Suppression of hypoxia-inducible factor 1a (HIF-1a) by tirapazamine is dependent on eiF2a phosphorylation rather than the mtorc1/4E-BP1 pathway

Abstract

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Tirapazamine (TPZ), a well-characterized bioreductive anticancer agent, is currently in Phase II and III clinical trials. A major aspect of the anticancer activity of TPZ is its identity as a tumor-specific topoisomerase IIa inhibitor. In the study, for the first time, we found that TPZ acts in a novel manner to inhibit HIF-1a accumulation driven by hypoxia or growth factors in human cancer cells and in HepG2 cell-derived tumors in athymic nude mice. We investigated the mechanism of TPZ on HIF-1a in HeLa human cervical cancer cells by western blot analysis, reverse transcription-PCR assay, luciferase reporter assay and small interfering RNA (siRNA) assay. Mechanistic studies demonstrated that neither HIF-1a mRNA levels nor HIF-1a protein degradation are affected by TPZ. However, TPZ was found to be involved in HIF-1a translational regulation. Further studies revealed that the inhibitory effect of TPZ on HIF-1a protein synthesis is dependent on the phosphorylation of translation initiation factor 2a (eIF2a) rather than the mTOR complex 1/eukaryotic initiation factor 4E-binding protein-1 (mTORC1/4E-BP1) pathway. Immunofluorescence analysis of tumor sections provide the in vivo evidences to support our hypothesis. Additionally, siRNA specifically targeting topoisomerase IIa did not reverse the ability of TPZ to inhibit HIF-1a expression, suggesting that the HIF-1a inhibitory activity of TPZ is independent of its topoisomerase IIa inhibition. In conclusion, our findings suggest that TPZ is a potent regulator of HIF-1a and provide new insight into the potential molecular mechanism whereby TPZ serves to reduce HIF-1a expression. © 2010 Zhang et al.