Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway

Many oncogenes induce expression of vascular endothelial growth factor (VEGF), a key factor in tumor angiogenesis. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF regulation. Because hypoxia is a major stimulus for VEGF production, we examined the effects of LY294002, a selective PI3K inhibitor, on hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1alpha induction and phosphorylation under hypoxia. However, HIF-2alpha expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1alpha and HIF-2alpha expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1alpha expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1alpha expression also showed down-regulation of HIF-1alpha after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes.     

Role of Rac1 and Cdc42 in hypoxia induced p53 and von Hippel-Lindau suppression and HIF1alpha activation

Low oxygen tension can influence tumor progression by enhancing angiogenesis, a process that may involve Rho GTPases whose activities have been implicated in tumorigenesis and metastasis. In the present study, we show that hypoxia can increase the mRNA levels and intracellular activities of Rac1 and Cdc42 in a time-dependent manner. The hypoxia-stimulated activities of Rac1 and Cdc42 could be blocked by the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and the protein tyrosine kinase (PTK) inhibitor genistein but were not affected by the p38MAPK inhibitor SB203580 or the MEK-1 inhibitor PD98059, suggesting that the hypoxia-mediated signals were through PI3K and PTK. Correlating with the increased activities of Rac1 and Cdc42, the expression of the pro-angiogenesis factors HIF-1alpha and vascular endothelial growth factor (VEGF) was upregulated by hypoxia, whereas the expression of the tumor suppressors von Hippel-Lindau and p53 was down-regulated. Dominant negative N17Rac1 and N17Cdc42 could upregulate the expression of p53 and pVHL but downregulate that of HIF-1alpha and VEGF under hypoxia. Furthermore, the preconditioned medium from N17Rac1 or N17Cdc42-expressing gastric cancer cells was able to inhibit the proliferation of HUVECs. Our results indicate that PI3K and PTK-mediated activations of Rac1 and Cdc42 are involved in the hypoxia-induced production of angiogenesis-promoting factors and tumor suppressors, and suggest that the Rho family GTPases Rac1 and Cdc42 may contribute to the hypoxia-mediated angiogenesis.     

Growth regulation and co-stimulation of human colorectal cancer cell lines by insulin-like growth factor I, II and transforming growth factor alpha.

We have tested growth factor responsiveness of a panel of eight human colorectal carcinoma cell lines. Insulin-like growth factors I and II (IGF-I and IGF-II) stimulated growth of five lines (HT-29, LS411N, LS513, SW480, WiDr). At 30 ng ml-1 both factors enhanced growth up to 3-fold. They induced half-maximal stimulation at 1.9-6.51 ng ml-1. Even after delayed addition IGF-I and II significantly enhanced growth in a short-term proliferation assay. They exerted maximal effects under limiting serum conditions (0.5% FCS) and at low cell density (1.25-5 x 10(4) ml-1). Using these conditions transforming growth factor alpha (TGF alpha) enhanced proliferation of all IGF-responsive cell lines, except SW480. 1.11-3.31 ng ml-1 were required to obtain a half-maximal response. With 10-20 ng ml-1 maximal stimulation occurred at plateau values different from those for IGF-I/II. Proliferation of all cell lines responsive to both IGF-I and TGF alpha was further enhanced by combining both factors, resulting a synergistic response of LS513, while the effects on HT-29, LS411N and WiDr were additive. With HT-29 and LS411N a 24 h exposure to TGF alpha was sufficient to obtain a full response in the co-stimulatory assay. Our results illustrate the importance of IGF-I/II and TGF alpha as stimulators of growth of colorectal carcinomas.