RESUMEN
Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.
Asunto(s)
Acetatos/metabolismo , Glucosa/metabolismo , Ácido Pirúvico/metabolismo , ATP Citrato (pro-S)-Liasa/fisiología , Acetilcoenzima A/biosíntesis , Acetilcoenzima A/metabolismo , Acetilación , Animales , Femenino , Glucólisis/fisiología , Lipogénesis/fisiología , Masculino , Mamíferos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Oxidorreductasas , Piruvato Descarboxilasa/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.
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Factores de Transcripción Forkhead/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Receptores Toll-Like/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Transportador de Glucosa de Tipo 1/genética , Glucólisis , Tolerancia Inmunológica , Diana Mecanicista del Complejo 1 de la Rapamicina , Metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Glutamine is an essential nutrient for cancer cell survival and proliferation. Enhanced utilization of glutamine often depletes its local supply, yet how cancer cells adapt to low glutamine conditions is largely unknown. Here, we report that IκB kinase ß (IKKß) is activated upon glutamine deprivation and is required for cell survival independently of NF-κB transcription. We demonstrate that IKKß directly interacts with and phosphorylates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major driver of aerobic glycolysis, at Ser269 upon glutamine deprivation to inhibit its activity, thereby down-regulating aerobic glycolysis when glutamine levels are low. Thus, due to lack of inhibition of PFKFB3, IKKß-deficient cells exhibit elevated aerobic glycolysis and lactate production, leading to less glucose carbons contributing to tricarboxylic acid (TCA) cycle intermediates and the pentose phosphate pathway, which results in increased glutamine dependence for both TCA cycle intermediates and reactive oxygen species suppression. Therefore, coinhibition of IKKß and glutamine metabolism results in dramatic synergistic killing of cancer cells both in vitro and in vivo. In all, our results uncover a previously unidentified role of IKKß in regulating glycolysis, sensing low-glutamine-induced metabolic stress, and promoting cellular adaptation to nutrient availability.
Asunto(s)
Glutamina/metabolismo , Quinasa I-kappa B/metabolismo , Fosfofructoquinasa-2/metabolismo , Adaptación Fisiológica/genética , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Glucólisis/genética , Células HEK293 , Células HeLa , Humanos , Quinasa I-kappa B/genética , Células MCF-7 , Ratones , FN-kappa B/metabolismo , FosforilaciónRESUMEN
We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.
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Conmoción Encefálica , Encefalopatías Metabólicas , Hidroliasas , Adulto , Niño , Preescolar , Humanos , Hidroliasas/metabolismo , Mitocondrias/metabolismo , NAD/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Proteómica , Conmoción Encefálica/complicaciones , Conmoción Encefálica/genética , Encefalopatías Metabólicas/etiología , Encefalopatías Metabólicas/genéticaRESUMEN
Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.
Asunto(s)
Células Madre Pluripotentes Inducidas , Lipofuscinosis Ceroideas Neuronales , Animales , Humanos , Ésteres del Colesterol , Glicoproteínas de Membrana/genética , Metabolómica , Chaperonas Moleculares , Lipofuscinosis Ceroideas Neuronales/genética , Pez Cebra/genéticaRESUMEN
BACKGROUND: SDS-PAGE followed by in-gel digestion (IGD) is a popular workflow in mass spectrometry-based proteomics. In GeLC-MS/MS, a protein lysate of a biological sample is separated by SDS-PAGE and each gel lane is sliced in 5-20 slices which, after IGD, are analyzed by LC-MS/MS. The database search results for all slices of a biological sample are combined yielding global protein identification and quantification for each sample. In large scale GeLC-MS/MS experiments the manual processing steps including washing, reduction and alkylation become a bottleneck. Here we introduce the whole gel (WG) procedure where, prior to gel slice cutting, the processing steps are carried out on the whole gel. RESULTS: In two independent experiments human HCT116 cell lysate and mouse tumor tissue lysate were separated by 1D SDS PAGE. In a back to back comparison of the IGD procedure and the WG procedure, both protein identification (>80% overlap) and label-free protein quantitation (R2=0.94) are highly similar between procedures. Triplicate analysis of the WG procedure of both HCT116 cell lysate and formalin-fixed paraffin embedded (FFPE) tumor tissue showed identification reproducibility of >88% with a CV<20% on protein quantitation. CONCLUSIONS: The whole gel procedure allows for reproducible large-scale differential GeLC-MS/MS experiments, without a prohibitive amount of manual processing and with similar performance as conventional in-gel digestion. This procedure will especially enable clinical proteomics for which GeLC-MS/MS is a popular workflow and sample numbers are relatively high.
RESUMEN
Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.
Asunto(s)
Microbioma Gastrointestinal , Trasplante de Células Madre Hematopoyéticas , Neoplasias , Neutropenia , Ratones , Animales , Propionatos , Verrucomicrobia , Moco/metabolismo , Mucinas/metabolismo , Dieta , Neutropenia/metabolismo , Neoplasias/metabolismoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Glutamina/metabolismo , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Linfocitos T/metabolismoRESUMEN
Novel biomarkers for HCC surveillance in cirrhotic patients are urgently needed. Exosomes and their lipid content in particular represent potentially valuable noninvasive diagnostic biomarkers. We isolated exosomes from plasma of 72 cirrhotic patients, including 31 with HCC. Exosomes and unfractionated plasma were processed for untargeted lipidomics using ultra-high-resolution mass spectrometry. A total of 2,864 lipid species, belonging to 52 classes, were identified. Both exosome fractionation and HCC diagnosis had significant impact on the lipid profiles. Ten lipid classes were enriched in HCC exosomes compared with non-HCC exosomes. Dilysocardiolipins were detected in 35% of the HCC exosomes but in none of the non-HCC exosomes (P < 0.001). Cardiolipins and sphingosines had the highest differential effects (fold change of 133.08, q = 0.001 and 38.57, q < 0.001, respectively). In logistic regression analysis, high abundances of exosomal sphingosines, dilysocardiolipins, lysophosphatidylserines, and (O-acyl)-1-hydroxy fatty acids were strongly associated with HCC [OR (95% confidence interval (CI)), 271.1 (14.0-5,251.9), P < 0.001; 46.5 (2.3-939.9), P = 0.012; 14.9 (4.3-51.2), P < 0.001; 10.3 (3.2-33.1), P < 0.001]. Four lipid classes were depleted in HCC exosomes compared with non-HCC exosomes. In logistic regression analysis, lack of detection of sulfatides and acylGlcSitosterol esters was strongly associated with HCC [OR (95% CI): 215.5 (11.5-4,035.9), P < 0.001; 26.7 (1.4-528.4), P = 0.031]. These HCC-associated changes in lipid composition of exosomes reflected alterations in glycerophospholipid metabolism, retrograde endocannabinoid signaling, and ferroptosis. In conclusion, this study identified candidate biomarkers for early detection of HCC as well as altered pathways in exosomes that may contribute to tumor development and progression. PREVENTION RELEVANCE: This study identifies lipids in circulating exosomes, that could serve as biomarkers for the early detection of hepatocellular carcinoma as well as altered pathways in exosomes that may contribute to tumor development and progression.
Asunto(s)
Carcinoma Hepatocelular/diagnóstico , Lípidos/sangre , Cirrosis Hepática/sangre , Neoplasias Hepáticas/diagnóstico , Anciano , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Detección Precoz del Cáncer/métodos , Exosomas/química , Exosomas/metabolismo , Exosomas/patología , Femenino , Humanos , Lipidómica , Cirrosis Hepática/complicaciones , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
BACKGROUND: Hispanics in South Texas have high rates of hepatocellular carcinoma (HCC) and nonalcoholic fatty liver disease (NAFLD). Liver fibrosis severity is the strongest predictive factor of NAFLD progression to HCC. We examined the association between free fatty acids (FA) and advanced liver fibrosis or HCC in this population. METHODS: We quantified 45 FAs in plasma of 116 subjects of the Cameron County Hispanic Cohort, 15 Hispanics with HCC, and 56 first/second-degree relatives of Hispanics with HCC. Liver fibrosis was assessed by FibroScan. RESULTS: Advanced liver fibrosis was significantly associated with low expression of very long chain (VLC) saturated FAs (SFA), odd chain SFAs, and VLC n-3 polyunsaturated FAs [PUFA; AOR; 95% confidence interval (CI), 10.4 (3.7-29.6); P < 0.001; 5.7 (2.2-15.2); P < 0.001; and 3.7 (1.5-9.3); P = 0.005]. VLC n3-PUFAs significantly improved the performance of the noninvasive markers for advanced fibrosis - APRI, FIB-4, and NFS. Plasma concentrations of VLC SFAs and VLC n-3 PUFAs were further reduced in patients with HCC. Low concentrations of these FAs were also observed in relatives of patients with HCC and in subjects with the PNPLA3 rs738409 homozygous genotype. CONCLUSIONS: Low plasma concentrations of VLC n-3 PUFAs and VLC SFAs were strongly associated with advanced liver fibrosis and HCC in this population. Genetic factors were associated with low concentrations of these FAs as well. IMPACT: These results have implications in identifying those at risk for liver fibrosis progression to HCC and in screening this population for advanced fibrosis. They also prompt the evaluation of VLC n-3 PUFA or VLC SFA supplementation to prevent cirrhosis and HCC.
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Carcinoma Hepatocelular/sangre , Ácidos Grasos/sangre , Cirrosis Hepática/sangre , Neoplasias Hepáticas/sangre , Enfermedad del Hígado Graso no Alcohólico/sangre , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/etiología , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Hispánicos o Latinos , Humanos , Cirrosis Hepática/etiología , Masculino , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Factores de Riesgo , TexasRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.
Asunto(s)
Ceguera/genética , Glicoproteínas de Membrana/deficiencia , Lipofuscinosis Ceroideas Neuronales/genética , Epitelio Pigmentado de la Retina/patología , Animales , Atrofia/genética , Atrofia/patología , Autofagia , Ceguera/patología , Línea Celular , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Glucógeno/metabolismo , Humanos , Lisosomas/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Microscopía Electrónica , Chaperonas Moleculares/genética , Mutación , Lipofuscinosis Ceroideas Neuronales/complicaciones , Lipofuscinosis Ceroideas Neuronales/patología , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro. T cells metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. We demonstrate that the ribose subunit of inosine can enter into central metabolic pathways to provide ATP and biosynthetic precursors, and that cancer cells display diverse capacities to utilize inosine as a carbon source. Moreover, the supplementation with inosine enhances the anti-tumour efficacy of immune checkpoint blockade and adoptive T-cell transfer in solid tumours that are defective in metabolizing inosine, reflecting the capability of inosine to relieve tumour-imposed metabolic restrictions on T cells.
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Linfocitos T CD8-positivos/metabolismo , Carbono/metabolismo , Glucosa/deficiencia , Inosina/metabolismo , Traslado Adoptivo , Animales , Línea Celular Tumoral , Células HeLa , Humanos , Hipoxantina/metabolismo , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Nutrientes , Purina-Nucleósido Fosforilasa/metabolismo , Ribosa/metabolismoRESUMEN
Patients with carcinoma of unknown primary (CUP) present with metastatic disease for which the primary site cannot be found, despite extensive standard investigation. Here, we describe the development and implementation of the first clinically available microarray-based test for this cancer type (CUPPrint), based on 633 individual tumors representing 30 carcinoma and 17 noncarcinoma classes. Tissue of origin prediction for either fresh frozen or paraffin-embedded tumor samples is achieved with the use of a custom 8-pack 1.9k microarray and robust classification algorithm. An expression profile of 495 genes was used to predict tumor origin by applying a k-nearest neighbor algorithm. Internal cross-validation and analysis of an independent, previously published, 229-sample dataset revealed that clinically informative predictions were made for up to 94% of samples analyzed. Analysis of 13 previously published CUP specimens yielded predicted tumor origins that supported the clinical suspicion in 12 cases (92%). Microarray profiling presents a promising tool to assist in the identification of the primary tumor and might direct a more tailored treatment for CUP patients.
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Pruebas Diagnósticas de Rutina , Perfilación de la Expresión Génica , Neoplasias Primarias Desconocidas/diagnóstico , Neoplasias Primarias Desconocidas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Algoritmos , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Estadificación de Neoplasias , Neoplasias Primarias Desconocidas/clasificación , Adhesión en Parafina , PronósticoRESUMEN
Exposure to ambient air particulate matter (PM2.5) is well established as a risk factor for cardiovascular and pulmonary disease. Both epidemiologic and controlled exposure studies in humans and animals have demonstrated an association between air pollution exposure and metabolic disorders such as diabetes. Given the central role of the liver in peripheral glucose homeostasis, we exposed mice to filtered air or PM2.5 for 16 weeks and examined its effect on hepatic metabolic pathways using stable isotope resolved metabolomics (SIRM) following a bolus of 13C6-glucose. Livers were analyzed for the incorporation of 13C into different metabolic pools by IC-FTMS or GC-MS. The relative abundance of 13C-glycolytic intermediates was reduced, suggesting attenuated glycolysis, a feature found in diabetes. Decreased 13C-Krebs cycle intermediates suggested that PM2.5 exposure led to a reduction in the Krebs cycle capacity. In contrast to decreased glycolysis, we observed an increase in the oxidative branch of the pentose phosphate pathway and 13C incorporations suggestive of enhanced capacity for the de novo synthesis of fatty acids. To our knowledge, this is one of the first studies to examine 13C6-glucose utilization in the liver following PM2.5 exposure, prior to the onset of insulin resistance (IR).
RESUMEN
The plasticity of a preexisting regulatory circuit compromises the effectiveness of targeted therapies, and leveraging genetic vulnerabilities in cancer cells may overcome such adaptations. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is characterized by oxidative phosphorylation (OXPHOS) deficiency caused by fumarate hydratase (FH) nullizyogosity. To identify metabolic genes that are synthetically lethal with OXPHOS deficiency, we conducted a genetic loss-of-function screen and found that phosphogluconate dehydrogenase (PGD) inhibition robustly blocks the proliferation of FH mutant cancer cells both in vitro and in vivo. Mechanistically, PGD inhibition blocks glycolysis, suppresses reductive carboxylation of glutamine, and increases the NADP+/NADPH ratio to disrupt redox homeostasis. Furthermore, in the OXPHOS-proficient context, blocking OXPHOS using the small-molecule inhibitor IACS-010759 enhances sensitivity to PGD inhibition in vitro and in vivo. Together, our study reveals a dependency on PGD in OXPHOS-deficient tumors that might inform therapeutic intervention in specific patient populations.
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Fosforilación Oxidativa , Fosfogluconato Deshidrogenasa/genética , Mutaciones Letales Sintéticas , Animales , Línea Celular Tumoral , Femenino , Fumarato Hidratasa/genética , Genómica/métodos , Glucólisis , Humanos , Mutación con Pérdida de Función , Ratones , Ratones DesnudosRESUMEN
Delivering isotopic tracers for metabolic studies in rodents without overt stress is challenging. Current methods achieve low label enrichment in proteins and lipids. Here, we report noninvasive introduction of 13C6-glucose via a stress-free, ad libitum liquid diet. Using NMR and ion chromatography-mass spectrometry, we quantify extensive 13C enrichment in products of glycolysis, the Krebs cycle, the pentose phosphate pathway, nucleobases, UDP-sugars, glycogen, lipids, and proteins in mouse tissues during 12 to 48 h of 13C6-glucose feeding. Applying this approach to patient-derived lung tumor xenografts (PDTX), we show that the liver supplies glucose-derived Gln via the blood to the PDTX to fuel Glu and glutathione synthesis while gluconeogenesis occurs in the PDTX. Comparison of PDTX with ex vivo tumor cultures and arsenic-transformed lung cells versus xenografts reveals differential glucose metabolism that could reflect distinct tumor microenvironment. We further found differences in glucose metabolism between the primary PDTX and distant lymph node metastases.
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Isótopos de Carbono/metabolismo , Glucosa/metabolismo , Neoplasias Pulmonares/metabolismo , Redes y Vías Metabólicas , Metabolómica/métodos , Animales , Isótopos de Carbono/química , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Femenino , Glucosa/química , Glucógeno/química , Glucógeno/metabolismo , Glucólisis , Xenoinjertos , Humanos , Hígado/química , Hígado/metabolismo , Neoplasias Pulmonares/química , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Vía de Pentosa FosfatoRESUMEN
During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.
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Epigénesis Genética/genética , Glucosa/metabolismo , Metástasis de la Neoplasia/genética , Neoplasias Pancreáticas/genética , Carcinogénesis/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Cromatina/genética , Epigenómica/métodos , Expresión Génica/genética , Heterocromatina/genética , Histonas/genética , Humanos , Neoplasias Pancreáticas/metabolismoRESUMEN
BACKGROUND: A 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis. RESULTS: To facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 probes suitable for high throughput processing. RNA of 162 patient samples from two previous studies was subjected to hybridization to this custom array to validate the prognostic value. Classification results obtained from the original analysis were then compared to those generated using the algorithms based on the custom microarray and showed an extremely high correlation of prognosis prediction between the original data and those generated using the custom mini-array (p < 0.0001). CONCLUSION: In this report we demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool. The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is therefore an excellent tool to predict outcome of disease in breast cancer patients.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Pronóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Cancer and stromal cell metabolism is important for understanding tumor development, which highly depends on the tumor microenvironment (TME). Cell or animal models cannot recapitulate the human TME. We have developed an ex vivo paired cancerous (CA) and noncancerous (NC) human lung tissue approach to explore cancer and stromal cell metabolism in the native human TME. This approach enabled full control of experimental parameters and acquisition of individual patient's target tissue response to therapeutic agents while eliminating interferences from genetic and physiological variations. In this two-case study of non-small-cell lung cancer, we performed stable isotope-resolved metabolomic (SIRM) experiments on paired CA and NC lung tissues treated with a macrophage activator ß-glucan and (13)C6-glucose, followed by ion chromatography-Fourier transform mass spectrometry (IC-FTMS) and nuclear magnetic resonance (NMR) analyses of (13)C-labeling patterns of metabolites. We demonstrated that CA lung tissue slices were metabolically more active than their NC counterparts, which recapitulated the metabolic reprogramming in CA lung tissues observed in vivo. We showed ß-glucan-enhanced glycolysis, Krebs cycle, pentose phosphate pathway, antioxidant production, and itaconate buildup in patient UK021 with chronic obstructive pulmonary disease (COPD) and an abundance of tumor-associated macrophages (TAMs) but not in UK049 with no COPD and much less macrophage infiltration. This metabolic response of UK021 tissues was accompanied by reduced mitotic index, increased necrosis, and enhaced inducible nitric oxide synthase (iNOS) expression. We surmise that the reprogrammed networks could reflect ß-glucan M1 polarization of human macrophages. This case study presents a unique opportunity for investigating metabolic responses of human macrophages to immune modulators in their native microenvironment on an individual patient basis.