Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines

Hypoxia is an important regulator of vascular endothelial growth factor (VE GF) expression, and VEGF is associated with poor prognosis in bladder cance r. To investigate further the mechanisms of VEGF regulation, we examined VE GF expression by mRNA and protein analysis in four human bladder cancer cel l lines, shelving a progression from well to poorly differentiated phenotyp es under varying conditions of confluence and hypoxia (0.1% O-2) and with c hemical mimic of hypoxia, Hypoxia significantly increased VEGF protein expr ession in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia in ducibility at confluence, whereas inducibility was maintained in the invasi ve cell lines 253J and EJ28, This pattern of VEGF expression in the invasiv e cell Lines correlated with the expression of the transcription factor hyp oxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible fact or-2 alpha. (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-ki nase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-indu cible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-depen dent pathway, which regulates basal amounts of VEGF, but does not affect in ducibility, Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regul ated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results sug gest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in blad der cancer and are therapeutic targets in this disease.