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-α), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α 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-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α 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-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive), 786-0 (HIF-2α positive) and VHL wild type head & neck cancer cells FaDu (HIF-1α) 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-1α by MSC.


RESULTS
We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-α (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-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with significant antitumor activity against ccRCC xenograft.


CONCLUSIONS
Our results show the role of PHD2/3 in stable expression of HIF-α in human ccRCC. Furthermore, HIF-1α degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-α regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.
Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. sreenivasulu.chintala@roswellpark.org
Sreenivasulu
S
Chintala
Tanbir
T
Najrana
Karoly
K
Toth
Shousong
S
Cao
Farukh A
FA
Durrani
Roberto
R
Pili
Youcef M
YM
Rustum
D000818:Animals; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D016566:Organoselenium Compounds / Q000494:pharmacology*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016566:Organoselenium Compounds; D042461:Vascular Endothelial Growth Factor A; C008493:methylselenic acid; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051794:Von Hippel-Lindau Tumor Suppressor Protein