Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from C-13-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies

Effects of metabolic cancer therapy on tumor microenvironment

Targeting tumor metabolism for cancer therapy is an old strategy. In fact, historically the first effective cancer therapeutics were directed at nucleotide metabolism. The spectrum of metabolic drugs considered in cancer increases rapidly – clinical trials are in progress for agents directed at glycolysis, oxidative phosphorylation, glutaminolysis and several others. These pathways are essential for cancer cell proliferation and redox homeostasis, but are also required, to various degrees, in other cell types present in the tumor microenvironment, including immune cells, endothelial cells and fibroblasts. How metabolism-targeted treatments impact these tumor-associated cell types is not fully understood, even though their response may co-determine the overall effectivity of therapy. Indeed, the metabolic dependencies of stromal cells have been overlooked for a long time. Therefore, it is important that metabolic therapy is considered in the context of tumor microenvironment, as understanding the metabolic vulnerabilities of both cancer and stromal cells can guide new treatment concepts and help better understand treatment resistance. In this review we discuss recent findings covering the impact of metabolic interventions on cellular components of the tumor microenvironment and their implications for metabolic cancer therapy

PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner

Background: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4- dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. Methods: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. Results: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. Conclusions: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress

Role of the Transforming Growth Factor-β in regulating hepatocellular carcinoma oxidative metabolism.

Transforming Growth Factor beta (TGF-β) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profle of hepatocellular carcinoma (HCC) cells that show diferences in TGF-β expression. Oxygen consumption rate (OCR), extracellular acidifcation rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without signifcant diferences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fuxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-β function was activated with extracellular TGF-β1 or inhibited through TGF-β receptor I silencing showed that TGF-β induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-β also regulated the expression of key genes involved in the fux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-β pathway regulates oxidative metabolism in HCC cells

Not to be picky: PEPCK-M ensures metabolic flexibility in cancer cells and neuronal progenitors

[eng] Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that catalyses decarboxylation of oxaloacetate to phosphoenolpyruvate and it is part of gluconeogenic/glyceroneogenic pathway. There are two known isoforms of PEPCK, the mitochondrial and the cytosolic isozyme that are catalysing chemically identical reactions, but they differ in regulation and expression pattern. Selective presence of mitochondrial isoform of this enzyme (PEPCK-M, PCK2) in all types of cancer examined and in cycling neuroprogenitors, suggests a functional relationship with the metabolic adaptations of these cells. This thesis has had as its main objectives the characterization of the role of PEPCK-M in tumour cells and in neuronal progenitor cells. Metabolism of cell in CNS is not completely elucidated yet. Here we demonstrate that Tbr2 positive neuronal progenitors are metabolically dependent on lactate, which is favouring maintenance of their undifferentiated state. Lactate as metabolite can feed anabolic pathways and sustain ATP production by its oxidation in the TCA cycle. However, essential pathways like PPP, glycerol synthesis or one carbon metabolism pathways require carbons to feed the glycolytic intermediate pool. PEPCK-M in this setting, with lactate as sole carbon source is the only known pathway to fulfil the above-mentioned anabolic requirements. By using inhibitor of PEPCK-M we were able to prove that Tbr2 positive neuronal progenitors are metabolically dependent on PEPCK-M activity and their number significantly decrease after inhibiting PEPCK-M in vitro and in vivo. PEPCK-M activity in tumour cells is necessary for survival and growth in 2D and in cultures on semi-solid agar (anchorage-independent growth), which suggests that this enzyme has a fundamental role in the survival program to cell stress. A Kaplan-Meier analysis from datasets available in the GEO database (> 5000 patients) shows that elevated PCK2 expression is significantly associated with a worse prognosis in patients with breast cancer. Despite its potential relevance for metabolic adaptations in cancer, the mechanisms responsible for its pro- survival activity are not known. Therefore, we have proposed to study these mechanisms through metabolomic analysis where we wanted to examine whether PEPCK-M feeds an alternative pathway to glucose using carbons from glutamine in an experimental model with reduced and overexpressed levels of PEPCK-M activity. We demonstrated the functionality of PEPCK-M driven cataplerosis in MCF7 cells grown under glucose deprivation by showing synthesis of serine and glycine from glutamine by observing contribution of 13C-labeled carbons from [U-13C] glutamine into these metabolites. In the absence of nutritional stress (high abundance of glucose and amino acids), the silencing of PEPCK-M induces oxidative stress and the accumulation of succinate, with the consequent induction of p21 and deficiencies in cell growth. Glutamine cataplerosis is not affected by alterations in PEPCK-M activity. However, a higher enrichment of all carbons with 13C in intermediates of the Krebs cycle (TCA cycle) suggests a reduction in flux through this pathway. Together, these data increase our understanding of metabolic adaptations in tumours and the role of PEPCK in providing alternative carbon fluxes to deal with nutritional stress. Finally, these studies allow us to propose PEPCK-M as a new target for the treatment of tumorigenic processes that will need to be validated in the future.[spa] El fosfoenolpiruvato carboxiquinasa mitocondrial (PEPCK-M; PCK2) se regula transcripcionalmente por limitación de aminoácidos y por ER-estrés, de una manera dependiente de ATF4, aumentando así la supervivencia de la célula. La presencia selectiva de esta isoenzima en todos los tipos de cáncer examinado y en células neuroprogenitoras, sugiere una relación funcional con las adaptaciones metabólicas de estas células. Esta tesis ha tenido como objetivos fundamentales la caracterización del rol de la PEPCK-M en célula tumoral y en célula neuroprogenitora En cultivos neuronales, los neuroprogenitores Tbr2 positivos requieren lactato como sustrato metabólico para el mantenimiento de su fenotipo y su metabolismo. La PEPCK-M se expresa a niveles altos en este tipo celular y su actividad es necesaria para mantener la viabilidad de estos progenitores y cumplir con los requerimientos anabólicos a partir de carbonos provenientes del lactato. La actividad PEPCK-M en célula tumoral es necesaria para la supervivencia y crecimiento. A pesar de su potencial relevancia para las adaptaciones metabólicas en cáncer, no se conocen los mecanismos responsables de su actividad pro-supervivencia. Por ello, nos hemos propuesto estudiar estos mecanismos mediante análisis de metabolómica con los que hemos querido examinar si la PEPCK-M alimenta una vía alternativa a la glucosa utilizando carbonos provenientes de glutamina en un modelo experimental con niveles de actividad PEPCK-M reducidos y sobreexpresados. La contribución de carbonos marcados con 13C a partir de [U- 13C] glutamina en los productos de ramificación de glicolisis como serina y glicina, esta correlacionando directamente con los niveles de actividad PEPCK-M en condiciones de estrés nutricional (baja glucosa). La cataplerosis de glutamina no se ve afectada por alteraciones en la actividad de PEPCK-M. Sin embargo, un mayor enriquecimiento de 13C en intermediarios del ciclo de Krebs sugieren una reducción del flujo a través de esta vía. En conjunto, estos datos aumentan nuestra comprensión de las adaptaciones metabólicas en los tumores y el papel de la PEPCK en la provisión de flujos de carbono alternativas para lidiar con el estrés nutricional. Finalmente, estos estudios nos permiten proponer a la PEPCK-M como una nueva diana para el tratamiento de procesos tumorogénicos que necesitará ser validada en el futuro

The effect of the composition of PLA films and lactate release on glial and neuronal maturation and the maintenance of the neuronal progenitor niche.

To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly l/dl lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. l-lactate in the medium recapitulated PLA70/30's maintenance of neuronal restricted progenitors but did not sustain bipotential or glial restricted progenitors in the cultures, as occurred when neural cells were grown on PLA70/30. Our results suggest that PLA70/30 may mimic some of the physical and biochemical characteristics of the NSC niche. Its mechanical and surface properties may act synergistically in the modulation of bipotential and glial restricted progenitor phenotypes, while it is l-lactate, either added to the medium or released by the film that drives the maintenance of neuronal restricted progenitor cell phenotypes.Peer ReviewedPostprint (published version

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism

The effect of the composition of PLA films and lactate release on glial and neuronal maturation and the maintenance of the neuronal progenitor niche.

To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly l/dl lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. l-lactate in the medium recapitulated PLA70/30's maintenance of neuronal restricted progenitors but did not sustain bipotential or glial restricted progenitors in the cultures, as occurred when neural cells were grown on PLA70/30. Our results suggest that PLA70/30 may mimic some of the physical and biochemical characteristics of the NSC niche. Its mechanical and surface properties may act synergistically in the modulation of bipotential and glial restricted progenitor phenotypes, while it is l-lactate, either added to the medium or released by the film that drives the maintenance of neuronal restricted progenitor cell phenotypes.Peer Reviewe

Tumors defective in homologous recombination rely on oxidative metabolism: relevance to treatments with PARP inhibitors

Inhibidors de la PARP; Metabolisme del càncer; MetforminaInhibidores de PARP, Metabolismo del cáncer; MetforminaPARP inhibitors; Cancer metabolism; MetforminMitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors.This study has been funded by the Ministerio de Ciencia, Innovación y Universidades, which is part of the Agencia Estatal de Investigación (AEI), through the project SAF2017-85869-R (cofunded by the European Regional Development Fund (ERDF), a way to build Europe) to FV and BFU2015-66030-R to JCP; by the FIS PI15/00854 and FIS PI16/01898 (Instituto Carlos III, cofunded by FEDER funds/European Regional Development Fund (ERDF), a way to build Europe) to MAP and AVillanueva and with the support of the Secretariat for Universities and Research of the Department of Business and Knowledge of the Government of Catalonia (2017SGR449) to FV. We thank the CERCA Program/Generalitat de Catalunya for their institutional support. We particularly wish to acknowledge the collaboration of the patients and the IDIBGI Biobank (Biobanc IDIBGI, B.0000872), which is part of the Spanish National Biobank Network and the Xarxa de Bancs de Tumors de Catalunya (XBTC), financed by the Pla Director d'Oncologia de Catalunya, Spain. We thank Cristina Saura (Breast Cancer & Melanoma Group) and people from the Experimental Therapeutics Group at VHIO and Sara González (Unitat de Diagnòstic Molecular, ICO-Duran i Reynals). We thank H. Simon, R. Bartrons, and A. Manzano (Universitat de Barcelona) and A. Vaquero (IDIBELL) for reagents