Ádám Nagy

Cross-validation of survival associated biomarkers in gastric cancer using transcriptomic data of 1,065 patients.

Introduction Multiple gene expression based prognostic biomarkers have been repeatedly identified in gastric carcinoma. However, without confirmation in an independent validation study, their clinical utility is limited. Our goal was to establish a robust database enabling the swift validation of previous and future gastric cancer survival biomarker candidates. Results The entire database incorporates 1,065 gastric carcinoma samples, gene expression data. Out of 29 established markers, higher expression of BECN1 (HR = 0.68, p = 1.5E-05), CASP3 (HR = 0.5, p = 6E-14), COX2 (HR = 0.72, p = 0.0013), CTGF (HR = 0.72, p = 0.00051), CTNNB1 (HR = 0.47, p = 4.3E-15), MET (HR = 0.63, p = 1.3E-05), and SIRT1 (HR = 0.64, p = 2.2E-07) correlated to longer OS. Higher expression of BIRC5 (HR = 1.45, p = 1E-04), CNTN1 (HR = 1.44, p = 3.5E- 05), EGFR (HR = 1.86, p = 8.5E-11), ERCC1 (HR = 1.36, p = 0.0012), HER2 (HR = 1.41, p = 0.00011), MMP2 (HR = 1.78, p = 2.6E-09), PFKB4 (HR = 1.56, p = 3.2E-07), SPHK1 (HR = 1.61, p = 3.1E-06), SP1 (HR = 1.45, p = 1.6E-05), TIMP1 (HR = 1.92, p = 2.2E- 10) and VEGF (HR = 1.53, p = 5.7E-06) were predictive for poor OS. MATERIALS AND METHODS We integrated samples of three major cancer research centers (Berlin, Bethesda and Melbourne datasets) and publicly available datasets with available follow-up data to form a single integrated database. Subsequently, we performed a literature search for prognostic markers in gastric carcinomas (PubMed, 2012–2015) and re-validated their findings predicting first progression (FP) and overall survival (OS) using uni- and multivariate Cox proportional hazards regression analysis. Conclusions The major advantage of our analysis is that we evaluated all genes in the same set of patients thereby making direct comparison of the markers feasible. The best performing genes include BIRC5, CASP3, CTNNB1, TIMP-1, MMP-2, SIRT, and VEGF.

Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage

Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis‐aconitate. In mitochondria, itaconate can be converted by succinate‐coenzyme A (CoA) ligase to itaconyl‐CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS‐induced stimulation of Irg1 in murine bone marrow‐derived macrophages (BMDM) and RAW‐264.7 cells. In rotenone‐treated macrophage cells, stimulation by LPS led to impairment in substrate‐level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW‐264.7 cells, the LPS‐induced impairment in SLP was reversed by short‐interfering RNA(siRNA)—but not scrambled siRNA—treatment directed against Irg1. LPS dose‐dependently inhibited oxygen consumption rates (61‐91%) and elevated glycolysis rates (>21%) in BMDM but not RAW‐264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose‐dependently (0.5‐2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP‐induced depolarizations (3‐19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a “CoA trap” in the form of itaconyl‐CoA that negatively affects the upstream supply of succinyl‐CoA from the α‐ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate‐CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate‐CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1‐expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.—Németh, B., Doczi, J., Csete, D., Kacso, G., Ravasz, D., Adams, D., Kiss, G., Nagy, A. M., Horvath, G., Tretter, L., Mócsai, A., Csépányi‐Kömi, R., Iordanov, I., Adam‐Vizi, V., Chinopoulos, C. Abolition of mitochondrial substrate‐level phosphorylation by itaconic acid produced by LPS‐induced Irg1 expression in cells of murine macrophage lineage. FASEB J. 30, 286‐300 (2016). www.fasebj.org

TP53 mutation hits energy metabolism and increases glycolysis in breast cancer.

Promising new hallmarks of cancer is alteration of energy metabolism that involves molecular mechanisms shifting cancer cells to aerobe glycolysis. Our goal was to evaluate the correlation between mutation in the commonly mutated tumor suppressor gene TP53 and metabolism. We established a database comprising mutation and RNA-seq expression data of the TCGA repository and performed receiver operating characteristics (ROC) analysis to compare expression of each gene between TP53 mutated and wild type samples. All together 762 breast cancer samples were evaluated of which 215 had TP53 mutation. Top up-regulated metabolic genes include glycolytic enzymes (e.g. HK3, GPI, GAPDH, PGK1, ENO1), glycolysis regulator (PDK1) and pentose phosphate pathway enzymes (PGD, TKT, RPIA). Gluconeogenesis enzymes (G6PC3, FBP1) were down-regulated. Oxygen consumption and extracellular acidification rates were measured in TP53 wild type and mutant breast cell lines with a microfluorimetric analyzer. Applying metabolic inhibitors in the presence and absence of D-glucose and L-glutamine in cell culture experiments resulted in higher glycolytic and mitochondrial activity in TP53 mutant breast cancer cell lines. In summary, TP53 mutation influences energy metabolism at multiple levels. Our results provide evidence for the synergistic activation of multiple hallmarks linking to these the mutation status of a key driver gene.

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

KRAS driven expression signature has prognostic power superior to mutation status in non-small cell lung cancer

KRAS is the most frequently mutated oncogene in non‐small cell lung cancer (NSCLC). However, the prognostic role of KRAS mutation status in NSCLC still remains controversial. We hypothesize that the expression changes of genes affected by KRAS mutation status will have the most prominent effect and could be used as a prognostic signature in lung cancer.

Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets

Multiple studies suggested using different miRNAs as biomarkers for prognosis of hepatocellular carcinoma (HCC). We aimed to assemble a miRNA expression database from independent datasets to enable an independent validation of previously published prognostic biomarkers of HCC. A miRNA expression database was established by searching the TCGA (RNA-seq) and GEO (microarray) repositories to identify miRNA datasets with available expression and clinical data. A PubMed search was performed to identify prognostic miRNAs for HCC. We performed a uni- and multivariate Cox regression analysis to validate the prognostic significance of these miRNAs. The Limma R package was applied to compare the expression of miRNAs between tumor and normal tissues. We uncovered 214 publications containing 223 miRNAs identified as potential prognostic biomarkers for HCC. In the survival analysis, the expression levels of 55 and 84 miRNAs were significantly correlated with overall survival in RNA-seq and gene chip datasets, respectively. The most significant miRNAs were hsa-miR-149, hsa-miR-139, and hsa-miR-3677 in the RNA-seq and hsa-miR-146b-3p, hsa-miR-584, and hsa-miR-31 in the microarray dataset. Of the 223 miRNAs studied, the expression was significantly altered in 102 miRNAs in tumors compared to normal liver tissues. In summary, we set up an integrated miRNA expression database and validated prognostic miRNAs in HCC.

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.

Author Correction: Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The impact of l-thyroxine treatment of donors and recipients on postoperative outcomes after heart transplantation

OBJECTIVE The effect of thyroid dysfunction on adverse outcomes has been studied in many different patient populations. The objective of this study was to investigate the effect of thyroid hormone supplementation of donors and recipients on postoperative outcomes after orthotopic heart transplantation. DESIGN Retrospective. SETTING Single center, university hospital. PARTICIPANTS Two-hundred and sixty-six consecutive patients undergoing heart transplantation. INTERVENTIONS No interventions. MEASUREMENTS AND MAIN RESULTS Demographic, hemodynamic, and clinical characteristics; donor and recipient United Network for Organ Sharing scores; and information on thyroid hormone support of donors and recipients were recorded. During the median follow-up of 4.59 years (interquartile range 4.26-4.92 y), 70 patients (26.3%) died. After adjustments were made for the United Network for Organ Sharing score, recipients who were treated preoperatively with l-thyroxine had a lower risk for all-cause mortality (adjusted hazard ratio [HR] 0.24, 95% confidence interval [CI] 0.06-0.98; p = 0.047) compared with recipients who were not treated with l-thyroxine. In addition, l-thyroxine treatment of donors was associated with a better recipient survival (HR 0.31, 95% CI 0.11-0.87; p = 0.025). CONCLUSIONS Pretransplantation thyroid hormone supplementation of donors and recipients was associated with improved long-term survival after heart transplantation.

Higher Transaminase Levels in the Postoperative Period After Orthotopic Heart Transplantation Are Associated With Worse Survival

OBJECTIVE Preoperative liver function in heart failure patients is associated with extensive functional, structural, and hemodynamic abnormalities. The authors hypothesized that perioperative liver dysfunction is associated with worse 2-year survival after orthotopic heart transplantation. DESIGN Retrospective study. SETTING Single-center, university hospital. PARTICIPANTS The study comprised 209 consecutive patients undergoing heart transplantation. INTERVENTIONS No interventions. MEASUREMENTS AND MAIN RESULTS Hepatobiliary markers, hemodynamic parameters, echocardiographic parameters, the need for mechanical cardiac support, demographic parameters, and United Network for Organ Sharing and Model for End-Stage Liver Disease (MELD) scores were investigated. Fifty-five patients (26.3%) died, and the mean survival time was 3.61 years after transplantation. In multivariate Cox regression analysis, in addition to the preoperative modified MELD score, the 4th quartiles of the maximum aspartate transaminase (AST) and alanine transaminase levels on the 4th through 7th postoperative days were independently associated with mortality (odds ratio [OR] 2.46, 95% confidence interval [CI] 1.09-5.55; p = 0.031 and OR 2.41, 95% CI 1.13-5.18; p = 0.024, respectively). By expressing the transaminase values as the multiplier of the sex-specific top normal value, the maximum AST and alanine transaminase levels (OR 1.02, 95% CI 1.01-1.02; p < 0.001 and OR 1.02, 95% CI 1.01-1.03; p = 0.001, respectively) were linked to worse survival. Among the postdischarge parameters, the modified MELD score (OR 1.17, 95% CI 1.09-1.27; p < 0.001) and the AST level were associated with postdischarge mortality (OR 1.002, 95% CI 1.001-1.003; p < 0.001 as a continuous variable; OR 1.07, 95% CI 1.05-1.10; p < 0.001, expressed as the multiplier of the sex-specific normal value, respectively). CONCLUSIONS The severity of postoperative liver dysfunction negatively influences survival after heart transplantation, and liver function should be closely assessed in these patients.