Mouse anti-Human EGLN3 monoclonal antibody, clone 4D6

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 -subunits (HIF-) and their downstream targets. HIF-2 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-2 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 down-regulation of HIF-2 protein and mRNA. Depletion of other PHD family members had no effect on HIF-2 expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2 protein expression. Accordingly, PHD3 knockdown decreased HIF-2 target gene expression in ccRCC cells and expression was restored upon forced HIF-2 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-2 in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2 expression and that of its target genes, which may enhance kidney cancer aggressiveness.

Renal cell carcinoma (RCC) is the most common cancer arising from the kidney in adults, with clear cell RCC(ccRCC) representing the majority of all RCCs. Expression of a human HIF1 alpha triple-mutant (P402A, P564A, and N803A) construct in the proximal tubule cells of C57BL/6 mice [TRAnsgenic model of Cancer of the Kidney (TRACK); ref. 1] mimics the histologic changes found in early stage human ccRCC. To better understand the genomic landscape, a high-throughput sequence analysis was performed with cDNA libraries (RNAseq) derived from TRACK transgenic positive (TG(+)) kidney cortex along with human ccRCC transcripts from the Oncomine and The Cancer Genome Atlas databases. Importantly, the expression profiles of TRACK TG(+) kidneys show significant similarities with those observed in human ccRCC, including increased expression of genes involved in glycolysis and the tricarboxylic acid cycle. Some of the transcripts overexpressed in both the TRACK mouse model and human ccRCC include ANKRD37, CA9, EGLN3, HK2, NDUFA4L2, and SLC16A3. These data suggest that constitutive activation of HIF1 alpha in kidney proximal tubule cells transcriptionally reprograms the regulation of metabolic pathways in the kidney and that HIF1a is a major contributor to the altered metabolism observed in human ccRCC. (C)2015 AACR.