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Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.
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Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1's protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.
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Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.
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Astrocitoma , Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Glioma , Humanos , Ratones , Animales , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/genética , Ácido Micofenólico/uso terapéutico , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/metabolismo , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Expresión Génica , Guanosina/uso terapéuticoRESUMEN
Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.
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Aminoácidos Esenciales , Genoma , Aminoácidos/genética , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli/genética , Mamíferos , ValinaRESUMEN
Brain metastases are the most common brain malignancy. This review discusses the studies presented at the third annual meeting of the Melanoma Research Foundation in the context of other recent reports on the biology and treatment of melanoma brain metastases (MBM). Although symptomatic MBM patients were historically excluded from immunotherapy trials, efforts from clinicians and patient advocates have resulted in more inclusive and even dedicated clinical trials for MBM patients. The results of checkpoint inhibitor trials were discussed in conversation with current standards of care for MBM patients, including steroids, radiotherapy, and targeted therapy. Advances in the basic scientific understanding of MBM, including the role of astrocytes and metabolic adaptations to the brain microenvironment, are exposing new vulnerabilities which could be exploited for therapeutic purposes. Technical advances including single-cell omics and multiplex imaging are expanding our understanding of the MBM ecosystem and its response to therapy. This unprecedented level of spatial and temporal resolution is expected to dramatically advance the field in the coming years and render novel treatment approaches that might improve MBM patient outcomes.
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Neoplasias Encefálicas , Melanoma , Neoplasias Primarias Secundarias , Humanos , Ecosistema , Melanoma/patología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/secundario , Inmunoterapia/métodos , Neoplasias Primarias Secundarias/patología , Encéfalo , Microambiente TumoralRESUMEN
Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.
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4-Hidroxifenilpiruvato Dioxigenasa/metabolismo , Ácidos Mandélicos/metabolismo , Metaboloma , Ubiquinona/análogos & derivados , Animales , Línea Celular , Femenino , Humanos , Ácidos Mandélicos/análisis , Ratones , Ratones Desnudos , Tirosina/metabolismo , Ubiquinona/biosíntesisRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
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Neuronas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biosíntesis de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Axones/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Codón/genética , Femenino , Glicina/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/metabolismo , Tejido Nervioso/patología , Consumo de Oxígeno , Neoplasias Pancreáticas/patología , Pirazoles , Pirimidinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , RatasRESUMEN
Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of SV content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type-specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.
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Encéfalo/metabolismo , Espectrometría de Masas/métodos , Metaboloma , Metabolómica/métodos , Vesículas Sinápticas/metabolismo , Animales , Femenino , Ácido Glutámico/metabolismo , Masculino , RatonesRESUMEN
Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
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Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Glucosa-6-Fosfato Isomerasa/metabolismo , Glucólisis/genética , Fosforilación Oxidativa , Vía de Pentosa Fosfato/fisiología , Células Th17/metabolismo , Animales , Hipoxia de la Célula/genética , Hipoxia de la Célula/inmunología , Quimera/genética , Cromatografía de Gases , Cromatografía Liquida , Infecciones por Clostridium/inmunología , Citocinas/deficiencia , Citocinas/genética , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucólisis/inmunología , Homeostasis/genética , Homeostasis/inmunología , Inflamación/genética , Inflamación/inmunología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Vía de Pentosa Fosfato/genética , Vía de Pentosa Fosfato/inmunología , RNA-Seq , Análisis de la Célula Individual , Células Th17/inmunología , Células Th17/patologíaRESUMEN
A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis.This article is highlighted in the In This Issue feature, p. 1241.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Encéfalo/patología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Encéfalo/metabolismo , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Resistencia a Antineoplásicos , Femenino , Técnicas de Silenciamiento del Gen , Glicina/análisis , Glicina/metabolismo , Humanos , Metabolómica , Ratones , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Proteómica , RNA-Seq , Serina/análisis , Serina/metabolismo , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.
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Mitocondrias/metabolismo , Serina/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas , Carbono/metabolismo , Pruebas Genéticas , Humanos , Células Jurkat , Células K562 , Transportador 1 de Sodio-Glucosa/genéticaRESUMEN
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).
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Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Piperazinas/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Piperazinas/síntesis química , Piperazinas/química , Relación Estructura-Actividad , Tiourea/síntesis química , Tiourea/químicaRESUMEN
Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.
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Fosfatidilinositol 3-Quinasa Clase I , Complejo Cetoglutarato Deshidrogenasa , Mutación , Proteínas de Neoplasias , Neoplasias , Animales , Línea Celular Tumoral , Ciclo del Ácido Cítrico/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Glucólisis/genética , Humanos , Complejo Cetoglutarato Deshidrogenasa/biosíntesis , Complejo Cetoglutarato Deshidrogenasa/genética , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
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Carbono/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/biosíntesis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carbono/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Femenino , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Purinas/biosíntesis , Serina/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Timidina/biosíntesis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.
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Leucina/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Serina-Treonina Quinasas TOR/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mutación , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/metabolismo , Glioblastoma/patología , Glicina Hidroximetiltransferasa/metabolismo , Glicina/metabolismo , Isquemia/metabolismo , Acetona/análogos & derivados , Acetona/metabolismo , Acetona/toxicidad , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/enzimología , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular , Femenino , Glioblastoma/irrigación sanguínea , Glioblastoma/enzimología , Glicina-Deshidrogenasa (Descarboxilante)/antagonistas & inhibidores , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Humanos , Isquemia/enzimología , Isquemia/patología , Ratones , Necrosis , Consumo de Oxígeno , Piruvaldehído/metabolismo , Piruvaldehído/toxicidad , Piruvato Quinasa/metabolismo , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.
Asunto(s)
Transición Epitelial-Mesenquimal , Pirimidinas/metabolismo , Animales , Carcinoma/metabolismo , Línea Celular Tumoral , Dihidrouracilo Deshidrogenasa (NADP)/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Mesodermo/citología , Mesodermo/metabolismo , Ratones , ARN Interferente Pequeño/metabolismoRESUMEN
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.