RESUMEN
BACKGROUND: LACTB was recently identified as a mitochondrial tumour suppressor that negatively affects cancer cell proliferation by inducing cell death and/or differentiation, depending on the cell type and tissue. However, the detailed mechanism underlying the LACTB-induced cancer cell death is largely unknown. METHODS: We used cell-based, either in 2D or 3D conditions, and in vivo experiments to understand the LACTB mechanisms. In this regard, protein array followed by an enrichment analysis, cell proliferation assays using different compounds, western blot analysis, flow cytometry and immunofluorescence were performed. Differences between quantitative variables following normal distribution were valuated using Student t test for paired or no-paired samples according to the experiment. For in vivo experiments differences in tumour growth were analyzed by 2-way ANOVA. RESULTS: We show, that LACTB expression leads to cell cycle arrest in G1 phase and increase of DNA oxidation that leads to activation of intrinsic caspase-independent cell death pathway. This is achieved by an increase of mitochondrial reactive oxygen species since early time points of LACTB induction. CONCLUSION: Our work provides a deeper mechanistic insight into LACTB-mediated cancer-cell death and shows the dynamics of the cellular responses a particular tumor suppressive stimulus might evoke under different genetic landscapes.
Asunto(s)
Neoplasias de la Mama , Caspasas , Humanos , Femenino , Caspasas/genética , Caspasas/metabolismo , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Puntos de Control del Ciclo Celular , Especies Reactivas de Oxígeno/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genéticaRESUMEN
The myocyte enhancer factor 2 and histone deacetylase (MEF2-HDAC) axis is a master regulator of different developmental programs and adaptive responses in adults. In this paper, we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is upregulated during acini formation, which coincides with exit from the proliferative phase. Upregulation of the transcription of MEF2 proteins is coupled to downregulation of HDAC7, which occurs independently from changes in mRNA levels, and proteasome- or autophagy-mediated degradation. During acini formation, the MEF2-HDAC axis contributes to the promotion of cell cycle exit, through the engagement of the CDK inhibitor CDKN1A. Only in proliferating cells can HDAC7 bind to the first intron of the CDKN1A gene, a region characterized by epigenetic markers of active promoters and enhancers. In cells transformed by the oncogene HER2 (ERBB2), acini morphogenesis is altered, MEF2 transcription is repressed and HDAC7 is continuously expressed. Importantly, reactivation of MEF2 transcriptional activity in these cells, through the use of a HER2 inhibitor or by enhancing MEF2 function, corrected the proliferative defect and re-established normal acini morphogenesis.
Asunto(s)
Células Acinares/metabolismo , Células Epiteliales/metabolismo , Histona Desacetilasas/metabolismo , Factores de Transcripción MEF2/metabolismo , Línea Celular , Proliferación Celular/genética , Proliferación Celular/fisiología , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente , Histona Desacetilasas/genética , Humanos , Immunoblotting , Factores de Transcripción MEF2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.
Asunto(s)
Transición Epitelial-Mesenquimal , Neoplasias Ováricas , Factores de Transcripción de la Familia Snail , beta-Lactamasas , Femenino , Humanos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Carcinogénesis , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Tumor suppressors represent a critical line of defense against tumorigenesis. Their mechanisms of action and the pathways they are involved in provide important insights into cancer progression, vulnerabilities, and treatment options. Although nuclear and cytosolic tumor suppressors have been extensively investigated, relatively little is known about tumor suppressors localized within the mitochondria. However, recent research has begun to uncover the roles of these important proteins in suppressing tumorigenesis. Here, we review this newly developing field and summarize available information on mitochondrial tumor suppressors.
Asunto(s)
Metabolismo Energético , Genes Mitocondriales , Genes Supresores de Tumor , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animales , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/patologíaRESUMEN
Background: In the breast, the pleiotropic epigenetic regulator HDAC7 can influence stemness. Materials & Methods: The authors used MCF10 cells knocked-out for HDAC7 to explore the contribution of HDAC7 to IGF1 signaling. Results: HDAC7 buffers H3K27ac levels at the IGFBP6 and IGFBP7 genomic loci and influences their expression. In this manner, HDAC7 can tune IGF1 signaling to sustain stemness. In HDAC7 knocked-out cells, RXRA promotes the upregulation of IGFBP6/7 mRNAs. By contrast, HDAC7 increases FABP5 expression, possibly through repression of miR-218. High levels of FABP5 can reduce the delivery of all-trans-retinoic acid to RXRA. Accordingly, the silencing of FABP5 increases IGFBP6 and IGFBP7 expression and reduces mammosphere generation. Conclusion: The authors propose that HDAC7 controls the uptake of all-trans-retinoic acid, thus influencing RXRA activity and IGF1 signaling.
Asunto(s)
Histona Desacetilasas/genética , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Línea Celular , Epigénesis Genética , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Glándulas Mamarias Humanas/citología , Receptor alfa X Retinoide/genéticaRESUMEN
HDAC7 is a pleiotropic transcriptional coregulator that controls different cellular fates. Here, we demonstrate that in human mammary epithelial cells, HDAC7 sustains cell proliferation and favours a population of stem-like cells, by maintaining a proficient microenvironment. In particular, HDAC7 represses a repertoire of cytokines and other environmental factors, including elements of the insulin-like growth factor signalling pathway, IGFBP6 and IGFBP7. This HDAC7-regulated secretome signature predicts negative prognosis for luminal A breast cancers. ChIP-seq experiments revealed that HDAC7 binds locally to the genome, more frequently distal from the transcription start site. HDAC7 can colocalize with H3K27-acetylated domains and its deletion further increases H3K27ac at transcriptionally active regions. HDAC7 levels are increased in RAS-transformed cells, in which this protein was required not only for proliferation and cancer stem-like cell growth, but also for invasive features. We show that an important direct target of HDAC7 is IL24, which is sufficient to suppress the growth of cancer stem-like cells.
Asunto(s)
Células Epiteliales/enzimología , Células Epiteliales/patología , Histona Desacetilasas/metabolismo , Glándulas Mamarias Humanas/patología , Células Madre Neoplásicas/patología , Microambiente Tumoral , Acetilación , Proteína Morfogenética Ósea 4/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Reprogramación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Histonas/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Pronóstico , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Transcriptoma/genéticaRESUMEN
The Ras-related GTPase Rap has been implicated in multiple cellular functions. In particular, Rap1 is a crucial regulator of both inside-out integrin activation and outside-in E-cadherin-mediated signaling. Thus, Rap1 was proposed as a fundamental regulator of the cross talk between cadherins and integrins. We provide microscopic techniques to study subcellular localization of Rap1 protein in the crosstalk between integrins and cadherins.
Asunto(s)
Espacio Intracelular/metabolismo , Microscopía Fluorescente/métodos , Proteínas de Unión al GTP rap1/metabolismo , Cadherinas/metabolismo , Línea Celular , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Integrinas/metabolismo , Transporte de ProteínasRESUMEN
Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.