. The regulatory effect of oncogenes on mediators of angiogenesis has some potentially important therapeutic consequences. For example, it strengthens the rationale of pharmacologically targeting oncogene products, such as mutant RAS proteins, as an anti-tumor therapeutic strategy. Such drugs may attack the source of one or more angiogenic growth factors and by doing so, function, at least in part, as anti-angiogenic agents in vivo.

Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells.

Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant 125I-VEGF165 to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.