Overproduction of vascular endothelial growth factor related to von Hippel-Lindau tumor suppressor gene mutations and hypoxia-inducible factor-1α expression in renal cell carcinomas

Purpose: The von Hippel-Lindau (VHL) tumor suppressor gene is frequently inactivated in the common type of sporadic clear cell renal cell carcinoma (RCC) as well as RCCs associated with VHL disease. The VHL protein targets hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor that can induce vascular endothelial growth factor (VEGF) expression, for ubiquitination and degradation. Accumulation of HIF-1alpha caused by mutant VHL protein in tumor cells may result in VEGF over expression, which has been used to explain the increased vascularity of RCC. However, quantitative analyses of VEGF production and its correlation with VHL mutations and HIF-1alpha expression in authentic tissues from patients with RCC are lacking. Materials and Methods: We analyzed VHL gene mutations by direct DNA sequencing and methylation specific polymerase chain reaction in 31 paired RCC tissue samples. HIF-1alpha protein expression detected by immunoblotting and immunohistochemical staining, and VEGF protein measured by enzyme-linked immunosorbent assay and immunohistochemical staining were performed using tumor and corresponding normal tissues. Results: VHL gene mutations were detected in 44% of clear cell RCCs but no differences in methylation patterns in the promoter or exon 1 were found. RCCs with VHL gene mutations or of advanced grade produced significantly higher concentrations of VEGF (p < 0.0001). HIF-1alpha protein expression was found in 40% of clear cell RCCs but 80% of them had VHL mutations (p < 0.006). HIF-1alpha expression correlated directly with higher levels of VEGF production (p < 0.0001). Conclusions: Our findings indicate that VHL gene alterations and HIF-1a protein expression correlate with a significant increase in VEGF production by RCC. In turn it is associated with a more aggressive tumor phenotype.