RESUMEN
The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.
Asunto(s)
Elementos E-Box , Modelos Moleculares , Factor de Transcripción 3/química , Proteína 1 Relacionada con Twist/química , Secuencia de Bases , Sitios de Unión , Línea Celular , Humanos , Enlace de Hidrógeno , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Factor de Transcripción 3/metabolismo , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
The ASPP (apoptosis-stimulating protein of p53) family of proteins can function in the nucleus to modulate the transcriptional activity of p53, with ASPP1 and ASPP2 contributing to the expression of apoptotic target genes. In this study, we describe a new function for cytoplasmic ASPP1 in controlling YAP (Yes-associated protein)/TAZ. ASPP1 can inhibit the interaction of YAP with LATS1 (large tumor suppressor 1), a kinase that phosphorylates YAP/TAZ and promotes cytoplasmic sequestration and protein degradation. This function of ASPP1 therefore enhances nuclear accumulation of YAP/TAZ and YAP/TAZ-dependent transcriptional regulation. The consequence of YAP/TAZ activation by ASPP1 is to inhibit apoptosis, in part through the down-regulation of Bim expression, leading to resistance to anoikis and enhanced cell migration. These results reveal a potential oncogenic role for cytoplasmic ASPP1, in contrast to the tumor-suppressive activity described previously for nuclear ASPP1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anoicis , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Western Blotting , Adhesión Celular , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Hidroxiurea/farmacología , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/genética , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción , Activación Transcripcional , Proteínas Señalizadoras YAPRESUMEN
In addition to acting as a transcriptional cofactor for p53, ASPP1 has been shown to function in the cytoplasm to regulate the nuclear localization and activity of YAP/TAZ. We show here that the ability of ASPP1 to activate YAP results in the decreased expression of LATS2, which lowers the ability of p53 to induce p21, cell-cycle arrest and senescence. ASPP1 expression peaks in S-phase, and down-regulation of ASPP1 leads to a reduction in DNA synthesis and enhanced senescence in response to drugs that impede DNA replication. These activities of cytoplasmic ASPP1 in opposing p53-mediated p21 expression are in contrast to the role of nuclear ASPP1 in cooperating with p53 to induce the expression of apoptotic target genes, and may help to dampen p53 activity in normal cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Reguladoras de la Apoptosis/fisiología , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Regulación de la Expresión Génica/fisiología , Proteína p53 Supresora de Tumor/fisiología , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma/patología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Senescencia Celular/efectos de los fármacos , Neoplasias del Colon/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Dactinomicina/farmacología , Doxorrubicina/farmacología , Humanos , Hidroxiurea/farmacología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Ubiquitin-specific peptidase 42 (USP42) is a deubiquitylating enzyme that can target p53 and contribute to the stabilization of p53 in response to stress. We now show that USP42 can also regulate transcription independently of p53. USP42 co-localized with RNA polymerase II (RNA Pol II) in nuclear foci, bound to histone H2B, and deubiquitylated H2B. Depletion of USP42 increased H2B ubiquitylation at a model promoter and decreased both basal and induced transcription from a number of promoters. These results are consistent with a role for USP42 in regulating transcription by deubiquitylating histones.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Tioléster Hidrolasas/metabolismo , Transcripción Genética , Ubiquitinación , Transporte Activo de Núcleo Celular , Línea Celular , Núcleo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas/genética , Tioléster Hidrolasas/deficiencia , Tioléster Hidrolasas/genéticaRESUMEN
The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.
Asunto(s)
Tioléster Hidrolasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Humanos , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Interferente Pequeño/genética , Estrés Fisiológico , Tioléster Hidrolasas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate pathway-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes the transcriptional activity of ERRα that could lead to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis or level in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.
RESUMEN
The study aims to combine the delivery of two anticancer drugs to target both proliferating cancer cells and dormant cancer stem cells (CSCs) present in colorectal cancer. Two drugs were selected and encapsulated in lipid nanocapsules: SN38, the active form of irinotecan, which is unstable in the plasma but active against replicating cells, and salinomycin, a highly toxic ionophore active against cancer stem cells that is not suitable for clinical use. Using an engineered medium that enhanced the ratio of CSCs in HCT116 cell cultures, we demonstrated by clonogenicity tests and in sphere assays that Salinomycin acts mainly on CSCs, while SN38 acts mainly on proliferating cancer cells. In a preclinical murine CRC model, encapsulation of both drugs in lipid nanocapsules reduced their toxicity, including hemolysis, and led to a higher survival than what was observed following treatment with single drugs or non-encapsulated drugs. Nanoparticles loaded with an anticancer drug and salinomycin were effective against the therapy-resistant dormant CSCs and cancer cells.
Asunto(s)
Neoplasias Colorrectales , Nanopartículas , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Irinotecán , Ratones , Nanomedicina , Células Madre Neoplásicas , PiranosRESUMEN
BACKGROUND: Aryl phosphate esters (APEs) are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives, are poorly characterized. OBJECTIVE: Our aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs than direct assessment of their concentration and determine its potential deleterious biological effects in chronically exposed mice. METHODS: Conventional animals (FVB mice) were acutely or chronically exposed to relevant doses of DPhP or to triphenyl phosphate (TPhP), one of its main precursors. Both molecules were measured in blood and other tissues by liquid chromatography-mass spectrometry (LC-MS). Effects of chronic DPhP exposure were addressed through liver multi-omics analysis to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses. RESULTS: Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1mg/mL in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty acid catabolic processes centered on acylcarnitine and mitochondrial ß-oxidation in mice exposed to DPhP in comparison with those treated with vehicle. An interesting finding was that in mice exposed to DPhP, mRNA, expression of genes involved in lipid catabolic processes and regulated by peroxisome proliferator-activated receptor alpha (PPARα) was lower than that in vehicle-treated mice. Immunohistochemistry analysis showed a specific down-regulation of HMGCS2, a kernel target gene of PPARα. Overall, DPhP absorption disrupted body weight-gain processes. CONCLUSIONS: Our results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver is essential for controlling fast and feast periods, with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular pathologies and metabolic disease incidence deserves, in light of our results, further investigations. https://doi.org/10.1289/EHP6826.
Asunto(s)
Contaminantes Ambientales/toxicidad , Fosfatos/toxicidad , Animales , Ésteres/toxicidad , Ratones , Modelos Químicos , Pruebas de ToxicidadRESUMEN
The purpose of this study was to investigate the feasibility of an intravenously administered combinational therapy using lipid nanocapsules (LNCs) as a drug delivery carrier for the treatment of different cancers. Therefore, we encapsulated 6 anticancer drugs within LNCs. Their size was approximately 50â¯nm. Except for oxaliplatin, their encapsulation efficiency, which was measured by different analytical methods, varied between 75% for SN38 to 100% for regorafenib. The in vitro studies showed a nonsignificant difference between the cytotoxicity of free and encapsulated drugs and a significant decrease in haemolysis by encapsulation in LNCs. Finally, the in vivo experiment showed that a combinational regimen of SN38-LNCs and regorafenib-LNCs abates CT26 murine colorectal cancer growth and increases median survival time.
Asunto(s)
Antineoplásicos/administración & dosificación , Irinotecán/administración & dosificación , Nanocápsulas/administración & dosificación , Neoplasias/tratamiento farmacológico , Compuestos de Fenilurea/administración & dosificación , Piridinas/administración & dosificación , Animales , Línea Celular Tumoral , Combinación de Medicamentos , Eritrocitos/efectos de los fármacos , Femenino , Hemólisis/efectos de los fármacos , Humanos , Lípidos/administración & dosificación , Ratones Endogámicos BALB CRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) is an attractive target for HER-2 positive breast cancer therapy because of its key role in protein translation regulation, cell growth and metabolism. We present here a metabolomic investigation exploring the impact of mTOR inhibition on serum metabolic profiles from patients with non-metastatic breast cancer overexpressing HER-2. Baseline, treatment-related and post-treatment serum samples were analyzed for 79 patients participating in the French clinical trial RADHER, in which randomized patients with HER-2 positive breast cancer received either trastuzumab alone (arm T) or a trastuzumab and everolimus combination (arm T+E). Longitudinal series of NMR serum metabolic profiles were exploited to investigate treatment effects on the patients metabolism over time, in both group. Trastuzumab and everolimus combination induces faster changes in patients metabolism than trastuzumab alone, visible after only one week of treatment as well as a residual effect detectable up to three weeks after ending the treatment. These metabolic fingerprints highlight the involvement of several metabolic pathways reflecting a systemic effect, particularly on the liver and visceral fat. Comparison of serum metabolic profiles between the two arms shows that everolimus, an mTORC1 inhibitor, is responsible for host metabolism modifications observed in arm T+E. In HER-2 positive breast cancer, our metabolomic approach confirms a fast and persistent host metabolism modification caused by mTOR inhibition.
RESUMEN
Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/genética , Metionina Sulfóxido Reductasas/genética , Células Madre/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Inmunoprecipitación de Cromatina , Daño del ADN , Células Epiteliales/citología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Glándulas Mamarias Humanas/citología , Metionina Sulfóxido Reductasas/metabolismo , Ratones Endogámicos NOD , Persona de Mediana Edad , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Células Madre/citología , Análisis de Matrices Tisulares , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto Joven , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.