Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1α

Hypoxia-inducible factor 1α (HIF-1α) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1α protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1α inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1α. protein via a VHL-independent mechanism and reduction of HIF-1α transcriptional activity. HIF-1α inhibition by LAQ824 was associated with HIF-1α acetylation and polyubiquitination. HIF-1α immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1α. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1α at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1α only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1α protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1α protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1α stability and provide a rationale for targeting HIF-1α with HDAC inhibitors against class II isozymes.