Hypoxia-inducible factor-2α:: effect on radiation sensitivity and differential regulation by an mTOR inhibitor

To determine the role of hypoxia-inducible factor-2 alpha (HIF2 alpha) on the sensitivity of renal cell carcinoma (RCC) cell lines to ionizing radiation and to determine if the mTOR antagonist, rapamycin, could decrease HIF2 alpha protein levels. Cell lines expressing stable short-hairpin RNAs (shRNAs) encoding HIF2 alpha shRNAs or an empty vector were transfected with a hypoxia responsive element (HRE)-driven firefly luciferase reporter gene. Two separate paired cell lines were assayed for their response to increasing doses of ionizing radiation. Proliferation and cell cycle kinetics were compared for cell lines expressing HIF2 alpha shRNAs and empty vectors. The effect of an mTOR antagonist, rapamycin on HIF1 alpha and HIF2 alpha proteins levels was also assessed. We confirmed that the 786-O RCC lines with stably integrated shRNAs against HIF2 alpha had decreased activation of a plasmid with a HRE-driven firefly luciferase reporter gene. Lines from two separate cell clones with decreased HIF2 alpha levels showed a significant increase in radiation sensitivity and an increase in G2 cell cycle arrest. Rapamycin, while effective in decreasing HIF1 alpha protein levels, did not affect HIF2 alpha levels in either of the RCC cell lines. These results show that decreasing levels of HIF2 alpha leads to an increased sensitivity to ionizing radiation. This finding may explain in part, the known resistance of RCC to radiation therapy. Although mTOR antagonists are approved for the treatment of RCC, these agents do not decrease HIF2 alpha levels and therefore might not be effective in enhancing the radio-sensitivity of these tumours.