Hypoxia and oxidative stress in breast cancer: Hypoxia signalling pathways

Hypoxia-inducible factor-1 (HIF), which is centrally involved in physiological oxygen homeostasis, is also activated in the majority of tumours. Activation of HIF can occur through genetic mechanisms or as a result of hypoxia within the tumour microenvironment. In some cases HIF activation appears to be intimately linked to the proliferative stimulus itself. HIF affects patterns of gene expression and tumour growth, although precise effects vary between tumour types. Modulation of HIF activity, if correctly applied, may be therapeutically beneficial in tumour therapy

Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1 signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by estrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.Research Foundation – Flanders (FWO: G.0A72.13, G.096414 and G0A4216N) and P.C. from long-term structural funding – Methusalem Funding by the Flemish Government

Cardiopulmonary phenotype associated with human PHD2 mutation.

Oxygen-dependent regulation of the erythropoietin gene is mediated by the hypoxia-inducible factor (HIF) family of transcription factors. When oxygen is plentiful, HIF undergoes hydroxylation by a family of oxygen-dependent prolyl hydroxylase domain (PHD) proteins, promoting its association with the von Hippel-Lindau (VHL) ubiquitin E3 ligase and subsequent proteosomal degradation. When oxygen is scarce, the PHD enzymes are inactivated, leading to HIF accumulation and upregulation not only of erythropoietin expression, but also the expression of hundreds of other genes, including those coordinating cardiovascular and ventilatory adaptation to hypoxia. Nevertheless, despite the identification of over 50 mutations in the PHD-HIF-VHL pathway in patients with previously unexplained congenital erythrocytosis, there are very few reports of associated cardiopulmonary abnormalities. We now report exaggerated pulmonary vascular and ventilatory responses to acute hypoxia in a 35-year-old man with erythrocytosis secondary to heterozygous mutation in PHD2, the most abundant of the PHD isoforms. We compare this phenotype with that reported in patients with the archetypal disorder of cellular oxygen sensing, Chuvash polycythemia, and discuss the possible clinical implications of our findings, particularly in the light of the emerging role for small molecule PHD inhibitors in clinical practice

A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

BACKGROUND: Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. METHODS: We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. RESULTS: We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. CONCLUSIONS: Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans

Statin-induced expression of CD59 on vascular endothelium in hypoxia: a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis

Hypoxia, which leads to dysfunctional cell metabolism, and complement activation both play central roles in the pathogenesis of rheumatoid arthritis (RA). Recent studies have reported that mice deficient for the complement-inhibitory protein CD59 show enhanced susceptibility to antigen-induced arthritis and reported that statins have anti-inflammatory effects in RA. We hypothesized that the anti-inflammatory effect of statins in RA relates in part to their ability to increase CD59 expression in hypoxic conditions and therefore to reduce complement activation. Flow-cytometric analysis showed that CD59 expression on endothelial cells (EC) was unaffected by atorvastatin in normoxia (21% O(2)), whereas in hypoxic conditions (1% O(2)) an up to threefold dose-dependent increase in CD59 expression was seen. This effect of hypoxia was confirmed by treatment of EC with chemical mimetics of hypoxia. The upregulation of CD59 protein expression in hypoxia was associated with an increase in steady-state mRNA. L-Mevalonate and geranylgeraniol reversed the response, confirming a role for inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase and geranylgeranylation. Likewise, inhibition by N(G)-monomethyl-L-arginine and N(G)-nitro-L-arginine methyl ester confirmed that CD59 upregulation in hypoxia was nitric oxide dependent. The expression of another complement-inhibitory protein, decay-accelerating factor (DAF), is known to be increased by atorvastatin in normoxia; this response was also significantly enhanced under hypoxic conditions. The upregulation of CD59 and DAF by atorvastatin in hypoxia prevented the deposition of C3, C9 and cell lysis that follows exposure of reoxygenated EC to serum. This cytoprotective effect was abrogated by inhibitory anti-CD59 and anti-DAF mAbs. The modulation of EC CD59 and DAF by statins under hypoxic conditions therefore inhibits both early and late complement activation and may contribute to the anti-inflammatory effects of statins in RA

Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.Thanks to Dr John Bomalaski, (Polaris Pharmaceuticals, Inc) for supplying the ADI-PEG20, to Dr Simon S Hoer for useful discussions and to members of Histopathology/ISH (CRUK Cambridge Institute, UK) for IHC and imaging assistance. This work was supported by the Wellcome Trust and the NIHR Cambridge Biomedical Research Centre Senior Investigator Awards (to P.H.M., supporting N.B.), EU FP7 Metoxia Grant agreement no. 222741 (to P.H.M., supporting G.C.), UCL Cancer Research UK Centre (to M.R.), King’s College London and UCL Comprehensive Cancer Imaging Centre, Cancer Research UK and EPSRC in association with the Medical Research Council (MRC), the DoH (England: to R.B.P.), MRC Cancer Unit Core Funding (to C.F., supporting E.G.).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2295

Developmentally arrested structures preceding cerebellar tumors in von Hippel-Lindau disease.

There is increasing evidence that suggests that knockout of tumor-suppressor gene function causes developmental arrest and protraction of cellular differentiation. In the peripheral nervous system of patients with the tumor-suppressor gene disorder, von Hippel-Lindau disease, we have demonstrated developmentally arrested structural elements composed of hemangioblast progenitor cells. Some developmentally arrested structural elements progress to a frank tumor, hemangioblastoma. However, in von Hippel-Lindau disease, hemangioblastomas are frequently observed in the cerebellum, suggesting an origin in the central nervous system. We performed a structural and topographic analysis of cerebellar tissues obtained from von Hippel-Lindau disease patients to identify and characterize developmentally arrested structural elements in the central nervous system. We examined the entire cerebella of five tumor-free von Hippel-Lindau disease patients and of three non-von Hippel-Lindau disease controls. In all, 9 cerebellar developmentally arrested structural elements were detected and topographically mapped in 385 blocks of von Hippel-Lindau disease cerebella. No developmentally arrested structural elements were seen in 214 blocks from control cerebella. Developmentally arrested structural elements are composed of poorly differentiated cells that express hypoxia-inducible factor (HIF)2α, but not HIF1α or brachyury, and preferentially involve the molecular layer of the dorsum cerebelli. For the first time, we identify and characterize developmentally arrested structural elements in the central nervous system of von Hippel-Lindau patients. We provide evidence that developmentally arrested structural elements in the cerebellum are composed of developmentally arrested hemangioblast progenitor cells in the molecular layer of the dorsum cerebelli

Inactivation of the von Hippel-Lindau tumour suppressor gene induces Neuromedin U expression in renal cancer cells.

BACKGROUND: 209 000 new cases of renal carcinoma are diagnosed each year worldwide and new therapeutic targets are urgently required. The great majority of clear cell renal cancer involves inactivation of VHL, which acts as a gatekeeper tumour suppressor gene in renal epithelial cells. However how VHL exerts its tumour suppressor function remains unclear. A gene expression microarray comparing RCC10 renal cancer cells expressing either VHL or an empty vector was used to identify novel VHL regulated genes. FINDINGS: NMU (Neuromedin U) is a neuropeptide that has been implicated in energy homeostasis and tumour progression. Here we show for the first time that VHL loss-of-function results in dramatic upregulation of NMU expression in renal cancer cells. The effect of VHL inactivation was found to be mediated via activation of Hypoxia Inducible Factor (HIF). Exposure of VHL expressing RCC cells to either hypoxia or dimethyloxalylglycine resulted in HIF activation and increased NMU expression. Conversely, suppression of HIF in VHL defective RCC cells via siRNA of HIF-α subunits or expression of Type 2C mutant VHLs reduced NMU expression levels. We also show that renal cancer cells express a functional NMU receptor (NMUR1), and that NMU stimulates migration of renal cancer cells. CONCLUSIONS: These findings suggest that NMU may act in an autocrine fashion, promoting progression of kidney cancer. Hypoxia and HIF expression are frequently observed in many non-renal cancers and are associated with a poor prognosis. Our study raises the possibility that HIF may also drive NMU expression in non-renal tumours