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

Abstract

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. This work was supported by Cancer Foundation Finland (to P.M. and P.M.J.), The Sigrid Juselius Foundation (to P.M.J.), Turku University Hospital (EVO1303) (to P.M., H.H. and P.M.J.), Helsinki University Hospital (to P.M. and P.M.J.), The University of Turku Graduate School (to P.M.), and Finnish Cultural Foundation Varsinais-Suomi Regional fund (to P.M.).

Circular RNAs in Clear Cell Renal Cell Carcinoma: Their Microarray-Based Identification, Analytical Validation, and Potential Use in a Clinico-Genomic Model to Improve Prognostic Accuracy

“…For the mentioned five up- and eleven down-regulated circRNAs, the circRNA IDs used in the databases ArrayStar and circBase [22] are summarized in Table 2. To identify circRNAs with a putative function in ccRCC initiation/progression, we selected circRNAs from host genes with putative roles in angiogenesis and hypoxia in ccRCC and other cancers including EGLN3 , NOX4 , and RHOBTB3 [23,24,25,26,27]. Thus, corresponding circRNAs of the three host genes that are named according to the database circBase hsa_circ_0101692, hsa_circ_0023984, and hsa_circ_000744 were selected for further validation (Figure 2C).…”

“…Interestingly, the in-silico analysis for circEGLN3 predicted a potential binding of miR-31-5p and miR-1205 to circEGLN3 and the corresponding linear RNA of EGLN3. EGLN3 is known to contribute to ccRCC initiation [25,26,54] by targeting HIF. The up-regulation of both circEGLN3 and linRNA of EGLN3 in ccRCC and the predicted interaction via miR-31-5p and miR-1205 could reflect a self-sustaining mechanism of the oncogene EGLN3.…”

<p>HIF1α/PD-L1 axis mediates hypoxia-induced cell apoptosis and tumor progression in follicular thyroid carcinoma</p>

“…In response to hypoxia situation, hypoxia-inducible factor (HIF) is upregulated to coordinate an appropriated adaptive response 3. HIF is a highly conserved transcription factor comprising a regulatory α subunit (HIF1α, HIF2α, or HIF3α) and a constitutive β subunit (HIF-β) 4. It has been reported that hypoxia-induced overexpression of HIF1α promotes epithelial–mesenchymal transition in FTC 5.…”

Circ_101341 Deteriorates the Progression of Clear Cell Renal Cell Carcinoma Through the miR- 411/EGLN3 Axis

“…The previous study reported that PHD3 was highly expressed in serum from RCC patients and could be a novel biomarker with the valuable diagnostic performance, 33 and its high level might reinforce ccRCC aggressiveness. 34 Moreover, a tissue-wide expression profiling was generated using cDNA subtraction and microarrays, and EGLN3 was discovered to be highly regulated in RCC samples. 35 These observations suggested that RCC was associated with a high level of EGLN3 .…”