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

HIF prolyl hydroxylase PHD3 regulates translational machinery and glucose metabolism in clear cell renal cell carcinoma.

BACKGROUND: A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear. METHODS: To gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis. RESULTS: Our data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling. CONCLUSIONS: Our findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.

ROTS: reproducible RNA-seq biomarker detector-prognostic markers for clear cell renal cell cancer.

Recent comprehensive assessments of RNA-seq technology support its utility in quantifying gene expression in various samples. The next step of rigorously quantifying differences between sample groups, however, still lacks well-defined best practices. Although a number of advanced statistical methods have been developed, several studies demonstrate that their performance depends strongly on the data under analysis, which compromises practical utility in real biomedical studies. As a solution, we propose to use a data-adaptive procedure that selects an optimal statistic capable of maximizing reproducibility of detections. After demonstrating its improved sensitivity and specificity in a controlled spike-in study, the utility of the procedure is confirmed in a real biomedical study by identifying prognostic markers for clear cell renal cell carcinoma (ccRCC). In addition to identifying several genes previously associated with ccRCC prognosis, several potential new biomarkers among genes regulating cell growth, metabolism and solute transport were detected.

The regulation, localization, and functions of oxygen-sensing prolyl hydroxylase PHD3.

The prolyl 4-hydroxylase domain protein 3 (PHD3) belongs to 2-oxoglutarate and iron-dependent dioxygenases. Together with the two closest paralogues, PHD1 and PHD2, these enzymes have been identified as cellular oxygen sensors that can mark the hypoxia-inducible factor α (HIF-α) for von Hippel-Lindau protein-mediated proteasomal destruction. Although having overlapping functions with PHD1 and PHD2, PHD3 markedly differs from the two isoforms. PHD3 shows a different expression pattern and subcellular localization as well as activity under low oxygen tension. Moreover, it has the widest range of non-HIF targets underlying its diverse functions. The functions of PHD3 differ depending on the cell type and also partially on the microenvironmental conditions it is expressed at. Under normoxia, PHD3 has been shown to be proapoptotic, but under hypoxia, it can have cell survival or proliferation-supporting functions. Here we discuss the regulation, targets, and functions of PHD3.

p62/SQSTM1 regulates cellular oxygen sensing by attenuating PHD3 activity through aggregate sequestration and enhanced degradation.

The hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 regulates cellular responses to hypoxia. In normoxia the expression of PHD3 is low and it occurs in cytosolic aggregates. SQSTM1/p62 (p62) recruits proteins into cytosolic aggregates, regulates metabolism and protein degradation and is downregulated by hypoxia. Here we show that p62 determines the localization, expression and activity of PHD3. In normoxia PHD3 interacted with p62 in cytosolic aggregates, and p62 was required for PHD3 aggregation that was lost upon transfer to hypoxia, allowing PHD3 to be expressed evenly throughout the cell. In line with this, p62 enhanced the normoxic degradation of PHD3. Depletion of p62 in normoxia led to elevated PHD3 levels, whereas forced p62 expression in hypoxia downregulated PHD3. The loss of p62 resulted in enhanced interaction of PHD3 with HIF-α and reduced HIF-α levels. The data demonstrate p62 is a critical regulator of the hypoxia response and PHD3 activity, by inducing PHD3 aggregation and degradation under normoxia.

Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells.

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.

Retention of prolyl hydroxylase PHD2 in the cytoplasm prevents PHD2-induced anchorage-independent carcinoma cell growth.

Cellular oxygen tension is sensed by a family of prolyl hydroxylases (PHD1-3) that regulate the degradation of hypoxia-inducible factors (HIF-1alpha and -2alpha). The PHD2 isoform is considered as the main downregulator of HIF in normoxia. Our previous results have shown that nuclear translocation of PHD2 associates with poorly differentiated tumor phenotype implying that nuclear PHD2 expression is advantageous for tumor growth. Here we show that a pool of PHD2 is shuttled between the nucleus and the cytoplasm. In line with this, accumulation of wild type PHD2 in the nucleus was detected in human colon adenocarcinomas and in cultured carcinoma cells. The PHD2 isoforms showing high nuclear expression increased anchorage-independent carcinoma cell growth. However, retention of PHD2 in the cytoplasm inhibited the anchorage-independent cell growth. A region that inhibits the nuclear localization of PHD2 was identified and the deletion of the region promoted anchorage-independent growth of carcinoma cells. Finally, the cytoplasmic PHD2, as compared with the nuclear PHD2, less efficiently downregulated HIF expression. Forced HIF-1alpha or -2alpha expression decreased and attenuation of HIF expression increased the anchorage-independent cell growth. However, hydroxylase-inactivating mutations in PHD2 had no effect on cell growth. The data imply that nuclear PHD2 localization promotes malignant cancer phenotype.

Prolyl hydroxylase PHD3 activates oxygen-dependent protein aggregation.

The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability.

Overexpression and nuclear translocation of hypoxia-inducible factor prolyl hydroxylase PHD2 in head and neck squamous cell carcinoma is associated with tumor aggressiveness.

PURPOSE: Hypoxia in tumors is associated with poor prognosis and resistance to treatment. The outcome of hypoxia is largely regulated by the hypoxia-inducible factors (HIF-1alpha and HIF-2alpha). HIFs in turn are negatively regulated by a family of prolyl hydroxylases (PHD1-3). The PHD2 isoform is the main down-regulator of HIFs in normoxia and mild hypoxia. This study was designed to analyze the correlation of the expression and subcellular localization of PHD2 with the pathologic features of human carcinomas and HIF-1alpha expression. EXPERIMENTAL DESIGN: The expression of PHD2 was studied from paraffin-embedded normal tissue (n = 21) and head and neck squamous cell carcinoma (HNSCC; n = 44) by immunohistochemistry. Further studies included PHD2 mRNA detection and HIF-1alpha immunohistochemistry from HNSCC specimens as well as PHD2 immunocytochemistry from HNSCC-derived cell lines. RESULTS: In noncancerous tissue, PHD2 is robustly expressed by endothelial cells. In epithelium, the basal proliferating layer also shows strong expression, whereas the more differentiated epithelium shows little or no PHD2 expression. In HNSCC, PHD2 shows strongly elevated expression both at the mRNA and protein level. Moreover, PHD2 expression increases in less differentiated phenotypes and partially relocalizes from the cytoplasm into the nucleus. Endogenously high nuclear PHD2 is seen in a subset of HNSCC-derived cell lines. Finally, although most of the tumor regions with high PHD2 expression show down-regulated HIF-1alpha, regions with simultaneous HIF-1alpha and PHD2 expression could be detected. CONCLUSIONS: Our results show that increased levels and nuclear translocation of the cellular oxygen sensor, PHD2, are associated with less differentiated and strongly proliferating tumors. Furthermore, they imply that even the elevated PHD2 levels are not sufficient to down-regulate HIF-1alpha in some tumors.