RESUMEN
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.
Asunto(s)
Ácido Aspártico , Proteína beta Potenciadora de Unión a CCAAT , Animales , Ratones , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Ácido Aspártico/metabolismo , Malatos/metabolismo , NAD/metabolismo , Fibroblastos/metabolismo , Glucólisis , DesoxiglucosaRESUMEN
Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alquinos/farmacología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Anemia de Fanconi/complicaciones , Anemia de Fanconi/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/complicaciones , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oligopéptidos/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPß-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the Cebpb-mRNA. Here, we describe that reduced C/EBPß-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBPßΔuORF mice display an extended lifespan. Since LIP levels increase upon aging in wild type mice, our data reveal an important role for C/EBPß in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBPß-LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.
Asunto(s)
Envejecimiento , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Expresión Génica , Animales , Regulación hacia Abajo , Femenino , Longevidad , Masculino , Ratones Endogámicos C57BLRESUMEN
Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Enfermedades Neurodegenerativas/enzimología , Péptidos/metabolismo , Agregado de Proteínas , Agregación Patológica de Proteínas , ARN Polimerasa III/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Animales , Animales Modificados Genéticamente , Sitios de Unión , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/enzimología , Citosol/enzimología , Modelos Animales de Enfermedad , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Polimerasa III/genética , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.