RESUMO
The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers.
Assuntos
Linfoma de Burkitt/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Células HEK293 , Xenoenxertos , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
Pluripotent stem cells are thought of as a surrogate of early developmental stages that sustain the capacity to generate all cell types in the body, thereby constituting an invaluable tool to address the mechanisms underlying cellular plasticity. In the mouse, cells resembling totipotent 2-cell-stage embryos (2-cell-like cells) arise at a very low frequency in embryonic stem cell (ESC) cultures. However, the extent to which these early-embryonic-like cells recapitulate the molecular features of the early embryo is unclear. Here, we have undertaken a characterization of some of the metabolic features of early-embryonic-like cells in culture. Our data indicate that early-embryonic-like cells exhibit decreased glycolytic and respiratory activity, lower levels of reactive oxygen species and increased glucose uptake, suggesting a shift of the metabolic programme during 2-cell-like cell reprogramming. Accordingly, we find that 2-cell-like cells can be induced by defined metabolites. Thus, in addition to their transcriptional and chromatin features, 2-cell-like cells recapitulate some of the metabolic features of their in vivo counterpart. Altogether, our work underscores a distinct metabolic state of early-embryonic-like cells and identifies compounds that can induce their emergence in vitro.
Assuntos
Células-Tronco Embrionárias , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Reprogramação Celular , Cromatina , Embrião de Mamíferos , CamundongosRESUMO
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Neoplasias Pancreáticas/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/prevenção & controle , Carcinoma Ductal Pancreático/secundário , Catepsinas/genética , Catepsinas/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genes ras , Heterozigoto , Homozigoto , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Transdução de Sinais , Carga Tumoral , Células Tumorais CultivadasRESUMO
The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.
Assuntos
Adipócitos/citologia , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , MicroRNAs/metabolismo , Adipócitos/metabolismo , Adipogenia , Animais , Corticosterona/metabolismo , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Inflamação , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/cirurgia , Sobrepeso/cirurgia , Fenótipo , RNA Interferente Pequeno/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Células-Tronco/citologia , TransfecçãoRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding proteins (4E-BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1-associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPß) mRNA into the C/EBPß-LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E-BPs is required for suppression of LIP. Intriguingly, mice lacking the cis-regulatory upstream open reading frame (uORF) in the C/EBPß-mRNA, which is required for mTORC1-stimulated translation into C/EBPß-LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPß-isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Metabolismo dos Lipídeos , Complexos Multiproteicos/metabolismo , RNA Mensageiro/genética , Serina-Treonina Quinases TOR/metabolismo , Adipogenia/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Restrição Calórica , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Fases de Leitura Aberta , Oxirredução , Fenótipo , Biossíntese de Proteínas , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
OBJECTIVE: Cancer cells use glycolysis for generation of metabolic intermediates and ATP needed for cell growth and proliferation. The transcription factor C/EBPß-LIP stimulates glycolysis and mitochondrial respiration in cancer cells. We initially observed that high expression of C/EBPß-LIP makes cells vulnerable to treatment with the glycolysis inhibitor 2-deoxyglucose. The aim of the study was to uncover the involved mechanisms of C/EBPß-LIP induced sensitivity to glycolysis inhibition. METHODS: We used genetically engineered cell lines to examine the effect of C/EBPß-LIP and -LAP protein isoforms on glycolysis and NADH/NAD+ metabolism in mouse embryonic fibroblasts (MEFs), and triple negative breast cancer (TNBC) cells that endogenously express high levels of C/EBPß-LIP. Analyses included assays of cell proliferation, cell survival and metabolic flux (OCR and ECAR by Seahorse XF96). Small molecule inhibitors were used to identify underlying metabolic pathways that mediate sensitivity to glycolysis inhibition induced by C/EBPß-LIP. RESULTS: The transcription factor C/EBPß-LIP stimulates both glycolysis and the malate-aspartate shuttle (MAS) and increases the sensitivity to glycolysis inhibition (2-deoxyglucose) in fibroblasts and breast cancer cells. Inhibition of glycolysis with ongoing C/EBPß-LIP-induced MAS activity results in NADH depletion and apoptosis that can be rescued by inhibiting either the MAS or other NAD+-regenerating processes. CONCLUSION: This study indicates that a low NADH/NAD+ ratio is an essential mediator of 2-deoxyglucose toxicity in cells with high cytoplasmic NAD+-regeneration capacity and that simultaneous inhibition of glycolysis and lowering of the NADH/NAD+ ratio may be considered to treat cancer.
Assuntos
Ácido Aspártico , Proteína beta Intensificadora de Ligação a CCAAT , Animais , Camundongos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ácido Aspártico/metabolismo , Malatos/metabolismo , NAD/metabolismo , Fibroblastos/metabolismo , Glicólise , DesoxiglucoseRESUMO
The transcription factors LAP1, LAP2 and LIP are derived from the Cebpb-mRNA through the use of alternative start codons. High LIP expression has been associated with human cancer and increased cancer incidence in mice. However, how LIP contributes to cellular transformation is poorly understood. Here we present that LIP induces aerobic glycolysis and mitochondrial respiration reminiscent of cancer metabolism. We show that LIP-induced metabolic programming is dependent on the RNA-binding protein LIN28B, a translational regulator of glycolytic and mitochondrial enzymes with known oncogenic function. LIP activates LIN28B through repression of the let-7 microRNA family that targets the Lin28b-mRNA. Transgenic mice overexpressing LIP have reduced levels of let-7 and increased LIN28B expression, which is associated with metabolic reprogramming as shown in primary bone marrow cells, and with hyperplasia in the skin. This study establishes LIP as an inducer of cancer-type metabolic reprogramming and as a regulator of the let-7/LIN28B regulatory circuit.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , MicroRNAs/genética , Neoplasias/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Códon , Fibroblastos/metabolismo , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Consumo de Oxigênio , Proteoma , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Transdução de SinaisRESUMO
Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.
Assuntos
Glicemia/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Adiposidade , Adulto , Animais , Autofagia , Colesterol/sangue , Feminino , Homeostase , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP) transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα). Protein lysine acetylation is a key post-translational modification (PTM) that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT) p300 and deacetylated by the lysine deacetylase (KDAC) sirtuin1 (SIRT1). SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+) and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply.
Assuntos
Proteína p300 Associada a E1A/genética , Mitocôndrias/metabolismo , Sirtuína 1/genética , Acetilação , Animais , Proteína p300 Associada a E1A/metabolismo , Humanos , Sirtuína 1/metabolismoRESUMO
DNA damage and telomere dysfunction shorten organismal lifespan. Here we show that oral glucose administration at advanced age increases health and lifespan of telomere dysfunctional mice. The study reveals that energy consumption increases in telomere dysfunctional cells resulting in enhanced glucose metabolism both in glycolysis and in the tricarboxylic acid cycle at organismal level. In ageing telomere dysfunctional mice, normal diet provides insufficient amounts of glucose thus leading to impaired energy homeostasis, catabolism, suppression of IGF-1/mTOR signalling, suppression of mitochondrial biogenesis and tissue atrophy. A glucose-enriched diet reverts these defects by activating glycolysis, mitochondrial biogenesis and oxidative glucose metabolism. The beneficial effects of glucose substitution on mitochondrial function and glucose metabolism are blocked by mTOR inhibition but mimicked by IGF-1 application. Together, these results provide the first experimental evidence that telomere dysfunction enhances the requirement of glucose substitution for the maintenance of energy homeostasis and IGF-1/mTOR-dependent mitochondrial biogenesis in ageing tissues.
Assuntos
Glucose/química , Telômero/ultraestrutura , Envelhecimento , Animais , Glicemia/metabolismo , Calorimetria , Cruzamentos Genéticos , Dano ao DNA , Dieta , Metabolismo Energético , Fibroblastos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glicólise , Heterozigoto , Homeostase , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/química , Sirolimo/química , Serina-Treonina Quinases TOR/metabolismo , Timo/metabolismoRESUMO
Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.