Hypoxia-inducible factor (HIF)-prolyl hydroxylase 3 (PHD3) maintains high HIF2A mRNA levels in clear cell renal cell carcinoma

Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) resulting in the accumulation of hypoxia-inducible factor a-subunits (HIF-a) and their downstream targets. HIF-2a expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2a protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to downregulation of HIF-2a protein and mRNA. Depletion of other PHD family members had no effect on HIF-2a expression and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2a protein expression. Accordingly, PHD3 knockdown decreased HIF-2a target gene expression in ccRCC cells and expression was restored upon forced HIF-2a expression. The effect of PHD3 depletion was pinpointed to HIF2A mRNA stability. In line with these in vitro results, a strong positive correlation of PHD3 and HIF2A mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2a in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2a expression and that of its target genes, which may enhance kidney cancer aggressiveness.