RESUMEN
Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.
Asunto(s)
Ácido Fólico/metabolismo , Mitocondrias/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Aminohidrolasas/metabolismo , Biocatálisis , Proteínas Portadoras/metabolismo , Codón/genética , Transporte de Electrón , Antagonistas del Ácido Fólico/farmacología , Proteínas de Unión al GTP/metabolismo , Glicina Hidroximetiltransferasa/deficiencia , Glicina Hidroximetiltransferasa/metabolismo , Guanosina/metabolismo , Células HCT116 , Células HEK293 , Humanos , Leucina/genética , Lisina/genética , Metilación/efectos de los fármacos , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Enzimas Multifuncionales/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , ARN de Transferencia/genética , Proteínas de Unión al ARN , Ribosomas/metabolismo , Sarcosina/metabolismo , Tetrahidrofolatos/metabolismo , Nucleótidos de Timina/biosíntesisRESUMEN
In this issue, Kim et al. (2015) show that ZMP (5-aminoimidazole-4-carboxamide ribonucleotide) binds to and activates a conserved riboswitch to regulate expression of one-carbon metabolism genes.
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Endosomas/enzimología , Complejos Multiproteicos/fisiología , Fagosomas/enzimología , Procesamiento Proteico-Postraduccional , Serina-Treonina Quinasas TOR/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
The enzyme serine hydroxymethyltransferse (SHMT) converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Folate one-carbon units support purine and thymidine synthesis, and thus cell growth. Mammals have both cytosolic SHMT1 and mitochondrial SHMT2, with the mitochondrial isozyme strongly up-regulated in cancer. Here we show genetically that dual SHMT1/2 knockout blocks HCT-116 colon cancer tumor xenograft formation. Building from a pyrazolopyran scaffold that inhibits plant SHMT, we identify small-molecule dual inhibitors of human SHMT1/2 (biochemical IC50 â¼ 10 nM). Metabolomics and isotope tracer studies demonstrate effective cellular target engagement. A cancer cell-line screen revealed that B-cell lines are particularly sensitive to SHMT inhibition. The one-carbon donor formate generally rescues cells from SHMT inhibition, but paradoxically increases the inhibitor's cytotoxicity in diffuse large B-cell lymphoma (DLBCL). We show that this effect is rooted in defective glycine uptake in DLBCL cell lines, rendering them uniquely dependent upon SHMT enzymatic activity to meet glycine demand. Thus, defective glycine import is a targetable metabolic deficiency of DLBCL.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Glicina/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Femenino , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/metabolismo , Conformación ProteicaRESUMEN
Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4â¯month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Ligasas de Carbono-Nitrógeno/genética , Epilepsia/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Microcefalia/genética , Enfermedades Mitocondriales/genética , Trastornos Psicomotores/genética , Sistemas de Transporte de Aminoácidos Acídicos/líquido cefalorraquídeo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiportadores/líquido cefalorraquídeo , Antiportadores/genética , Antiportadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ligasas de Carbono-Nitrógeno/líquido cefalorraquídeo , Ligasas de Carbono-Nitrógeno/deficiencia , Ligasas de Carbono-Nitrógeno/metabolismo , Epilepsia/líquido cefalorraquídeo , Epilepsia/complicaciones , Epilepsia/patología , Femenino , Receptor 1 de Folato/deficiencia , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/líquido cefalorraquídeo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Masculino , Enfermedades Metabólicas/líquido cefalorraquídeo , Enfermedades Metabólicas/complicaciones , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/patología , Microcefalia/líquido cefalorraquídeo , Microcefalia/complicaciones , Microcefalia/patología , Enfermedades Mitocondriales/líquido cefalorraquídeo , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/metabolismo , Malformaciones del Sistema Nervioso/líquido cefalorraquídeo , Malformaciones del Sistema Nervioso/complicaciones , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Distrofias Neuroaxonales , Trastornos Psicomotores/líquido cefalorraquídeo , Trastornos Psicomotores/complicaciones , Trastornos Psicomotores/metabolismo , Tetrahidrofolatos/líquido cefalorraquídeo , Tetrahidrofolatos/metabolismoRESUMEN
The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.
Asunto(s)
Cromatografía Liquida/métodos , Leucovorina/análisis , Espectrometría de Masas/métodos , Tetrahidrofolatos/análisis , Técnicas de Cultivo de Célula , Antagonistas del Ácido Fólico/farmacología , Células HEK293 , Humanos , Leucovorina/metabolismo , Metotrexato/farmacología , Tetrahidrofolatos/metabolismoRESUMEN
Myc is one of the most commonly deregulated oncogenes in human cancer, yet therapies directly targeting Myc hyperactivation are not presently available in the clinic. The evolutionarily conserved function of Myc in modulating protein synthesis control is critical to the Myc oncogenic program. Indeed, enhancing the protein synthesis capacity of cancer cells directly contributes to their survival, proliferation, and genome instability. Therefore, inhibiting enhanced protein synthesis may represent a highly relevant strategy for the treatment of Myc-dependent human cancers. However, components of the translation machinery that can be exploited as therapeutic targets for Myc-driven cancers remain poorly defined. Here, we uncover a surprising and important functional link between Myc and mammalian target of rapamycin (mTOR)-dependent phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 (4EBP1), a master regulator of protein synthesis control. Using a pharmacogenetic approach, we find that mTOR-dependent phosphorylation of 4EBP1 is required for cancer cell survival in Myc-dependent tumor initiation and maintenance. We further show that a clinical mTOR active site inhibitor, which is capable of blocking mTOR-dependent 4EBP1 phosphorylation, has remarkable therapeutic efficacy in Myc-driven hematological cancers. Additionally, we demonstrate the clinical implications of these results by delineating a significant link between Myc and mTOR-dependent phosphorylation of 4EBP1 and therapeutic response in human lymphomas. Together, these findings reveal that an important mTOR substrate is found hyperactivated downstream of Myc oncogenic activity to promote tumor survival and confers synthetic lethality, thereby revealing a unique therapeutic approach to render Myc druggable in the clinic.
Asunto(s)
Linfocitos B/fisiología , Benzoxazoles/farmacología , Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Benzoxazoles/administración & dosificación , Western Blotting , Proteínas de Ciclo Celular , Factores Eucarióticos de Iniciación , Everolimus , Humanos , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Fosforilación , Pirimidinas/administración & dosificación , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.
Asunto(s)
Colesterol/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Factores Eucarióticos de Iniciación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Células 3T3 NIH , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.
Asunto(s)
Glicina Hidroximetiltransferasa , Glicina , Glicina Hidroximetiltransferasa/genética , Homeostasis , Carbono , SerinaRESUMEN
Activating mutations in the CTNNB1 gene encoding ß-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). Profound alterations in lipid metabolism, including increases in fatty acid oxidation and transformation of the phospholipidome, occur in HCC with CTNNB1 mutations, but it is unclear what mechanisms give rise to these changes. We employed untargeted lipidomics and targeted isotope tracing to measure phospholipid synthesis activity in an inducible human liver cell line expressing mutant ß-catenin, as well as in transgenic zebrafish with activated ß-catenin-driven HCC. In both models, activated ß-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid isotope tracing analysis in human cells revealed a reduction in phosphatidylcholine (PC) production rates as assayed by choline incorporation. We developed lipid isotope tracing analysis for zebrafish tumors and observed reductions in phosphatidylcholine synthesis by both the CDP-choline and PEMT pathways. The observed changes in the ß-catenin-driven HCC phospholipidome suggest that zebrafish can recapitulate conserved features of HCC lipid metabolism and may serve as a model for identifying future HCC-specific lipid metabolic targets.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Fosfatidilcolinas , Pez Cebra , beta Catenina , beta Catenina/metabolismo , beta Catenina/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Pez Cebra/metabolismo , Pez Cebra/genética , Humanos , Animales , Fosfatidilcolinas/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metabolismo de los Lípidos/genética , Animales Modificados Genéticamente , Fosfolípidos/metabolismo , Línea Celular Tumoral , Lipidómica/métodosRESUMEN
The established clinical therapy for the treatment of acute myocardial infarction is primary percutaneous coronary intervention (PPCI) to restore blood flow to the ischemic myocardium. PPCI is effective at reperfusing the ischemic myocardium, however the rapid re-introduction of oxygenated blood also can cause ischemia-reperfusion (I/R) injury. Reperfusion injury is the culprit for up to half of the final myocardial damage, but there are no clinical interventions to reduce I/R injury. We previously demonstrated that inhibiting the lactate exporter, monocarboxylate transporter 4 (MCT4), and re-directing pyruvate towards oxidation can blunt isoproterenol-induced hypertrophy. Based on this finding, we hypothesized that the same pathway might be important during I/R. Here, we establish that the pyruvate-lactate metabolic axis plays a critical role in determining myocardial salvage following injury. Post-I/R injury, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium following I/R injury. MPC loss in cardiomyocytes caused more cell death with less myocardial salvage, which was associated with an upregulation of MCT4 in the myocardium at risk of injury. We deployed a pharmacological strategy of MCT4 inhibition with a highly selective compound (VB124) at the time of reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (Δψ), and Ca 2+ , increased pyruvate entry to TCA cycle, and improved myocardial salvage and functional outcomes following I/R injury. Altogether, our data suggest that normalizing the pyruvate-lactate metabolic axis via MCT4 inhibition is a promising pharmacological strategy to mitigate I/R injury.
RESUMEN
The clinical therapy for treating acute myocardial infarction is primary percutaneous coronary intervention (PPCI). PPCI is effective at reperfusing the heart, however the rapid re-introduction of blood can cause ischemia-reperfusion (I/R). Reperfusion injury is responsible for up to half of the final myocardial damage, but there are no pharmacological interventions to reduce I/R. We previously demonstrated that inhibiting monocarboxylate transporter 4 (MCT4) and re-directing pyruvate towards oxidation can blunt hypertrophy. We hypothesized this pathway might be important during I/R. Here, we establish that the pyruvate-lactate axis plays a role in determining myocardial salvage following injury. Post-I/R, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium. In cardiomyocytes lacking the MPC, there was increased cell death and less salvage after I/R, which was associated with an upregulation of MCT4. To determine the importance of pyruvate oxidation, we inhibited MCT4 with a small-molecule drug (VB124) at reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψ), and Ca2+, increased pyruvate entry to TCA cycle, increased oxygen consumption, improved myocardial salvage and functional outcomes following I/R. Our data suggests normalizing pyruvate-lactate metabolism by inhibiting MCT4 is a promising therapy to mitigate I/R injury.
RESUMEN
Non-small-cell lung cancer (NSCLC) with concurrent mutations in KRAS and the tumour suppressor LKB1 (KL NSCLC) is refractory to most therapies and has one of the worst predicted outcomes. Here we describe a KL-induced metabolic vulnerability associated with serine-glycine-one-carbon (SGOC) metabolism. Using RNA-seq and metabolomics data from human NSCLC, we uncovered that LKB1 loss enhanced SGOC metabolism via serine hydroxymethyltransferase (SHMT). LKB1 loss, in collaboration with KEAP1 loss, activated SHMT through inactivation of the salt-induced kinase (SIK)-NRF2 axis and satisfied the increased demand for one-carbon units necessary for antioxidant defence. Chemical and genetic SHMT suppression increased cellular sensitivity to oxidative stress and cell death. Further, the SHMT inhibitor enhanced the in vivo therapeutic efficacy of paclitaxel (first-line NSCLC therapy inducing oxidative stress) in KEAP1-mutant KL tumours. The data reveal how this highly aggressive molecular subtype of NSCLC fulfills their metabolic requirements and provides insight into therapeutic strategies.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP , Antioxidantes , Carcinoma de Pulmón de Células no Pequeñas , Glicina Hidroximetiltransferasa , Proteína 1 Asociada A ECH Tipo Kelch , Neoplasias Pulmonares , Mutación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas p21(ras) , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Antioxidantes/metabolismo , Animales , Estrés Oxidativo , Ratones , Línea Celular Tumoral , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genéticaRESUMEN
Mechanical unloading and circulatory support with left ventricular assist devices (LVADs) mediate significant myocardial improvement in a subset of advanced heart failure (HF) patients. The clinical and biological phenomena associated with cardiac recovery are under intensive investigation. Left ventricular (LV) apical tissue, alongside clinical data, were collected from HF patients at the time of LVAD implantation (n=208). RNA was isolated and mRNA transcripts were identified through RNA sequencing and confirmed with RT-qPCR. To our knowledge this is the first study to combine transcriptomic and clinical data to derive predictors of myocardial recovery. We used a bioinformatic approach to integrate 59 clinical variables and 22,373 mRNA transcripts at the time of LVAD implantation for the prediction of post-LVAD myocardial recovery defined as LV ejection fraction (LVEF) ≥40% and LV end-diastolic diameter (LVEDD) ≤5.9cm, as well as functional and structural LV improvement independently by using LVEF and LVEDD as continuous variables, respectively. To substantiate the predicted variables, we used a multi-model approach with logistic and linear regressions. Combining RNA and clinical data resulted in a gradient boosted model with 80 features achieving an AUC of 0.731±0.15 for predicting myocardial recovery. Variables associated with myocardial recovery from a clinical standpoint included HF duration, pre-LVAD LVEF, LVEDD, and HF pharmacologic therapy, and LRRN4CL (ligand binding and programmed cell death) from a biological standpoint. Our findings could have diagnostic, prognostic, and therapeutic implications for advanced HF patients, and inform the care of the broader HF population.
RESUMEN
Background and Aims: Activating mutations in the CTNNB1 gene encoding ß-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). HCC with CTNNB1 mutations show profound alterations in lipid metabolism including increases in fatty acid oxidation and transformation of the phospholipidome, but it is unclear how these changes arise and whether they contribute to the oncogenic program in HCC. Methods: We employed untargeted lipidomics and targeted isotope tracing to quantify phospholipid production fluxes in an inducible human liver cell line expressing mutant ß-catenin, as well as in transgenic zebrafish with activated ß-catenin-driven HCC. Results: In both models, activated ß-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid flux analysis in human cells revealed a large reduction in phosphatidylcholine (PC) production rates as assayed by choline tracer incorporation. We developed isotope tracing lipid flux analysis for zebrafish and observed similar reductions in phosphatidylcholine synthesis flux accomplished by sex-specific mechanisms. Conclusions: The integration of isotope tracing with lipid abundances highlights specific lipid class transformations downstream of ß-catenin signaling in HCC and suggests future HCC-specific lipid metabolic targets.
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ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.
Asunto(s)
Antineoplásicos/administración & dosificación , Camptotecina/análogos & derivados , Fluorouracilo/administración & dosificación , Indoles/administración & dosificación , Compuestos Organoplatinos/administración & dosificación , Purinas/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenosina Trifosfato , Camptotecina/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Combinación de Medicamentos , Humanos , Irinotecán , Mutación , Proteínas Nucleares/genética , Oxaliplatino , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Factores de Transcripción/genética , Proteínas ras/genéticaRESUMEN
Folate metabolism enables cell growth by providing one-carbon (1C) units for nucleotide biosynthesis. The 1C units are carried by tetrahydrofolate, whose production by the enzyme dihydrofolate reductase is targeted by the important anticancer drug methotrexate. 1C units come largely from serine catabolism by the enzyme serine hydroxymethyltransferase (SHMT), whose mitochondrial isoform is strongly upregulated in cancer. Here we report the SHMT inhibitor SHIN2 and demonstrate its in vivo target engagement with 13C-serine tracing. As methotrexate is standard treatment for T-cell acute lymphoblastic leukemia (T-ALL), we explored the utility of SHIN2 in this disease. SHIN2 increases survival in NOTCH1-driven mouse primary T-ALL in vivo. Low dose methotrexate sensitizes Molt4 human T-ALL cells to SHIN2, and cells rendered methotrexate resistant in vitro show enhanced sensitivity to SHIN2. Finally, SHIN2 and methotrexate synergize in mouse primary T-ALL and in a human patient-derived xenograft in vivo, increasing survival. Thus, SHMT inhibition offers a complementary strategy in the treatment of T-ALL.
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Sinergismo Farmacológico , Regulación Leucémica de la Expresión Génica , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Metotrexato/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the mitochondrial pyruvate carrier (MPC) and the cellular lactate exporter monocarboxylate transporter 4 (MCT4) as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.
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Proteínas de Transporte de Anión/metabolismo , Cardiomegalia/patología , Insuficiencia Cardíaca/patología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Animales , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/genética , Cardiomegalia/inducido químicamente , Cardiomegalia/complicaciones , Insuficiencia Cardíaca/etiología , Corazón Auxiliar , Humanos , Ácido Láctico/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/genética , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/antagonistas & inhibidores , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Ácido Pirúvico/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Función Ventricular Izquierda/fisiologíaRESUMEN
Brown adipose tissue (BAT) is composed of thermogenic cells that convert chemical energy into heat to maintain a constant body temperature and counteract metabolic disease. The metabolic adaptations required for thermogenesis are not fully understood. Here, we explore how steady state levels of metabolic intermediates are altered in brown adipose tissue in response to cold exposure. Transcriptome and metabolome analysis revealed changes in pathways involved in amino acid, glucose, and TCA cycle metabolism. Using isotopic labeling experiments, we found that activated brown adipocytes increased labeling of pyruvate and TCA cycle intermediates from U13C-glucose. Although glucose oxidation has been implicated as being essential for thermogenesis, its requirement for efficient thermogenesis has not been directly tested. We show that mitochondrial pyruvate uptake is essential for optimal thermogenesis, as conditional deletion of Mpc1 in brown adipocytes leads to impaired cold adaptation. Isotopic labeling experiments using U13C-glucose showed that loss of MPC1 led to impaired labeling of TCA cycle intermediates. Loss of MPC1 in BAT increased 3-hydroxybutyrate levels in blood and BAT in response to the cold, suggesting that ketogenesis provides an alternative fuel source to compensate. Collectively, these studies highlight that complete glucose oxidation is essential for optimal brown fat thermogenesis.
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Tejido Adiposo Pardo/fisiología , Proteínas de Transporte de Anión/genética , Frío , Glucosa/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Transportadores de Ácidos Monocarboxílicos/genética , Termogénesis , Adipocitos Marrones/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oxidación-Reducción , Suero/químicaRESUMEN
Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.
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Neoplasias de la Mama/genética , Carbono/metabolismo , Metabolómica/métodos , Mitocondrias/metabolismo , Serina/metabolismo , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , RatonesRESUMEN
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.