Bart Ghesquière

The miR-17-92 microRNA cluster regulates multiple components of the TGF-β pathway in neuroblastoma.

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-β signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-β signaling cascade as well as direct inhibition of TGF-β-responsive genes.

Partial and Transient Reduction of Glycolysis by PFKFB3 Blockade Reduces Pathological Angiogenesis

Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.

A roadmap for interpreting (13)C metabolite labeling patterns from cells.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.

Metabolism of stromal and immune cells in health and disease

Cancer cells have been at the centre of cell metabolism research, but the metabolism of stromal and immune cells has received less attention. Nonetheless, these cells influence the progression of malignant, inflammatory and metabolic disorders. Here we discuss the metabolic adaptations of stromal and immune cells in health and disease, and highlight how metabolism determines their differentiation and function.

Fatty acid carbon is essential for dNTP synthesis in endothelial cells

The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.

Tumour hypoxia causes DNA hypermethylation by reducing TET activity

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylation.

Stable isotopic labeling in proteomics.

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon‐13, nitrogen‐15, and oxygen‐18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications.

Redox Proteomics of Protein-bound Methionine Oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.

Upper and lower gastrointestinal evaluation of elderly inpatients who are iron deficient.

PURPOSE Iron deficiency anemia is commonly caused by chronic gastrointestinal blood loss, and a thorough examination of the gastrointestinal tract has become standard practice. In contrast, iron deficiency without anemia has hardly been studied, and its causes are less certain. The aim of the present study was to determine the diagnostic value of upper and lower gastrointestinal evaluation in elderly hospitalized patients with iron deficiency, irrespective of the hemoglobin level. PATIENTS AND METHODS In a prospective study, 151 consecutive elderly patients with iron deficiency (serum ferritin level < 50 microg/L at two separate occasions) were investigated using esophagogastroduodenoscopy with colonoscopy (n = 90) or barium enema (n = 61). RESULTS A potential upper gastrointestinal tract lesion was found in 47 (49%) of the 96 anemic patients and in 31 (56%) of the 55 nonanemic patients (P = 0.38). Nonanemic patients had a greater prevalence of erosive gastritis or duodenitis. Anemic patients (72%) were more frequently investigated with a colonoscopy than nonanemic patients (38%, P = 0.001), and a lower gastrointestinal lesion was found in 32% of the anemic patients and 16% of the nonanemic patients (P = 0.03). Cancer was the most common lesion in the colon; 11 of the 18 patients were asymptomatic. Site-specific symptoms, fecal occult blood loss, and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) were not associated with the detection of gastrointestinal lesions. In 9.5% of the patients with a benign upper gastrointestinal lesion, a synchronous colonic tumor was found. CONCLUSION Elderly patients with iron deficiency should undergo endoscopic examination, irrespective of the hemoglobin level. The presence of gastrointestinal symptoms, a positive fecal occult blood test, and the use of NSAIDs are of limited value in guiding the diagnostic procedure.

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.