Clear renal cell carcinoma (ccRCC) is a highly vascularized and proliferative tumor. ccRCC can be sporadic or familiar, usually associated with von Hippel-Lindau (VHL) gene mutations. VHL protein is part of the E3 ubiquitin ligase complex that regulates hypoxia-inducible factor (HIF). When a defective and inactive VHL protein is produced, HIF is not degraded, resulting in over-expression of hypoxia related genes including erythropoietin (EPO) and vascular endothelial growth factor (VEGF), thus promoting angiogenesis, proliferation and tumorogenesis. We analyzed tumor and surrounding healthy tissue from radical nephrectomy of 43 ccRCC patients. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) of the VHL gene in tumors and surrounding healthy tissues revealed somatic mutations in 27/43 (62.8%) of ccRCC samples. By testing genetic alteration or loss of heterozygosity (LOH), we detected 23 biallelic and 4 monoallelic VHL inactivations among 27 samples with genetic alteration in VHL gene. We also detected an increase in VEGF mRNA expression in ccRCC by 30 fold compared to healthy tissue. To assess how HIF-1 affects VEGF mRNA, tumors were divided in HIF-1 negative and HIF-1 positive samples. Increased VEGF mRNA expression was observed in both groups with the most prominent induction in HIF-1 positive tumor cells in comparison to normal renal tissue. We observed a significant induction of VEGF mRNA expression in wild type tumor samples compared to normal renal tissue, and a progressive increase in monoallelic and biallelic inactivation of VHL. Western blotting confirmed higher expression of VEGF protein in ccRCC. Beyond VEGF, EPO is also a HIF-responsive gene. Western blots revealed that EPO protein expression was significantly decreased in tumors without HIF-1 expression, while HIF-1 presence in tumor indicated a strong upregulation compared to HIF-1 negative tumors. Alterations in VHL gene did not change the expression of EPO protein. EPO acts by binding to its receptor-EPOR. Alike EPO, the expression of EPOR was decreased in tumors with the lack of HIF-1 protein, while HIF-1 presence induced EPOR expression. Alterations in VHL gene influenced the expression of EPOR protein. The expression of EPOR protein was very similar among control healthy tissue, tumors without mutations and tumors with bialllelic inactivation of VHL gene expression. The only change was observed in tumor with monoallelic inactivation of VHL as a downregulation of EPOR protein expression compared to other groups. We demonstrated increased VEGF level in ccRCC supported by HIF-1, while EPO/EPOR expression were decreased in ccRCC reverted by HIF-1 presence.
18 - 21 May 2019
European Society of Endocrinology