RESUMEN
Traditional cancer models rely on 2D cell cultures or 3D spheroids, which fail to recapitulate cell-extracellular matrix (ECM) interactions, a key element of tumor development. Existing hydrogel-based 3D alternatives lack mechanical support for cell growth and often suffer from low reproducibility. Here we report a novel strategy to make 3D models of breast cancer using a tissue-like, well-defined network environment based on recombinant spider silk, functionalized with a cell adhesion motif from fibronectin (FN-silk). With this approach, the canonical cancer cells SK-BR-3, MCF-7, and MDA-MB-231, maintain their characteristic expression of markers (i.e., ERα, HER2, and PGR) while developing distinct morphology. Transcriptomic analyses demonstrate how culture in the FN-silk networks modulates the biological processes of cell adhesion and migration while affecting physiological events involved in malignancy, such as inflammation, remodeling of the ECM, and resistance to anticancer drugs. Finally, we show that integration in FN-silk networks promotes the viability of cells obtained from the superficial scraping of patients' breast tumors.
RESUMEN
AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proteínas Proto-Oncogénicas/fisiología , Serina-Treonina Quinasas TOR/fisiología , Proteínas Supresoras de Tumor/fisiología , Transporte Activo de Núcleo Celular , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Fosforilación , Ribonucleótidos/farmacología , Pez CebraRESUMEN
PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK) 16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been identified in human patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only a few cellular targets/substrates have been proposed with no clear functional significance. We and others recently showed that cyclin Y binds and activates PCTAIRE-1 via phosphorylation and 14-3-3 binding. In order to understand the physiological role that PCTAIRE-1 plays in brain, we have performed a chemical genetic screen in vitro using an engineered PCTAIRE-1/cyclin Y complex and mouse brain extracts. Our screen has identified potential PCTAIRE-1 substrates (AP2-Associated Kinase 1 (AAK1), dynamin 1, and synaptojanin 1) in brain that have been shown to regulate crucial steps of receptor endocytosis, and are involved in control of neuronal synaptic transmission. Furthermore, mass spectrometry and protein sequence analyses have identified potential PCTAIRE-1 regulated phosphorylation sites on AAK1 and we validated their PCTAIRE-1 dependence in a cellular study and/or brain tissue lysates. Our results shed light onto the missing link between PCTAIRE-1 regulation and proposed physiological functions, and provide a basis upon which to further study PCTAIRE-1 function in vivo and its potential role in neuronal/brain disorders.
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Encéfalo/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Dinamina I/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Encéfalo/citología , Células COS , Chlorocebus aethiops , Ciclinas/genética , Dinamina I/genética , Pruebas Genéticas , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Especificidad por SustratoRESUMEN
AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.
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Proteínas Quinasas Activadas por AMP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Animales , Hepatocitos/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Metabolismo de los Lípidos/genética , Espectrometría de Masas/métodos , Ratones Noqueados , Fosforilación , Especificidad por SustratoRESUMEN
Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the NrasQ61KINK4a-/- mouse melanoma model to show that c-Myc is essential for tumor initiation, maintenance, and metastasis. c-Myc-expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c-Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c-Myc-positive melanoma cells activated and became dependent on the metabolic energy sensor AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c-Myc-expressing melanoma cells, while AMPK activation protected against cell death of c-Myc-depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early-stage human melanoma samples revealed an inverse correlation between C-MYC and patient survival, suggesting that C-MYC expression levels could serve as a prognostic marker for early-stage disease.
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Proteínas Quinasas Activadas por AMP/metabolismo , Transformación Celular Neoplásica/genética , Melanoma/patología , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanocitos/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4-/- mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.
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Proteínas Quinasas Activadas por AMP/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ácidos Grasos/metabolismo , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal , Proteínas Quinasas Activadas por AMP/genética , Animales , Quinasa 4 Dependiente de la Ciclina/genética , Embrión de Mamíferos/metabolismo , Ácidos Grasos/genética , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Mutación , Oxidación-ReducciónRESUMEN
Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.
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Proteínas Quinasas Activadas por AMP/metabolismo , Diferenciación Celular , Autorrenovación de las Células , Homeostasis , L-Lactato Deshidrogenasa/metabolismo , Músculos/citología , Células Madre/metabolismo , Animales , Glucólisis , Ratones , Ratones NoqueadosRESUMEN
AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory ß (ß1 and ß2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 µM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.