RESUMEN

In the present study, we investigated the consequences of deleting the glutaredoxin-2 gene (Glrx2-/-) on the development of non-alcoholic fatty liver disease (NAFLD) in male and female C57BL6N mice fed a control (CD) or high-fat diet (HFD). We report that the HFD induced a significant increase in body mass in the wild-type (Wt) and Glrx2-/- male, but not female, mice, which was associated with the hypertrophying of the abdominal fat. Interestingly, while the Wt male mice fed the HFD developed NAFLD, the deletion of the Glrx2 gene mitigated vesicle formation, intrahepatic lipid accumulation, and fibrosis in the males. The protective effect associated with ablating the Glrx2 gene in male mice was due to enhancement of mitochondrial redox buffering capacity. Specifically, liver mitochondria from male Glrx2-/- fed a CD or HFD produced significantly less hydrogen peroxide (mtH2O2), had lower malondialdehyde levels, greater activities for glutathione peroxidase and thioredoxin reductase, and less protein glutathione mixed disulfides (PSSG) when compared to the Wt male mice fed the HFD. These effects correlated with the S-glutathionylation of α-ketoglutarate dehydrogenase (KGDH), a potent mtH2O2 source and key redox sensor in hepatic mitochondria. In comparison to the male mice, both Wt and Glrx2-/- female mice displayed almost complete resistance to HFD-induced body mass increases and the development of NAFLD, which was attributed to the superior redox buffering capacity of the liver mitochondria. Together, our findings show that modulation of mitochondrial S-glutathionylation signaling through Glrx2 augments resistance of male mice towards the development of NAFLD through preservation of mitochondrial redox buffering capacity. Additionally, our findings demonstrate the sex dimorphisms associated with the manifestation of NAFLD is related to the superior redox buffering capacity and modulation of the S-glutathionylome in hepatic mitochondria from female mice.

RESUMEN

Importance: Prophylactic administration of antibiotics before skin incision is an important component in the prevention of periprosthetic joint infection in arthroplasty surgery. For antibiotics to be effective, the local tissue concentration (LTC) must exceed the minimum inhibitory concentration of typical infecting organisms; however, the LTC of cefazolin during arthroplasty is poorly understood. Objective: To compare the systemic concentration of cefazolin in serum with the LTC in fat, synovium, and bone during primary total knee arthroplasty (TKA) while assessing the effect of tourniquet inflation. Design, Setting, and Participants: This prospective randomized clinical trial was conducted from March 1, 2022, to June 30, 2023, in patients undergoing TKA at a single academic center. Intervention: Total knee arthroplasty with or without a limb tourniquet. Main Outcomes and Measures: Systemic blood and local tissues from the surgical site (fat, synovium, and bone) were harvested at regular intervals during the surgery. The primary outcome was the LTC of cefazolin, quantified using the liquid chromatography-tandem mass spectrometry technique. Results: A total of 59 patients were included in the study, with 29 in the tourniquet group (mean [SD] age, 69.3 [9.6] years; 23 [79.3%] female) and 30 in the no tourniquet group (mean [SD] age, 69.9 [9.7] years; 21 [70.0%] female). In patients undergoing TKA without a tourniquet, the mean concentration of cefazolin in serum was 71.9 µg/mL (95% CI, 66.4-77.5 µg/mL), whereas the mean LTCs were 13.9 µg/g (95% CI, 12.1-15.7 µg/g) in fat, 27.7 µg/g (95% CI, 24.3-31.0 µg/g) in synovium, and 17.7 µg/g (95% CI, 14.8-20.5 µg/g) in bone. For patients undergoing TKA with a tourniquet, the mean concentration of cefazolin in serum was 72.0 µg/mL (95% CI, 66.3-77.7 µg/mL), and the mean LTCs were 9.9 µg/g (95% CI, 8.7-11.1 µg/g) in fat, 21.8 µg/g (95% CI, 18.7-25.0 µg/g) in synovium, and 13.0 µg/g (95% CI, 10.8-15.2 µg/g) in bone. The use of a tourniquet resulted in significantly lower mean LTCs by 60 minutes after cefazolin infusion (10.8 µg/g [95% CI, 9.1-12.4 µg/g] vs 16.9 µg/g [95% CI, 14.1-19.6 µg/g], P = .001 in fat; 18.9 µg/g [95% CI, 14.1-23.6 µg/g] vs 25.8 µg/g [95% CI, 21.4-30.3 µg/g], P = .03 in synovium; and 11.8 µg/g [95% CI, 9.3-14.2 µg/g] vs 19.4 µg/g [95% CI, 14.5-24.4 µg/g], P = .007 in bone). Conclusions and Relevance: In this randomized clinical trial, the concentration of cefazolin was lower in local tissues (fat, synovium, and bone) than in systemic blood, and the use of a limb tourniquet further significantly reduced these concentrations. Although the current prophylactic dosing regimen for cefazolin provides sufficient serum concentrations, the levels in the periarticular tissue during TKA may be insufficient to prevent periprosthetic joint infection. Trial Registration: ClinicalTrials.gov Identifier: NCT05604157.

Asunto(s)

Antibacterianos , Artroplastia de Reemplazo de Rodilla , Cefazolina , Torniquetes , Humanos , Cefazolina/farmacocinética , Cefazolina/administración & dosificación , Cefazolina/sangre , Femenino , Masculino , Anciano , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Antibacterianos/análisis , Antibacterianos/uso terapéutico , Persona de Mediana Edad , Estudios Prospectivos , Infecciones Relacionadas con Prótesis/prevención & control , Profilaxis Antibiótica/métodos , Infección de la Herida Quirúrgica/prevención & control

RESUMEN

Metabolic reprogramming, a hallmark of tumorigenesis, involves alterations in glucose and fatty acid metabolism. Here, we investigate the role of Carnitine palmitoyl transferase 1a (Cpt1a), a key enzyme in long-chain fatty acid (LCFA) oxidation, in ErbB2-driven breast cancers. In ErbB2+ breast cancer models, ablation of Cpt1a delays tumor onset, growth, and metastasis. However, Cpt1a-deficient cells exhibit increased glucose dependency that enables survival and eventual tumor progression. Consequently, these cells exhibit heightened oxidative stress and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Inhibiting Nrf2 or silencing its expression reduces proliferation and glucose consumption in Cpt1a-deficient cells. Combining the ketogenic diet, composed of LCFAs, or an anti-ErbB2 monoclonal antibody (mAb) with Cpt1a deficiency significantly perturbs tumor growth, enhances apoptosis, and reduces lung metastasis. Using an immunocompetent model, we show that Cpt1a inhibition promotes an antitumor immune microenvironment, thereby enhancing the efficacy of anti-ErbB2 mAbs. Our findings underscore the importance of targeting fatty acid oxidation alongside HER2-targeted therapies to combat resistance in HER2+ breast cancer patients.

Asunto(s)

Neoplasias de la Mama , Carnitina O-Palmitoiltransferasa , Ácidos Grasos , Factor 2 Relacionado con NF-E2 , Oxidación-Reducción , Receptor ErbB-2 , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/antagonistas & inhibidores , Ácidos Grasos/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Microambiente Tumoral/efectos de los fármacos , Dieta Cetogénica , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Glucosa/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología

RESUMEN

Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.

Asunto(s)

Neoplasias de la Mama , Glutamato-Cisteína Ligasa , Glutatión , Homeostasis , Neoplasias Hepáticas , Oxidación-Reducción , Especies Reactivas de Oxígeno , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Humanos , Glutatión/metabolismo , Animales , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/genética , Ratones , Línea Celular Tumoral , Glutamato-Cisteína Ligasa/metabolismo , Glutamato-Cisteína Ligasa/genética , Glucólisis , Metástasis de la Neoplasia , Regulación Neoplásica de la Expresión Génica , Microambiente Tumoral

RESUMEN

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Asunto(s)

Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Código de Histonas , Histonas/genética , Histonas/metabolismo , Resistencia a Múltiples Medicamentos

RESUMEN

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.

RESUMEN

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

Asunto(s)

Neoplasias de la Mama , Humanos , Femenino , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Neoplasias de la Mama/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Estrés Oxidativo/fisiología , Fenotipo , Línea Celular Tumoral , Microambiente Tumoral

RESUMEN

SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.

Asunto(s)

Glutamina , Neoplasias , Humanos , Transportador de Glucosa de Tipo 1 , Adenosina Trifosfatasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Suplementos Dietéticos , ADN Helicasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.

Asunto(s)

Aminoácidos , Dinámicas Mitocondriales , Aminas/metabolismo , Aminoácidos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Purinas/metabolismo

RESUMEN

Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.

Asunto(s)

Carcinoma Ductal Pancreático , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas , Hormonas Tiroideas/metabolismo , Carcinoma Ductal Pancreático/genética , Humanos , Metionina , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/metabolismo , Oxidación-Reducción , Neoplasias Pancreáticas/genética , Piruvato Quinasa/metabolismo , Proteínas de Unión a Hormona Tiroide , Neoplasias Pancreáticas

RESUMEN

Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin-like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP-activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Asunto(s)

Complejo IV de Transporte de Electrones , Chaperonas Moleculares , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Células HCT116 , Humanos , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo

RESUMEN

Kidney cancer is one of the top ten cancer diagnosed worldwide and its incidence has increased the last 20 years. Clear Cell Renal Cell Carcinoma (ccRCC) are characterized by mutations that inactivate the von Hippel-Lindau (VHL) tumor suppressor gene and evidence indicated alterations in metabolic pathways, particularly in glutamine metabolism. We previously identified a small molecule, STF-62247, which target VHL-deficient renal tumors by affecting late-stages of autophagy and lysosomal signaling. In this study, we investigated ccRCC metabolism in VHL-deficient and proficient cells exposed to the small molecule. Metabolomics profiling using 1H NMR demonstrated that STF-62247 increases levels of glucose, pyruvate, glycerol 3-phosphate while glutamate, asparagine, and glutathione significantly decreased. Diminution of glutamate and glutamine was further investigated using mass spectrometry, western blot analyses, enzymatic activities, and viability assays. We found that expression of SLC1A5 increases in VHL-deficient cells treated with STF-62247, possibly to stimulate glutamine uptake intracellularly to counteract the diminution of this amino acid. However, exogenous addition of glutamine was not able to rescue cell viability induced by the small molecule. Instead, our results showed that VHL-deficient cells utilize glutamine to produce fatty acid in response to STF-62247. Surprisingly, this occurs through oxidative phosphorylation in STF-treated cells while control cells use reductive carboxylation to sustain lipogenesis. We also demonstrated that STF-62247 stimulated expression of stearoyl-CoA desaturase (SCD1) and peripilin2 (PLIN2) to generate accumulation of lipid droplets in VHL-deficient cells. Moreover, the carnitine palmitoyltransferase 1A (CPT1A), which control the entry of fatty acid into mitochondria for ß-oxidation, also increased in response to STF-62247. CPT1A overexpression in ccRCC is known to limit tumor growth. Together, our results demonstrated that STF-62247 modulates cellular metabolism of glutamine, an amino acid involved in the autophagy-lysosome process, to support lipogenesis, which could be implicated in the signaling driving to cell death.

RESUMEN

The maintenance of a strong IL21 production in memory CD4 T cells, especially in HIV-1-specific cells, represents a major correlate of natural immune protection against the virus. However, the molecular mechanisms underlying IL21 production during HIV-1 infection, which is only elevated among the naturally protected elite controllers (EC), are still unknown. We recently found out that lipophagy is a critical immune mediator that control an antiviral metabolic state following CD8A T cell receptor engagement, playing an important role in the natural control of HIV-1 infection. This led us to investigate whether the beneficial role of a strong macroautophagy/autophagy, could also be used to ensure effective IL21 production as well. Herein, we confirm that after both polyclonal and HIV-1-specific activation, memory CD4 T cells (Mem) from EC display enhanced activity of the autophagy-mediated proteolysis compared to ART. Our results indicate that the enhanced autophagy activity in EC was controlled by the energy-sensing PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1). We further confirmed the critical role of the autophagy-mediated proteolysis in the strong IL21 production in EC by using BECN1 gene silencing as well as protease, PRKAA1, and lysosomal inhibitors. Finally, we established that high autophagy-mediated proteolysis in EC fuels their cellular rates of mitochondrial respiration due to glutaminolysis. Our data confirm the critical role of autophagy in dictating the metabolic input, which is required not only to ensure protective cytotoxic CD8A T cell responses, but also to provide strong IL21 production among antiviral CD4 T cells.Abbreviations: AKG: alpha-ketoglutarate; ART: patients under antiretroviral therapy; ATG7: autophagy related 7; BaF: bafilomycin A1; BECN1: beclin 1; Chloro.: chloroquine; EC: elite controllers; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FOXO3: forkhead box O3; GLS: glutaminase; GLUD1: glutamate dehydrogenase 1; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MTOR: mechanistic target of rapamycin kinase; PBMC: peripheral blood mononuclear cells; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; SQSTM1: sequestosome 1; TCA: tricarboxylic acid cycle; ULK1: unc-51 like autophagy activating kinase.

Asunto(s)

Autofagia , VIH-1 , Adenosina Monofosfato , Antivirales/farmacología , Autofagia/fisiología , Linfocitos T CD4-Positivos , Humanos , Interleucinas , Leucocitos Mononucleares , Proteínas Quinasas

RESUMEN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).

Asunto(s)

Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Proteínas del Citoesqueleto/metabolismo , Degeneración Nerviosa/patología , Paclitaxel/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Animales , Canales de Calcio/metabolismo , ADP-Ribosa Cíclica/antagonistas & inhibidores , Femenino , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratas Sprague-Dawley

RESUMEN

Glucose metabolism modulates the islet ß cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.

Asunto(s)

Glucosa/metabolismo , Glutatión/biosíntesis , Piruvato Carboxilasa/metabolismo , Adulto , Animales , Antioxidantes/fisiología , Femenino , Glutatión/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo/fisiología , Cultivo Primario de Células

RESUMEN

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Fenformina/farmacología , Factor de Transcripción STAT1/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Metabolismo Energético/efectos de los fármacos , Femenino , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Humanos , Interferón gamma/administración & dosificación , Interferón gamma/deficiencia , Interferón gamma/metabolismo , Células MCF-7 , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Fenformina/administración & dosificación , Poli I-C/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT1/agonistas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.

Asunto(s)

Epigénesis Genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nucleótidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Azetidinas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones Endogámicos NOD , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Fase S/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (BpgmL166P) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in BpgmL166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of BpgmL166P involves a dual mechanism that enhances the host's stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target.

Asunto(s)

Anemia/etiología , Anemia/prevención & control , Bisfosfoglicerato Mutasa/deficiencia , Malaria Cerebral/enzimología , Malaria Cerebral/prevención & control , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Bisfosfoglicerato Mutasa/química , Bisfosfoglicerato Mutasa/genética , Bisfosfoglicerato Mutasa/metabolismo , Estabilidad de Enzimas , Eritrocitos/enzimología , Eritrocitos/parasitología , Eritropoyesis , Matriz Extracelular/metabolismo , Femenino , Células HEK293 , Humanos , Malaria Cerebral/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Parásitos/crecimiento & desarrollo , Plasmodium/crecimiento & desarrollo , Policitemia

RESUMEN

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Resveratrol/farmacología , Sistemas CRISPR-Cas , Línea Celular , Resistencia a Medicamentos/genética , Humanos , Hidroxiurea/farmacología , Células Jurkat , Nucleótidos/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Sirtuina 1/metabolismo , Estilbenos/farmacología

RESUMEN

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet ß-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

Asunto(s)

Arginina/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Inflamación/prevención & control , Células Secretoras de Insulina/efectos de los fármacos , Trastornos Innatos del Ciclo de la Urea/patología , Urea/metabolismo , Adolescente , Adulto , Anciano , Ácido Aspártico/metabolismo , Supervivencia Celular , Ciclo del Ácido Cítrico/efectos de los fármacos , Femenino , Humanos , Inflamación/patología , Células Secretoras de Insulina/patología , Masculino , Metabolómica , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Piruvato Carboxilasa/metabolismo , Trastornos Innatos del Ciclo de la Urea/metabolismo , Adulto Joven