Background: Hypoxia correlates with poor prognosis in several cancer types, including lung cancer. Prolyl hydroxylase domain proteins (PHDs) play a role in cell oxygen sensing, negatively regulating ...the hypoxia-inducible factor (HIF) pathway. Our study aim was to evaluate PHD1, PHD2 and PHD3 mRNA expression levels in primary tumours and normal lungs of non-small-cell lung cancer (NSCLC) patients and to correlate it with selected regulators of HIF signalling, with clinicopathological characteristics and overall survival (OS). Methods: Tumour tissue samples were obtained from 60 patients with surgically resected NSCLC who were treated with radical surgery. In 22 out of 60 cases, matching morphologically normal lung tissue was obtained. PHD1, PHD2 and PHD3 mRNA expressions were measured using RT-qPCR. Results: The PHD1 and PHD2 mRNA levels in primary tumours were significantly decreased compared to those in normal lungs (both p < 0.0001). PHD1 and PHD2 expression in tumours was positively correlated (rs = 0.82; p < 0.0001) and correlated well with HIF pathway downstream genes HIF1A, PKM2 and PDK1. Decreased PHD1 and PHD2 were associated with larger tumour size, higher tumour stage (PHD1 only) and squamous cell carcinoma. Patients with low PHD1 and patients with low PHD2 expression had shorter OS than patients with high PHD1 (p = 0.02) and PHD2 expression (p = 0.01). PHD1 showed borderline independent prognostic values in multivariate analysis (p = 0.06). In contrast, we found no associations between PHD3 expression and any of the observed parameters. Conclusions: Our results show that reduced expression of PHD1 and PHD2 is associated with the development and progression of NSCLC. PHD1 could be further assessed as a prognostic marker in NSCLC.
Increasing evidence suggests that high‐density lipoproteins (HDLs) promote hypoxia‐induced angiogenesis. The hypoxia‐inducible factor 1α (HIF‐1α)/vascular endothelial growth factor (VEGF) pathway is ...important in hypoxia and is modulated post‐translationally by prolyl hydroxylases (PHD1–PHD3) and E3 ubiquitin ligases (Siah1 and Siah2). We aimed to elucidate the mechanisms by which HDLs augment hypoxia‐induced angiogenesis. Preincubation (16 h) of human coronary artery endothelial cells with reconstituted high‐density lipoprotein (rHDL) containing apolipoprotein A‐I (apoA‐I) and phosphatidylcholine (20 μM, final apoA‐I concentration), before hypoxia, increased Siah1 (58%) and Siah2 (88%) mRNA levels and suppressed PHD2 (32%) and PHD3 (45%) protein levels compared with hypoxia‐induced control levels. After Siah1/2 small interfering RNA knockdown, rHDL was unable to suppress PHD2/3 and failed to induce HIF‐1α, VEGF, and tubulogenesis in hypoxia. Inhibition of the upstream phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathway also abrogated the effects of rHDL. Furthermore, knockdown of the scavenger receptor SR‐BI attenuated rHDL‐induced elevations in Siah1/2 and tubulogenesis in hypoxia, indicating that SR‐BI plays a key role. Finally, the importance of VEGF in mediating the ability of rHDL to drive hypoxia‐induced angiogenesis was confirmed using a VEGF‐neutralizing antibody. In summary, rHDL augments the HIF‐1α/VEGF pathway via SR‐BI and modulation of the post‐translational regulators of HIF‐1α (PI3K/Siahs/PHDs). HDL‐induced augmentation of angiogenesis in hypoxia may have implications for therapeutic modulation of ischemic injury.—Tan, J. T. M., Prosser, H. C. G., Vanags, L. Z., Monger, S. A., Ng, M. K. C., Bursill, C. A. High‐density lipoproteins augment hypoxia‐induced angiogenesis via regulation of post‐translational modulation of hypoxia inducible factor 1α. FASEB J. 28, 206–217 (2014). www.fasebj.org
Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the ...prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation‐dependent manner. Following p53–p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy‐induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5‐FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1‐specific inhibitors and their use in combination with chemotherapy.
Synopsis
Resistance to the commonly used chemotherapeutic drugs 5‐FU, irinotecan and oxaliplatin in colorectal cancer remains a major clinical problem. Here, it is shown that PHD1 (EGLN2) can cause resistance to chemotherapy through the regulation of p53‐mediated DNA repair.
Chemotherapy (5‐FU) effectiveness was improved upon silencing of PHD1 in a xenograft colorectal cancer model.
p38α‐mediated p53 phosphorylation upon chemotherapy was facilitated by PHD1 prolyl hydroxylase function and interaction with p53.
The interaction of p53 with the nucleotide excision repair component XPB and thus DNA repair upon chemotherapy‐induced damage was blocked by the reduction of p53 phosphorylation at Ser15.
DNA contact mutant p53 (R248) can still be modulated by PHD1 and regulate DNA repair.
Resistance to the commonly used chemotherapeutic drugs 5‐FU, irinotecan and oxaliplatin in colorectal cancer remains a major clinical problem. Here, it is shown that PHD1 (EGLN2) can cause resistance to chemotherapy through the regulation of p53‐mediated DNA repair.
The hypoxic response is mediated by two transcription factors, hypoxia-inducible factor (HIF)-1α and HIF-2α. These highly homologous transcription factors are induced in hypoxic foci and regulate ...cell metabolism, angiogenesis, cell proliferation, and cell survival. HIF-1α and HIF-2α are activated early in cancer progression and are important in several aspects of tumor biology. HIF-1α and HIF-2α have overlapping and distinct functions. In the intestine, activation of HIF-2α increases inflammation and colon carcinogenesis in mouse models. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1α is beneficial and can reduce intestinal inflammation. HIF-1α is a critical transcription factor regulating epithelial barrier function following inflammation. The beneficial value of pharmacological agents that chronically activate HIF-1α is decreased due to the tumorigenic potential of HIFs. The present study tested the hypothesis that chronic activation of HIF-1α may enhance colon tumorigenesis. Two models of colon cancer were assessed, a sporadic and a colitis-associated colon cancer model. Activation of HIF-1α in intestinal epithelial cells does not increase carcinogenesis or progression of colon cancer. Together, the data provide proof of principle that pharmacological activation of HIF-1α could be a safe therapeutic strategy for inflammatory bowel disease.
Oxidative stress and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of diabetic cardiovascular and renal diseases. Reactive oxygen species (ROS) mediate physiological and ...pathophysiological processes, being involved in the modulation of cell signaling, differentiation, and survival, but also in cyto- and genotoxic damage. As master regulators of glycolytic metabolism and oxygen homeostasis, HIFs have been largely studied for their role in cell survival in hypoxic conditions. However, in addition to hypoxia, other stimuli can regulate HIFs stability and transcriptional activity, even in normoxic conditions. Among these, a regulatory role of ROS and their byproducts on HIFs, particularly the HIF-1α isoform, has received growing attention in recent years. On the other hand, HIF-1α and HIF-2α exert mutually antagonistic effects on oxidative damage. In diabetes, redox-mediated HIF-1α deregulation contributes to the onset and progression of cardiovascular and renal complications, and recent findings suggest that deranged HIF signaling induced by hyperglycemia and other cellular stressors associated with metabolic disorders may cause mitochondrial dysfunction, oxidative stress, and inflammation. Understanding the mechanisms of mutual regulation between HIFs and redox factors and the specific contribution of the two main isoforms of HIF-α is fundamental to identify new therapeutic targets for vascular complications of diabetes.
Sprouty (Spry) proteins modulate the actions of receptor tyrosine kinases during development and tumorigenesis. Decreases in cellular levels of Spry, especially Sprouty2 (Spry2), have been implicated ...in the growth and progression of tumors of the breast, prostate, lung, and liver. During development and tumor growth, cells experience hypoxia. Therefore, we investigated how hypoxia modulates the levels of Spry proteins. Hypoxia elevated the levels of all four expressed Spry isoforms in HeLa cells. Amounts of endogenous Spry2 in LS147T and HEP3B cells were also elevated by hypoxia. Using Spry2 as a prototype, we demonstrate that silencing and expression of prolyl hydroxylase domain proteins (PHD1–3) increase and decrease, respectively, the cellular content of Spry2. Spry2 also preferentially interacted with PHD1–3 and von Hippel-Lindau protein (pVHL) during normoxia but not in hypoxia. Additionally, Spry2 is hydroxylated on Pro residues 18, 144, and 160, and substitution of these residues with Ala enhanced stability of Spry2 and abrogated its interactions with pVHL. Silencing of pVHL increased levels of Spry2 by decreasing its ubiquitylation and degradation and thereby augmented the ability of Spry2 to inhibit FGF-elicited activation of ERK1/2. Thus, prolyl hydroxylase mediated hydroxylation and subsequent pVHL-elicited ubiquitylation of Spry2 target it for degradation and, consequently, provide a novel mechanism of regulating growth factor signaling.
Background: Sprouty2 (Spry2) inhibits the actions of receptor tyrosine kinases (RTK) during development and disease.
Results: Stability of Spry2 is regulated by prolyl hydroxylation and binding to von Hippel-Lindau protein-associated E3 ligase.
Conclusion: PHD- and pVHL-mediated regulation of cellular levels of Spry2 modulates its ability to inhibit signaling by RTKs.
Significance: These findings provide new insights into modulation of levels of Spry2 to regulate RTK actions in disease.
With every heart beat blood rushes through a complex network of tubes to deliver essential ingredients of life, oxygen and nutrients. Consequently, this network of blood vessels is an indispensable ...part of vertebrate physiology. Its organization and architecture is highly dynamic in its form and function. Understanding how blood vessels develop, a process referred to as angiogenesis, is equally important as to know how they function considering that failure or misalignment of this process results in disorder and disease, in many cases of which death is inevitable. Much has been learned about the angiogenic process and the critical contributors of blood vessel function. A central determinant is oxygen, an evident contributor given the fact that oxygen delivery is a primary feature of blood vessel function. Not only is oxygen however essential for mitochondrial energy production, it also serves as a key molecule in various biochemical reactions, such as the formation of nitric oxide (NO), on its part a critical regulator of vascular tone and vessel homeostasis. Hence, oxygen abundance relates to the production of NO, and NO in turn regulates oxygen delivery and consumption. Given the importance of the intrinsic link these two molecules exert on angiogenesis and vessel function; this review shall highlight our current understanding on how these two molecules cooperate to form blood vessels.
► Oxygen abundance steers the angiogenic process via the oxygen sensing system. ► Endothelial and macrophage O2 sensors exert different roles in angiogenesis and arteriogenesis. ► Nitric oxide (NO) and oxygen abundance converge to regulate angio- and arteriogenesis. ► HIF-1α stimulates NO production by induction of iNOS steering angiogenesis. ► HIF-2α antagonizes NO production and angiogenesis by stimulating arginase expression.
The present study compares negative Ets transcription factor (Net) and hypoxia-inducible factor 1α (HIF1α) regulation by hypoxia. Their protein stabilities are differently regulated by hypoxia, ...defining three periods in the kinetics: normoxia (high Net levels and low HIF1α levels), early hypoxia (high levels of Net and HIF1α), and late hypoxia (degradation of Net and HIF1α). Modulators of prolyl hydroxylase domain protein (PHD) activity induce a mobility shift of Net, similar to HIF1α, suggesting that post-translational modifications of both factors depend on PHD activity. The three PHDs have different roles in the regulation of Net protein levels; PHD1 and PHD3 are involved in the stabilization of Net, whereas PHD2 controls its degradation in late hypoxia. Net physically interacts with PHD2 in hypoxia, whereas PHD1 and PHD3 bind to Net in normoxia and hypoxia. Under the same conditions, PHD2 and PHD3 regulate both HIF1α stabilization in early hypoxia and its degradation at late hypoxia, whereas PHD1 is involved in HIF1α degradation in late hypoxia. We describe interconnections between the regulation of both Net and HIF1α at the protein level. Evidence is provided for a direct physical interaction between Net and HIF1α and indirect transcriptional regulation loops that involve the PHDs. Taken together our results indicate that Net and HIF1α are components of distinct signaling pathways that are intricately linked.
Previously published data from our laboratory demonstrated that pharmacological inhibition of a family of enzymes known as prolyl hydroxylase domain proteins prevents neurotoxicity associated with ...the acute 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine intoxication model of Parkinson's disease in young animals. In this study, we assessed whether prolyl hydroxylase domain inhibition was neuroprotective in an inducible genetic dopaminergic glutathione depletion model previously characterized by our laboratory that more closely recapitulates the age-related and progressive nature of the human disease. Pharmacological prolyl hydroxylase domain inhibition via 3,4-dihydroxybenzoate was found to significantly attenuate hallmark mitochondrial dysfunction and loss of dopaminergic substantia nigral pars compacta neurons associated with this model. These studies further validate the possibility that prolyl hydroxylase domain inhibition may constitute a viable therapy for Parkinson's disease.
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