Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition

Background: Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL) gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-alpha), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1 alpha by Se-Methylselenocysteine (MSC) via stabilization of prolyl hydroxylases 2 and 3 (PHDs) and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-alpha, and VEGF-A in selected solid cancers, the mechanism of HIF-alpha inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts. Methods: Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon) were utilized to determine the incidence of PHD2/3, HIF-alpha, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-alpha inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1 alpha positive), 786-0 (HIF-2 alpha positive) and VHL wild type head & neck cancer cells FaDu (HIF-1 alpha) were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1 alpha by MSC. Results: We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-alpha (92%), and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-alpha in ccRCC tumors. MSC did not inhibit HIF-1 alpha protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1 alpha was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-alpha were associated with significant antitumor activity against ccRCC xenograft. Conclusions: Our results show the role of PHD2/3 in stable expression of HIF-alpha in human ccRCC. Furthermore, HIF-1 alpha degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-alpha regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.