ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

Metabolic Adjustments following Glutaminase Inhibition by CB-839 in Glioblastoma Cell Lines.

Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes: GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.

The hallucal interphalangeal ossicle: anatomy and basis for ultrasound-guided surgical shaving.

Painful lesions on the plantar aspect of the first interphalangeal joint (IPJ) of the hallux can be attributed to structures called ossicles, nodules, or sesamoids. The aims of the present study were first to verify that ultrasonography (US) is a high-sensitivity tool for diagnosing an interphalangeal ossicle (IO), and second to prove that US-guided-shaving surgery ("milling") is a safe and feasible technique for remodeling the IO. The study is divided into three parts. In the first part, the prevalence of IOs was estimated in 12 cadaver feet using US, anatomical dissection, and fluoroscopy. In the second, a detailed US and morphological description of the IO was obtained. In the third, six cadaver feet were subjected to surgical milling. IO prevalence was 41.6% in gross anatomy, 41.6% in US examination and just 16.6% in fluoroscopy. The ossicles had a mean length of 4 mm (± 2 mm) and a width of 7 mm (± 2 mm). The ossicles could be completely shaved in all specimens without injuring important anatomical structures. Our results indicate that US is a more precise tool for diagnosing an IO than X-ray. Moreover, our US-guided mini-invasive surgical technique appears feasible and safe.

Clinical and molecular characteristics of pediatric low-grade glioma complicated with ventriculo-peritoneal shunt related ascites.

INTRODUCTION: Ventriculo-peritoneal shunt (VPS) related ascites is a rare complication of pediatric low grade gliomas (pLGG). Physiopathology of this complication is not fully understood and there is paucity of data regarding the molecular profile of pLGG gliomas complicating with ascites and the optimal management of this unusual event. METHODS: International multi-institutional retrospective analysis of patients diagnosed with BRAF altered pLGG and ascites arising as a complication of VPS. Demographics, tumor characteristics, therapeutic approaches and outcomes were recorded. RESULTS: Nineteen patients were identified. Median age at diagnosis was 14 months (R: 2-144). Most patients (17; 89.4%) presented with lesions involving the optic pathway. Mean tumor standard volume was 34.8 cm2 (R: 12.5-85.4). Pilocytic Astrocytoma was the most frequent histological diagnosis (14;7 3.7%). Eight (42.1%) tumors harbored BRAF V600-E mutation and seven (36.8%) KIAA1549 fusion. The onset of ascites was documented at a median time of 5 months following VPS insertion. Four (21%) patients were managed with paracentesis only, 7(36.8%) required both paracentesis and shunt diversion, 7(36.8%) required only a shunt diversion and 1 (5.2%) patient was managed conservatively. Chemotherapy regimen was changed in 10 patients following ascites. Eight patients received targeted therapy (4 dabrafenib/4 trametinib) and 5 were radiated. There were eleven survivors with a median OS of 69 months (R: 3-144). CONCLUSIONS: Ascites is an early feature in the clinical course of young patients with midline BRAF altered pLGG, with high mortality rate observed in our cohort. The hypothesis of ascites as an adverse prognostic factor in pLGG warrants further prospective research.

Antioxidant responses related to temozolomide resistance in glioblastoma.

Glioblastoma remains one of the most challenging and devastating cancers, with only a very small proportion of patients achieving 5-year survival. The current standard of care consists of surgery, followed by radiation therapy with concurrent and maintenance chemotherapy with the alkylating agent temozolomide. To date, this drug is the only one that provides a significant survival benefit, albeit modest, as patients end up acquiring resistance to this drug. As a result, tumor progression and recurrence inevitably occur, leading to death. Several factors have been proposed to explain this resistance, including an upregulated antioxidant system to keep the elevated intracellular ROS levels, a hallmark of cancer cells, under control. In this review, we discuss the mechanisms of chemoresistance -including the important role of glioblastoma stem cells-with emphasis on antioxidant defenses and how agents that impair redox balance (i.e.: sulfasalazine, erastin, CB-839, withaferin, resveratrol, curcumin, chloroquine, and hydroxychloroquine) might be advantageous in combined therapies against this type of cancer.

Glutaminase isoforms expression switches microRNA levels and oxidative status in glioblastoma cells.

BACKGROUND: Glutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells. METHODS: Microarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G. RESULTS: MiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression. CONCLUSIONS: Glutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.

Tumor Metabolome: Therapeutic Opportunities Targeting Cancer Metabolic Reprogramming.

The study of cancer metabolism is regaining center stage and becoming a hot topic in tumor biology and clinical research, after a period where such kind of experimental approaches were somehow forgotten or disregarded in favor of powerful functional genomic and proteomic studies [...].

Anatomic basis for a new ultrasound-guided, mini-invasive technique for release of the deep transverse metatarsal ligament.

INTRODUCTION: Morton's neuroma is an entrapment neuropathy of the third common plantar digital nerve, caused by the deep transverse metatarsal ligament (DTML). Minimally invasive or percutaneous surgery is a very common procedure, but surgical effectivity of this technique remains controversial. The goal of our study was to prove the effectiveness and safety of a new ultrasound-guided technique for DTML-release in a cadaver model. MATERIALS, METHODS, AND RESULTS: The DTML was visualized in 10 fresh frozen donated body to science-feet (eight male and two females, five left and five right) using an US device (GE Logic R7; 13 MHz linear probe, Madrid, Spain). Consecutively, minimally invasive ultrasound-guided surgery was performed. Exclusion criteria of the donated bodies to science were previous history of forefoot surgery and space occupying mass lesions. The complete release of the ligament was achieved in all specimens without damage of any important anatomical structures as proven by anatomical dissection. CONCLUSIONS: The results of this study indicate that our novel approach of an ultrasound-guided release of the DTML is safer and more effective compared to blind techniques. The DTML could reliably be visualized and securely cut through a dorsal, minimally invasive surgical incision of only 2 mm.

Erratum: Majewska, E. et al. Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway. Cancers 2019, 11, 115.

The authors wish to make the following corrections to this paper [...].

Glutaminases regulate glutathione and oxidative stress in cancer.

Targeted therapies against cancer have improved both survival and quality of life of patients. However, metabolic rewiring evokes cellular mechanisms that reduce therapeutic mightiness. Resistant cells generate more glutathione, elicit nuclear factor erythroid 2-related factor 2 (NRF2) activation, and overexpress many anti-oxidative genes such as superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin reductase, providing stronger antioxidant capacity to survive in a more oxidative environment due to the sharp rise in oxidative metabolism and reactive oxygen species generation. These changes dramatically alter tumour microenvironment and cellular metabolism itself. A rational design of therapeutic combination strategies is needed to flatten cellular homeostasis and accomplish a drop in cancer development. Context-dependent glutaminase isoenzymes show oncogenic and tumour suppressor properties, being mainly associated to MYC and p53, respectively. Glutaminases catalyze glutaminolysis in mitochondria, regulating oxidative phosphorylation, redox status and cell metabolism for tumour growth. In addition, the substrate and product of glutaminase reaction, glutamine and glutamate, respectively, can work as signalling molecules moderating redox and bioenergetic pathways in cancer. Novel synergistic approaches combining glutaminase inhibition and redox-dependent modulation are described in this review. Pharmacological or genetic glutaminase regulation along with oxidative chemotherapy can help to improve the design of combination strategies that escalate the rate of therapeutic success in cancer patients.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Therapeutic targeting of glutaminolysis as an essential strategy to combat cancer.

Metabolic reprogramming in cancer targets glutamine metabolism as a key mechanism to provide energy, biosynthetic precursors and redox requirements to allow the massive proliferation of tumor cells. Glutamine is also a signaling molecule involved in essential pathways regulated by oncogenes and tumor suppressor factors. Glutaminase isoenzymes are critical proteins to control glutaminolysis, a key metabolic pathway for cell proliferation and survival that directs neoplasms' fate. Adaptive glutamine metabolism can be altered by different metabolic therapies, including the use of specific allosteric inhibitors of glutaminase that can evoke synergistic effects for the therapy of cancer patients. We also review other clinical applications of in vivo assessment of glutaminolysis by metabolomic approaches, including diagnosis and monitoring of cancer.

Metabolic Reprogramming of Cancer by Chemicals that Target Glutaminase Isoenzymes.

BACKGROUND: Metabolic reprogramming of tumours is a hallmark of cancer. Among the changes in the metabolic network of cancer cells, glutaminolysis is a key reaction altered in neoplasms. Glutaminase proteins control the first step in glutamine metabolism and their expression correlates with malignancy and growth rate of a great variety of cancers. The two types of glutaminase isoenzymes, GLS and GLS2, differ in their expression patterns and functional roles: GLS has oncogenic properties and GLS2 has been described as a tumour suppressor factor. RESULTS: We have focused on glutaminase connections with key oncogenes and tumour suppressor genes. Targeting glutaminase isoenzymes includes different strategies aimed at deactivating the rewiring of cancer metabolism. In addition, we found a long list of metabolic enzymes, transcription factors and signalling pathways dealing with glutaminase. On the other hand, a number of chemicals have been described as isoenzyme-specific inhibitors of GLS and/or GLS2 isoforms. These molecules are being characterized as synergic and therapeutic agents in many types of tumours. CONCLUSION: This review states the metabolic pathways that are rewired in cancer, the roles of glutaminase isoforms in cancer, as well as the metabolic circuits regulated by glutaminases. We also show the plethora of anticancer drugs that specifically inhibit glutaminase isoenzymes for treating several sets of cancer.

The Epithelial to Mesenchymal Transition Promotes Glutamine Independence by Suppressing GLS2 Expression.

Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths; therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of GLS2, which encodes a glutaminase, are inversely associated with EMT. GLS2 down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of GLS2 expression in GLS2-negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in GLS2-negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high GLS2 expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies.

Dysregulation of glutaminase and glutamine synthetase in cancer.

Besides fast glucose catabolism, many types of cancers are characterized by elevated glutamine consumption. Medical oncology pursuits to block specific pathways, mainly glycolysis and glutaminolysis, in tumor cells to arrest cancer development. This strategy frequently induces adaptive metabolic resistance that must be countered. Combination therapy is an anticancer synergistic tool to overcome both cancer growth and resistance mechanisms. Dysregulation of glutaminase and glutamine synthetase are key events that allow anabolic adaptation of tumors. Several specific drugs that inhibit metabolic enzymes dealing with glutamine metabolism have been able to eliminate some neoplasms. Targeting the tumor microenvironment can be also another essential factor to be taken into account when single or combined cancer metabolic therapy fails.

Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway.

GLS-encoded glutaminase promotes tumorigenesis, while GLS2-encoded glutaminase displays tumor-suppressive properties. In glioblastoma (GBM), the most aggressive brain tumor, GLS is highly expressed and in most cases GLS2 is silenced. Previously, it was shown that transfection with a sequence encoding GAB, the main GLS2 isoform, decreased the survival, growth, and ability to migrate of human GBM cells T98G and increased their sensitivity towards an alkylating agent temozolomide (TMZ) and oxidative stress compared to the controls, by a not well-defined mechanism. In this study we report that GAB transfection inhibits growth and increases susceptibility towards TMZ and H2O2-mediated oxidative stress of two other GBM cell lines, U87MG and LN229. We also show that in GAB-transfected cells treated with H2O2, the PI3K/AKT pathway is less induced compared to the pcDNA-transfected counterparts and that pretreatment with PDGF-BB, an activator of AKT, protects GAB-transfected cells from death caused by the H2O2 treatment. In conclusion, our results show that (i) GAB suppresses the malignant phenotype of the GBM cells of different tumorigenic potentials and genetic backgrounds and (ii) the GAB-mediated increase of sensitivity to oxidative stress is causally related to the inhibition of the PI3K/AKT pathway. The upregulation of the GLS2 expression and the inhibition of the PI3K/AKT pathway may become a novel combined therapeutic strategy for anti-glioma preclinical investigations.

Glutaminase isoenzymes in the metabolic therapy of cancer.

Altered cellular metabolism is a hallmark of cancer. Cancer cells express isoforms of metabolic enzymes that may constitute therapeutic targets. Glutaminase controls glutamine metabolism and their expression correlate with malignancy of tumours. The two types of glutaminase isoenzymes, GLS and GLS2, differ in their expression patterns and functional roles: GLS has oncogenic properties and GLS2 has been described as a tumour suppressor factor. Selective genomic and epigenomic intervention over glutaminase affects the metabolic reprogramming of cancer. This review highlights the molecular metabolic vulnerabilities in various types of cancer, to be used for biomarker development, drug design, and in personalized oncology.

Necrólisis epidérmica tóxica y lupus eritematoso sistémico / Toxic epidermal necrolysis and systemic lupus erythematosus

Resumen El lupus eritematoso sistémico con manifestación tipo necrólisis epidérmica tóxica es una entidad descrita recientemente y cada vez aparecen más reportes en la literatura. Se describe el caso de una paciente de 15 años con lupus eritematoso sistémico quien presentó una necrólisis epidérmica tóxica extensa, cuyas lesiones iniciales eran tipo eritema multiforme. Se discute el caso a la luz de la literatura actual sobre esta nueva entidad.

Abstract Toxic epidermal necrolysis (TEN)-like systemic lupus erythematosus is a recently described entity and more cases are being published in the literature today. We describe the case of a 15-year old patient with systemic lupus erythematosus who developed TEN that initially started with erythema multiforme (EM)-like lesiones. We discuss this case according to the published literature on this new entity.

Miocardiopatía diabética: un punto de vista retrospectivo / Diabetic cardiomyopathy: a retrospective point of view

Resumen Objetivo: Estimar la prevalencia de pacientes con probable miocardiopatía diabética (sin exigir biopsia endomiocárdica), analizando sus características y comparándolas con pacientes no diabéticos con insuficiencia cardiaca idiopática. Métodos: Estudio retrospectivo sobre una cohorte de 270 pacientes que ingresaron de manera consecutiva por insuficiencia cardiaca. De estos se excluyeron del estudio todos aquellos que tuvieran una causa conocida de insuficiencia cardiaca (cardiopatía isquémica, hipertensiva, hipertrófica, valvular u otras). En total 58 pacientes, 18 de los cuales eran diabéticos (la causa de la insuficiencia cardiaca pudo ser la miocardiopatía diabética) y 40 sin diabetes (insuficiencia cardiaca idiopática). Se procedió a comparar las características entre ambos grupos. El análisis estadístico y la comparación de las distintas variables se realizó mediante el programa SPSS versión 20.0. Resultados: De 270 pacientes estudiados, 18 podrían haber sido diagnosticados con miocardiopatía diabética, lo que supone hasta un 6% de la insuficiencia cardiaca en esta serie. Conclusiones: Se considera que este estudio puede hacer reflexionar sobre la prevalencia global de la miocardiopatía diabética, sus características y sus dificultades diagnósticas.

Abstract Objective: To determine the prevalence of patients with probable diabetic cardiomyopathy (without requesting an endomyocardial biopsy), as well as to analyse its characteristics and comparing them with non-diabetic patients with idiopathic heart failure. Methods: A retrospective study was conducted on a cohort of 270 patients consecutively admitted to hospital due to heart failure. Excluded from the study were those who had a known cause of heart failure (ischaemic, hypertensive, hypertrophic, valvular, or other causes of heart disease). This left a total of 58 patients, 18 of whom were diabetics (the cause of heart failure could be due to diabetic cardiomyopathy) and 40 non-diabetics (idiopathic heart failure). A comparison was made between the characteristics of both groups. The statistical analysis and the comparison of the different variables were performed using Version 20.0 of the SPSS program. Results: Of the 270 patients studied, 18 could have been diagnosed with diabetic cardiomyopathy, which would be 6% of the cardiac failures in this series. Conclusions: It is believed that this study can lead to reflecting on the overall prevalence of diabetic cardiomyopathy, its characteristics, and the difficulties in its diagnosis.