Hypoxia-Inducible Factor 1 Regulates Vascular Endothelial Growth Factor Expression in Human Pancreatic Cancer

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Abstract
The microenvironment of low oxygen that is present in human pancreatic cancer in vivo may actively influence tumor growth as well as neovascularization. To determine whether hypoxia-inducible factor 1 (HIF-1) is specifically activated by hypoxia in vitro in pancreatic cancer cells and correlated these findings with tumor specimens. Hypoxic regulation of vascular endothelial growth factor (VEGF) was studied by northern blot analysis and enzyme-linked immunosorbent assay. Electrophoretic mobility shift assays and western blot analysis were used to demonstrate hypoxic activation of HIF-1. The relationship between HIF-1 and VEGF in human pancreatic cancer specimens was studied by immunohistochemical analysis, northern blot analysis, and in situ hybridization. Studies in vivo of human pancreatic cancer tissue showed co-localization of VEGF mRNA, which is produced in ductal cancer cells, and HIF-1α protein, which was detectable in cell nuclei of the same cells. HIF-1α mRNA expression was dramatically upregulated (≈13-fold) in these specimens as well. In vitro, all pancreatic cancer cell lines increased VEGF production when exposed to low oxygen levels, by highly specific activation of HIF-1 DNA binding activity to the VEGF promoter. Cancer cell lines with high constitutive levels of HIF-1α protein were found to produce higher basal levels of VEGF. We conclude that HIF-1 is the regulatory link between tumor hypoxia and VEGF production in pancreatic cancer, thus establishing a biochemical pathway between tumor hypoxia and neoangiogenesis in this highly aggressive neoplasm.