EGLN1

Gene Summary

Gene:EGLN1; egl-9 family hypoxia-inducible factor 1
Aliases: HPH2, PHD2, SM20, ECYT3, HALAH, HPH-2, HIFPH2, ZMYND6, C1orf12, HIF-PH2
Location:1q42.1
Summary:The protein encoded by this gene catalyzes the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins. HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. This protein functions as a cellular oxygen sensor, and under normal oxygen concentration, modification by prolyl hydroxylation is a key regulatory event that targets HIF subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Mutations in this gene are associated with erythrocytosis familial type 3 (ECYT3). [provided by RefSeq, Nov 2009]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:egl nine homolog 1
HPRD
Source:NCBIAccessed: 25 June, 2015

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 25 June 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 25 June, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: EGLN1 (cancer-related)

Brinkhuizen T, Weijzen CA, Eben J, et al.
Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.
PLoS One. 2014; 9(9):e106427 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Basal cell carcinoma (BCC) is the most common cancer in Caucasians. Trichoepithelioma (TE) is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF) tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1) and mechanistic/mammalian target of rapamycin (mTOR) are key players in these pathways.
OBJECTIVES: To determine whether HIF1/mTOR signalling is involved in BCC and TE.
METHODS: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45) and TE (n = 35) samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, <30%, 30-80% and >80%).
RESULTS: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3), 73% and 75% (CAIX), 79% and 86% (GLUT1), 50% and 19% (HIF1α), 89% and 88% (pAKT), 55% and 61% (pS6), 15% and 25% (pMTOR), 44% and 63% (PHD2) and 44% and 49% (VEGF-A). CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included.
CONCLUSIONS: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.

Bandara V, Michael MZ, Gleadle JM
Hypoxia represses microRNA biogenesis proteins in breast cancer cells.
BMC Cancer. 2014; 14:533 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins.
METHODS: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays.
RESULTS: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production.
CONCLUSION: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs.

Tóth K, Chintala S, Rustum YM
Constitutive expression of HIF-α plays a major role in generation of clear-cell phenotype in human primary and metastatic renal carcinoma.
Appl Immunohistochem Mol Morphol. 2014; 22(9):642-7 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
The extensive lipid accumulation occurring in clear-cell renal cell carcinoma (ccRCC) results in a clear-cell cytoplasm. Hypoxia-inducible factor α (HIF-α) is constitutively expressed in many ccRCC and transcriptionally regulates >100 genes. In a recent breakthrough study, HIF-1α induced ccRCC in transgenic mice. On the basis of these findings, we developed a hypothesis that accounted for HIF-α generation of the clear-cell phenotype. The aim of the present study was to use immunohistochemical staining methods in tissue microarray to determine the extent to which the clear-cell phenotype coincided with HIF-α expression in primary and metastatic ccRCC. In addition, we studied whether the prolyl-hydroxylases (PHD2,3) play a role in promoting the elevated expression of HIF-α in tumor cells. The clear-cell phenotype was observed in all primary and metastatic cases of ccRCC examined. A total of 168 renal cell carcinomas were evaluated by immunohistochemical methods; 141 of the 168 (84%) tumors expressed HIF-α (HIF-1α and/or HIF-2α). In contrast, HIF-α was expressed in only 1 of the 23 (4%) non-ccRCCs. These data supported the hypothesis that in the majority of the tumors HIF-α expression overlapped with the clear-cell phenotype and was indicative of an HIF-α-mediated lipid accumulation. In a smaller percentage of ccRCC cases (16%), HIF-α was not detected in the tumor cells and suggested that lipid accumulation by HIF-α-lipid-independent process. PHD3 was undetectable in both primary and metastatic ccRCC cases. We concluded that the undetectable PHD3 could contribute to the higher HIF-α expression in ccRCC.

Sun W, Jelkmann W, Depping R
Prolyl-4-hydroxylase 2 enhances hypoxia-induced glioblastoma cell death by regulating the gene expression of hypoxia-inducible factor-α.
Cell Death Dis. 2014; 5:e1322 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
Oxygen deprivation (hypoxia) is a common feature of solid tumors in advanced stages. The primary cellular transcriptional responses to hypoxia are mainly mediated by the transcription factor hypoxia-inducible factor (HIF). HIF consists of an oxygen-labile α-subunit (HIF-1α, -2α) and a stable β-subunit (ARNT). Prolyl-4-hydroxylase 2 (PHD2) is known as an important mediator of the oxygen-dependent degradation of HIF-α subunits. As HIF-α subunits are not confirmed to be the only substrates of PHD2, it is unknown whether PHD2 regulates HIF-1α and HIF-2α by interacting with other intracellular molecules. In this study, we found that in the glioblastoma cells, PHD2 maintains the gene expression of HIF-1α in dependence of nuclear factor κB and suppresses the gene expression of HIF-2α through HIF-1α. The PHD2-mediated degradation of HIF-1α and HIF-2α seems less important. Furthermore, PHD2 enhances hypoxia-induced glioblastoma cell death by modulating the expression of the HIF target genes glucose transporter 1, vascular endothelial growth factor-A and Bcl-2 binding protein 3. Our findings show that PHD2 inhibits the adaptation of glioblastoma cells to hypoxia by regulating the HIF-α subunits in a non-canonical way. Modulation of PHD2 activity might be considered as a new way to inhibit glioblastoma progression.

Welander J, Andreasson A, Juhlin CC, et al.
Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma.
J Clin Endocrinol Metab. 2014; 99(7):E1352-60 [PubMed] Related Publications
CONTEXT: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes.
OBJECTIVE: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes.
DESIGN: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bβ, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. RESULTS were verified in tumor and constitutional DNA with Sanger sequencing.
RESULTS: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1Bβ, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1Bβ, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation.
CONCLUSIONS: Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bβ, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.

Ellinghaus P, Heisler I, Unterschemmann K, et al.
BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I.
Cancer Med. 2013; 2(5):611-24 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

Bagnall J, Leedale J, Taylor SE, et al.
Tight control of hypoxia-inducible factor-α transient dynamics is essential for cell survival in hypoxia.
J Biol Chem. 2014; 289(9):5549-64 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.

Rawluszko AA, Bujnicka KE, Horbacka K, et al.
Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer.
BMC Cancer. 2013; 13:526 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
BACKGROUND: Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH).
METHODS: PHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n=90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines.
RESULTS: We found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p=0.00026; p<0.00001; p<0.00001) and proteins (p=0.004164; p=0.0071; p<0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p=0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p<0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2'-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells.
CONCLUSION: We demonstrated that reduced PHD3 expression in cancerous tissue was accompanied by methylation of the CpG rich region located within the first exon and intron of the PHD3 gene. The diminished expression of PHD1 and PHD2 and elevated level of FIH protein in cancerous tissue compared to histopathologically unchanged colonic mucosa was not associated with DNA methylation within the CpG islands of the PHD1, PHD2 and FIH genes.

Andreasson A, Kiss NB, Caramuta S, et al.
The VHL gene is epigenetically inactivated in pheochromocytomas and abdominal paragangliomas.
Epigenetics. 2013; 8(12):1347-54 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
Pheochromocytoma (PCC) and abdominal paraganglioma (PGL) are neuroendocrine tumors that present with clinical symptoms related to increased catecholamine levels. About a third of the cases are associated with constitutional mutations in pre-disposing genes, of which some may also be somatically mutated in sporadic cases. However, little is known about inactivating epigenetic events through promoter methylation in these very genes. Using bisulphite pyrosequencing we assessed the methylation density of 11 PCC/PGL disease genes in 96 tumors (83 PCCs and 13 PGLs) and 34 normal adrenal references. Gene expression levels were determined by quantitative RT-PCR. Both tumors and normal adrenal samples exhibited low methylation index (MetI) in the EGLN1 (PDH2), MAX, MEN1, NF1, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127 promoters, not exceeding 10% in any of the samples investigated. Aberrant RET promoter methylation was observed in two cases only. For the VHL gene we found increased MetI in tumors as compared with normal adrenals (57% vs. 27%; P<0.001), in malignant vs. benign tumors (63% vs. 55%; P<0.05), and in PGL vs. PCC (66% vs. 55%; P<0.0005). Decreased expression of the VHL gene was observed in all tumors compared with normal adrenals (P<0.001). VHL MetI and gene expressions were inversely correlated (R = -0.359, P<0.0001). Our results show that the VHL gene promoter has increased methylation compared with normal adrenals (MetI>50%) in approximately 75% of PCCs and PGLs investigated, highlighting the role of VHL in the development of these tumors.

McInerney-Leo AM, Marshall MS, Gardiner B, et al.
Whole exome sequencing is an efficient and sensitive method for detection of germline mutations in patients with phaeochromcytomas and paragangliomas.
Clin Endocrinol (Oxf). 2014; 80(1):25-33 [PubMed] Related Publications
BACKGROUND: Genetic testing is recommended when the probability of a disease-associated germline mutation exceeds 10%. Germline mutations are found in approximately 25% of individuals with phaeochromcytoma (PCC) or paraganglioma (PGL); however, genetic heterogeneity for PCC/PGL means many genes may require sequencing. A phenotype-directed iterative approach may limit costs but may also delay diagnosis, and will not detect mutations in genes not previously associated with PCC/PGL.
OBJECTIVE: To assess whether whole exome sequencing (WES) was efficient and sensitive for mutation detection in PCC/PGL.
METHODS: Whole exome sequencing was performed on blinded samples from eleven individuals with PCC/PGL and known mutations. Illumina TruSeq (Illumina Inc, San Diego, CA, USA) was used for exome capture of seven samples, and NimbleGen SeqCap EZ v3.0 (Roche NimbleGen Inc, Basel, Switzerland) for five samples (one sample was repeated). Massive parallel sequencing was performed on multiplexed samples. Sequencing data were called using Genome Analysis Toolkit and annotated using annovar. Data were assessed for coding variants in RET, NF1, VHL, SDHD, SDHB, SDHC, SDHA, SDHAF2, KIF1B, TMEM127, EGLN1 and MAX. Target capture of five exome capture platforms was compared.
RESULTS: Six of seven mutations were detected using Illumina TruSeq exome capture. All five mutations were detected using NimbleGen SeqCap EZ v3.0 platform, including the mutation missed using Illumina TruSeq capture. Target capture for exons in known PCC/PGL genes differs substantially between platforms. Exome sequencing was inexpensive (<$A800 per sample for reagents) and rapid (results <5 weeks from sample reception).
CONCLUSION: Whole exome sequencing is sensitive, rapid and efficient for detection of PCC/PGL germline mutations. However, capture platform selection is critical to maximize sensitivity.

Jung CL, Kim HJ, Park JH, et al.
Synergistic activation of the Nrf2-signaling pathway by glyceollins under oxidative stress induced by glutathione depletion.
J Agric Food Chem. 2013; 61(17):4072-8 [PubMed] Related Publications
Oxidative stress state such as depletion of the intracellular glutathione (GSH) is associated with the development of cancer. Some dietary phytochemicals have been shown to possess a cancer preventive effect, although the understanding of the involved mechanisms is still limited. Recent study has shown that glyceollins, phytoalexins derived from soybean by biotic elicitor, might have a cancer preventive effect through induction of detoxifying/antioxidant enzymes. The objective of this study was to investigate the effects of glyceollins on the Nrf2 signaling pathway under excessive oxidative stress induced by GSH depletion. In mouse hepatoma cells (Hepa1c1c7) subjected to the buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (γGCS), the intracellular GSH content was significantly lowered. On the other hand, incubation with glyceollins in the presence of BSO increased the level of GSH, expression of γGCS, and nuclear translocation of NF-E2-related factor-2 (Nrf2), compared to the cells treated with BSO only. Nrf2-antioxidant responsive element (ARE)-reporter activity assay in HepG2-C8 showed that BSO increased the ARE-reporter activity in a dose-dependent manner, compared to vehicle-treated cells, whereas cotreatment with glyceollins caused further increase in reporter luciferase activity relative to BSO alone. Taken together, glyceollins synergistically activated the Nrf2 signaling pathway and subsequently the expression of phase 2/antioxidant enzymes in the presence of BSO, suggesting that BSO-induced oxidative stress and that glyceollins regulate the expression of phase 2/antioxidant enzymes through different mechanisms from each other.

Taïeb D, Yang C, Delenne B, et al.
First report of bilateral pheochromocytoma in the clinical spectrum of HIF2A-related polycythemia-paraganglioma syndrome.
J Clin Endocrinol Metab. 2013; 98(5):E908-13 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
CONTEXT: Molecular genetic research has so far resulted in the identification of 10 well-characterized susceptibility genes for hereditary pheochromocytoma (PHEO) or paraganglioma (PGL). Recently, a new syndrome characterized by multiple PGLs and somatostatinomas associated with congenital polycythemia due to somatic mutations in HIF2A has been reported.
OBJECTIVE: The aim of the study was to define the genetic defect in a new case of bilateral PHEO and multiple PGLs associated with congenital polycythemia.
PATIENT: A female patient presented with neonatal polycythemia (treated by phlebotomies, 1 session approximately every 4 mo), mildly enlarged cerebral ventricles, and bilateral PHEO and multiple PGLs. There was no family history of any neuroendocrine tumor or polycythemia. Surgical removal of the tumors only temporarily normalized plasma erythropoietin (Epo) levels and discontinued phlebotomies. No germline mutations were initially detected in the SDHB, SDHC, SDHD, VHL, and PHD2 genes, known to be associated with polycythemia. The PHEOs presented with a typical noradrenergic biochemical phenotype.
RESULTS: A heterozygous missense mutation (c.1589C>T) was identified in exon 12 of HIF2A, resulting in an alanine 530 substitution in the HIF-2α protein with valine (A530V). This somatic mutation was detected in the tissue from 1 PHEO and 1 PGL, with no HIF2A germline mutation found. This mutation led to stabilization of HIF-2α and hence a gain-of-function phenotype, as in previously published studies.
CONCLUSION: This case represents the first association of a somatic HIF2A gain-of-function mutation with PHEO and congenital polycythemia, and it alerts physicians to perform proper genetic screening in patients presenting with multiple norepinephrine-producing PHEOs and polycythemia. This report also extends the previous findings of a new syndrome of only multiple PGLs, somatostatinomas, and polycythemia to multiple PHEOs.

Lu N, Hui H, Yang H, et al.
Gambogic acid inhibits angiogenesis through inhibiting PHD2-VHL-HIF-1α pathway.
Eur J Pharm Sci. 2013; 49(2):220-6 [PubMed] Related Publications
Our previous studies revealed that gambogic acid (GA), the major active ingredient of gamboge, possessed antiangiogenic activities. In this study, we further explored the mechanism of inhibition effects of GA in tumor angiogenesis. The results of luciferase, RT-PCR, and ELISA assays indicated that GA significantly decreased transcription activation, mRNA expression, and secretion of VEGF in hypoxia. We detected that GA had no effect on mRNA level of HIF-1α which targets VEGF gene, but the increase of HIF-1α protein expression in hypoxia was repressed by GA, which can be reversed by proteasomal inhibitor MG132 and siRNA of VHL. But GA exhibited no effect on expression of VHL both in normoxia and hypoxia. HIF prolyl hydroxylases (PHD enzymes) act as oxygen sensors regulating HIF, and hence angiogenesis. Our results showed that GA potentially enhanced level of PHD2, the most important HIF hydroxylase, and showed no effect on PHD1 and PHD3. Transient transfection of siRNA of PHD2 could eliminate GA-induced VEGF secretion increase. Growth of HepG2 xenografts in BALB/cA nude mice was inhibited by GA and angiogenesis was repressed significantly in tumor xenografts by immunohistochemical staining of CD-31, a vascular endothelial marker, accompanied with decrease of HIF-1α and increase of PHD2 expression in tissue extracts. This work provides the demonstration that GA shows anti-angiogenic effects via inhibiting PHD2-VHL-HIF-1α pathway.

Bozóky B, Savchenko A, Csermely P, et al.
Novel signatures of cancer-associated fibroblasts.
Int J Cancer. 2013; 133(2):286-93 [PubMed] Related Publications
Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer-associated fibroblasts, in cancer development and progression. In our study, we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth-modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal versus tumor-associated fibroblasts. Twelve new proteins differentially expressed in cancer-associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A and PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence among the different tumors. Many of them were also positive in normal myofibroblast-like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment.

Yum S, Doh HJ, Hong S, et al.
Piceatannol, a hydroxystilbene natural product, stabilizes HIF-1α protein by inhibiting HIF prolyl hydroxylase.
Eur J Pharmacol. 2013; 699(1-3):124-31 [PubMed] Related Publications
To investigate the mechanisms underlying the biological activity of piceatannol (PCT), a hydroxystilbene natural product that has anti-colitic properties, we examined whether PCT could modulate hypoxia-inducible factor (HIF)-1 activity in human colon carcinoma cells. PCT induced HIF-1α protein, leading to induction of its target gene products, vascular endothelial growth factor and heme oxygenase-1, which are involved in amelioration of colitis. PCT induction of HIF-1α resulted from HIF-1α protein stabilization, which occurred through inhibition of HIF-prolyl hydroxylase-2 (HPH-2). PCT inhibition of HPH-2 was reversed by addition of ascorbate, a cofactor of HPH-2, but not the cosubstrate, 2-ketoglutarate, to the reaction mixture of an in vitro von Hippel-Lindau (VHL) capture assay, and pretreatment with ascorbate abrogated PCT induction of cellular HIF-1α. Moreover, PCT prevented hydroxylation of cellular HIF-1α and attenuated coimmunoprecipitation of Flag-VHL protein and HA-HIF-1α over-expressed in human embryonic kidney 293 cells. Structural analysis using derivatives of PCT revealed that the catechol moiety in PCT was required for the stabilization of HIF-1α protein. Taken together, PCT activation of HIF-1 resulting from inhibition of HPH-2 may be a molecular mechanism for an anti-colitic effect of the natural product.

Wottawa M, Leisering P, Ahlen Mv, et al.
Knockdown of prolyl-4-hydroxylase domain 2 inhibits tumor growth of human breast cancer MDA-MB-231 cells by affecting TGF-β1 processing.
Int J Cancer. 2013; 132(12):2787-98 [PubMed] Related Publications
The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-β1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-β1 target gene expression by altering the TGF-β1 processing capacity.

Lorenzo FR, Yang C, Ng Tang Fui M, et al.
A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma.
J Mol Med (Berl). 2013; 91(4):507-12 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2A (F374Y) (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2A (F374Y) may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

Galan SR, Kann PH
Genetics and molecular pathogenesis of pheochromocytoma and paraganglioma.
Clin Endocrinol (Oxf). 2013; 78(2):165-75 [PubMed] Related Publications
Although most pheochromocytomas (PCCs) and paragangliomas (PGLs) are sporadic, molecular genetic medicine has revealed that a considerable number of patients with apparently sporadic PCC actually have a genetic predisposition to the development of these tumors. After decades of intensive research, several genes are now known to play an important role in the pathogenesis of PCC. At present, these are RET proto-oncogene, von Hippel-Lindau disease tumor suppressor gene (VHL), neurofibromatosis type 1 tumor suppressor gene (NF1), genes encoding the succinate dehydrogenase (SDH) complex subunits SDHB, SDHC, and SDHD, but also SDHA, the gene encoding the enzyme responsible for the flavination of SDHA (SDHAF2 or hSDH5), and the newly described TMEM127 and MAX tumor suppressor genes. In addition to these ten PCC susceptibility genes, two other genes, KIF1B and PHD2, have also been associated with PCC. Studying the pathogenesis and the molecular correlation of these mutations has revealed the existence of two main transcription signatures: a pseudohypoxic cluster (VHL and SDH mutations) and a cluster rich in kinase receptor signaling and their downstream pathways (RET, NF1, TMEM127, and MAX mutations). However, the general mechanism in the pathogenesis of a syndrome does not entirely apply in the particular pathogenesis of PCC as a manifestation of that syndrome. A better understanding of the complexity and high genetic diversity of PCC and PGL may lead to more efficient diagnosis and management of the disease.

Zhdanov AV, Dmitriev RI, Papkovsky DB
Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling.
Biosci Rep. 2012; 32(6):587-95 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0-10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.

Chintala S, Najrana T, Toth K, et al.
Prolyl hydroxylase 2 dependent and Von-Hippel-Lindau independent degradation of Hypoxia-inducible factor 1 and 2 alpha by selenium in clear cell renal cell carcinoma leads to tumor growth inhibition.
BMC Cancer. 2012; 12:293 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) accounts for more than 80% of the cases of renal cell carcinoma. In ccRCC deactivation of Von-Hippel-Lindau (VHL) gene contributes to the constitutive expression of hypoxia inducible factors 1 and 2 alpha (HIF-α), transcriptional regulators of several genes involved in tumor angiogenesis, glycolysis and drug resistance. We have demonstrated inhibition of HIF-1α by Se-Methylselenocysteine (MSC) via stabilization of prolyl hydroxylases 2 and 3 (PHDs) and a significant therapeutic synergy when combined with chemotherapy. This study was initiated to investigate the expression of PHDs, HIF-α, and VEGF-A in selected solid cancers, the mechanism of HIF-α inhibition by MSC, and to document antitumor activity of MSC against human ccRCC xenografts.
METHODS: Tissue microarrays of primary human cancer specimens (ccRCC, head & neck and colon) were utilized to determine the incidence of PHD2/3, HIF-α, and VEGF-A by immunohistochemical methods. To investigate the mechanism(s) of HIF-α inhibition by MSC, VHL mutated ccRCC cells RC2 (HIF-1α positive), 786-0 (HIF-2α positive) and VHL wild type head & neck cancer cells FaDu (HIF-1α) were utilized. PHD2 and VHL gene specific siRNA knockdown and inhibitors of PHD2 and proteasome were used to determine their role in the degradation of HIF-1α by MSC.
RESULTS: We have demonstrated that ccRCC cells express low incidence of PHD2 (32%), undetectable PHD3, high incidence of HIF-α (92%), and low incidence of VEGF-A compared to head & neck and colon cancers. This laboratory was the first to identify MSC as a highly effective inhibitor of constitutively expressed HIF-α in ccRCC tumors. MSC did not inhibit HIF-1α protein synthesis, but facilitated its degradation. The use of gene knockdown and specific inhibitors confirmed that the inhibition of HIF-1α was PHD2 and proteasome dependent and VHL independent. The effects of MSC treatment on HIF-α were associated with significant antitumor activity against ccRCC xenograft.
CONCLUSIONS: Our results show the role of PHD2/3 in stable expression of HIF-α in human ccRCC. Furthermore, HIF-1α degradation by MSC is achieved through PHD2 dependent and VHL independent pathway which is unique for HIF-α regulation. These data provide the basis for combining MSC with currently used agents for ccRCC.

Liu Y, Jiang W, Liu J, et al.
IDH1 mutations inhibit multiple α-ketoglutarate-dependent dioxygenase activities in astroglioma.
J Neurooncol. 2012; 109(2):253-60 [PubMed] Related Publications
The mechanism of tumorigenesis associated with nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent isocitrate dehydrogenase 1 (IDH1) mutations in gliomas is not fully understood. Loss of catalytic activity leading to a decrease in α-ketoglutarate (αKG) and gain of novel catalytic activity leading to production of D: -2-hydroxylglutarate (D: -2-HG) are both found in IDH1-mutated glioma cells. Both the decrease of αKG and accumulation of D: -2-HG inhibit the activity of multiple dioxygenases including prolyl hydroxylase domain-2 (PHD2), collagen prolyl-4-hydroxylase, histone demethylases, and the ten-eleven translocation (TET) family of 5-methylcytosine hydroxylases. Here we correlated the products of these dioxygenases after IDH1 gene mutations with tumorigenesis in human astroglioma samples. DNA sequencing was carried out for 253 astroglioma samples to identify IDH1 mutations. Immunohistochemistry analysis was employed to verify the levels of endostatin, dimethylated H3k79 (H3k79me2), and 5-hydroxymethylcytosine (5hmC) in these astroglioma samples. IDH1 mutations occurred frequently in low grades of astrocytoma. One case bearing both IDH1 and IDH2 mutations was identified. IDH1-mutated cases displayed more frontal lobe location and p53-positive immunostaining than wild-type cases. IDH1 mutations were associated with increased histone methylation and decreased 5hmC. By inhibiting endostatin expression, IDH1 mutations indirectly promoted angiogenesis in gliomas. All these changes were same in astroglioma at different malignancy grade. IDH1 mutations showed wide regulation of angiogenesis and genome-wide change of histone and DNA methylation, which were not suppressed as the malignancy level progressed, suggesting an early role of IDH1 mutations in astrocytoma tumorigenesis.

Shang B, Cao Z, Zhou Q
Progress in tumor vascular normalization for anticancer therapy: challenges and perspectives.
Front Med. 2012; 6(1):67-78 [PubMed] Related Publications
Antitumor angiogenic therapy has been shown promising in the treatment of several advanced cancers since the approval of the first antiangiogenic drug Avastin in 2004. Although the current antiangiogenic drugs reduce the density of tumor blood vessels and result in tumor shrinkage at the early stage of treatment, recent studies have shown that antiangiogenic therapy has transient and insufficient efficacy, resulting in tumor recurrence in patients after several months of treatment. Blockage of blood and oxygen supplies creates a hypoxic and acidic microenvironment in the tumor tissues, which fosters tumor cells to become more aggressive and metastatic. In 2001, Jain proposed tumor vascular normalization as an alternative approach to treating cancers based on the pioneering work on tumor blood vessels by several other researchers. At present, normalizing the disorganized tumor vasculature, rather than disrupting or blocking them, has emerged as a new option for anticancer therapy. Preclinical and clinical data have shown that tumor vascular normalization using monoclonal antibodies, proteins, peptides, small molecules, and pericytes resulted in decreased tumor size and reduced metastasis. However, current tumor vascular normalizing drugs display moderate anticancer efficacy. Accumulated data have shown that a variety of vasculogenic/angiogenic tumor cells and genes play important roles in tumor neovascularization, growth, and metastasis. Therefore, multiple-targeting of vasculogenic tumor cells and genes may improve the efficacy of tumor vascular normalization. To this end, the combination of antiangiogenic drugs with tumor vascular normalizing therapeutics, as well as the integration of Western medicine with traditional Chinese medicine, may provide a good opportunity for discovering novel tumor vascular normalizing drugs for an effective anticancer therapy.

Heindryckx F, Kuchnio A, Casteleyn C, et al.
Effect of prolyl hydroxylase domain-2 haplodeficiency on the hepatocarcinogenesis in mice.
J Hepatol. 2012; 57(1):61-8 [PubMed] Related Publications
BACKGROUND & AIMS: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains--the key oxygen sensor responsible for the degradation of hypoxia inducible factors--tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied.
METHODS: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation.
RESULTS: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype.
CONCLUSIONS: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds.

Koivunen P, Lee S, Duncan CG, et al.
Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation.
Nature. 2012; 483(7390):484-8 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

Högel H, Rantanen K, Jokilehto T, et al.
Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells.
PLoS One. 2011; 6(11):e27112 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
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.

Wollenick K, Hu J, Kristiansen G, et al.
Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling.
Nucleic Acids Res. 2012; 40(5):1928-43 [PubMed] Article available free on PMC after 01/10/2015 Related Publications
The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.

Welander J, Söderkvist P, Gimm O
Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas.
Endocr Relat Cancer. 2011; 18(6):R253-76 [PubMed] Related Publications
Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors of the adrenal glands and the sympathetic and parasympathetic paraganglia. They can occur sporadically or as a part of different hereditary tumor syndromes. About 30% of PCCs and PGLs are currently believed to be caused by germline mutations and several novel susceptibility genes have recently been discovered. The clinical presentation, including localization, malignant potential, and age of onset, varies depending on the genetic background of the tumors. By reviewing more than 1700 reported cases of hereditary PCC and PGL, a thorough summary of the genetics and clinical features of these tumors is given, both as part of the classical syndromes such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease, neurofibromatosis type 1, and succinate dehydrogenase-related PCC-PGL and within syndromes associated with a smaller fraction of PCCs/PGLs, such as Carney triad, Carney-Stratakis syndrome, and MEN1. The review also covers the most recently discovered susceptibility genes including KIF1Bβ, EGLN1/PHD2, SDHAF2, TMEM127, SDHA, and MAX, as well as a comparison with the sporadic form. Further, the latest advances in elucidating the cellular pathways involved in PCC and PGL development are discussed in detail. Finally, an algorithm for genetic testing in patients with PCC and PGL is proposed.

Peurala E, Koivunen P, Bloigu R, et al.
Expressions of individual PHDs associate with good prognostic factors and increased proliferation in breast cancer patients.
Breast Cancer Res Treat. 2012; 133(1):179-88 [PubMed] Related Publications
Tumor hypoxia-inducible transcription factor (HIF) overexpression often associates with a poor prognosis. Stability of the HIF-α subunits is regulated by HIF prolyl 4-hydroxylases, PHD1-3. We assessed here immunohistochemical expression of PHD1-3 and HIF-1α and 2α in patients with invasive ductal breast carcinoma (n = 102) and correlated their expression levels with main clinical prognostic factors and survival. PHD1 expression correlated with high proliferation, and these tumors were mainly estrogen receptor-negative. PHD3 expression declined in tumors of large size, poor differentiation, and high proliferation. There was a tendency for increased breast cancer-specific survival and longer disease-free survival among patients with high tumor PHD2 expression. Surprisingly, PHD1-3 expression did not correlate with HIF-1α or HIF-2α downregulation. However, HIF-2α expression correlated independently with low tumor stage and HIF-1α expression had a tendency for decreased breast cancer-specific survival. PHD1 and 3 appear to be HIF-independent factors in breast cancer. Not all PHD1 associated proliferation is estrogen-dependent and it is associated with a poor prognosis of cancer. PHD3 may be an important regulator of apoptosis and it is mainly found in tumors with good prognosis. PHD2 expression is likely to be involved in increased survival.

Fieuw A, Kumps C, Schramm A, et al.
Identification of a novel recurrent 1q42.2-1qter deletion in high risk MYCN single copy 11q deleted neuroblastomas.
Int J Cancer. 2012; 130(11):2599-606 [PubMed] Related Publications
Neuroblastoma is an aggressive embryonal tumor that accounts for ∼15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (∼115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.

Su Y, Loos M, Giese N, et al.
Prolyl hydroxylase-2 (PHD2) exerts tumor-suppressive activity in pancreatic cancer.
Cancer. 2012; 118(4):960-72 [PubMed] Related Publications
BACKGROUND: Pancreatic cancer is 1 of the most common and poorly treated tumors. In search of new therapeutic approaches, the oxygen sensors prolyl hydroxylases (PHD) are potential targets. PHD2 is considered the key oxygen sensor-regulating hypoxia-inducible factor (HIF). Currently, there is conflicting evidence regarding the exact role of PHD2 in tumorigenesis. The objective of this study was to investigate the role of PHD2 in pancreatic cancer growth and progression.
METHODS: PHD2 expression was analyzed by quantitative real-time polymerase chain reaction analysis and immunohistochemistry in human tissue specimens and cell lines. Knockdown of PHD2 was done by using short-interfering RNAs (siRNAs) specific against PHD2, and PHD2 overexpression was achieved by stable combinational DNA transfection. In vivo, an orthotopic murine model was used. Angiogenic cytokines were assessed with enzyme-linked immunosorbent assays, and invasion was studied with Matrigel assays.
RESULTS: PHD2 expression was not altered substantially in cancer tissues and their metastases. Lymph node-negative tissues had higher levels of PHD2 than lymph node-positive tissues. PHD2 was hypoxia-inducible in pancreatic cancer cell lines and regulated cell growth through cyclin D1 down-regulation samples with PHD2 suppression and through p21 up-regulation in samples with of PHD2 overexpression. In vivo, PHD2 caused tumor growth retardation and reduced tumor invasion by inhibiting angiogenesis. This observation was caused by the suppression of angiogenic cytokines and tumor invasion.
CONCLUSIONS: The current results indicated that PHD2 plays an important role in pancreatic tumorigenesis. In summary, the authors concluded that PHD2 may function as a tumor suppressor gene in pancreatic cancer and, thus, may define a potential target for the treatment of pancreatic cancer.

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