RESUMEN
Sirtuin 7 (SIRT7) is a member of the mammalian family of nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylases, known as sirtuins. It acts as a potent oncogene in numerous malignancies, but the molecular mechanisms employed by SIRT7 to sustain lung cancer progression remain largely uncharacterized. We demonstrate that SIRT7 exerts oncogenic functions in lung cancer cells by destabilizing the tumor suppressor alternative reading frame (ARF). SIRT7 directly interacts with ARF and prevents binding of ARF to nucleophosmin, thereby promoting proteasomal-dependent degradation of ARF. We show that SIRT7-mediated degradation of ARF increases expression of protumorigenic genes and stimulates proliferation of non-small-cell lung cancer (NSCLC) cells both in vitro and in vivo in a mouse xenograft model. Bioinformatics analysis of transcriptome data from human lung adenocarcinomas revealed a correlation between SIRT7 expression and increased activity of genes normally repressed by ARF. We propose that disruption of SIRT7-ARF signaling stabilizes ARF and thus attenuates cancer cell proliferation, offering a strategy to mitigate NSCLC progression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Progresión de la Enfermedad , Neoplasias Pulmonares , Sirtuinas , Humanos , Sirtuinas/metabolismo , Sirtuinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Línea Celular TumoralRESUMEN
Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.
Asunto(s)
Cardiotónicos/metabolismo , Infarto del Miocardio/prevención & control , Oncostatina M/metabolismo , Receptores OSM-LIF/metabolismo , Animales , Hipoxia de la Célula/genética , Supervivencia Celular , Células Cultivadas , Humanos , Cinética , Factor Inhibidor de Leucemia/metabolismo , Ratones , Contracción Miocárdica , Infarto del Miocardio/genética , Miocitos Cardíacos/metabolismo , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Oncostatina M/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
The pleiotropic interleukin-6 (IL-6)-type cytokine oncostatin M (OSM) signals in multiple cell types, affecting processes such as cell differentiation, hematopoiesis, and inflammation. In humans, OSM exerts its effects through activation of either of two different heterodimeric receptor complexes, formed by glycoprotein 130 (gp130) and either OSM receptor (OSMR) or leukemia inhibitory factor receptor (LIFR). In contrast, the mouse OSM orthologue acts mainly through dimers containing OSMR and gp130 and shows limited activity through mouse LIFR. Despite their structural similarity, neither human nor mouse OSM signal through the other species' OSMR. The molecular basis for such species-specific signaling, however, remains poorly understood. To identify key molecular features of OSM that determine receptor activation in humans and mice, we generated chimeric mouse-human cytokines. Replacing regions within binding site III of murine OSM with the human equivalents showed that the cytokine's AB loop was critical for receptor selection. Substitutions of individual amino acids within this region demonstrated that residues Asn-37, Thr-40, and Asp-42 of the murine cytokine were responsible for limited LIFR activation and absence of human OSMR/LIFR signaling. In human OSM, Lys-44 appeared to be the main residue preventing mouse OSMR activation. Our data reveal that individual amino acids within the AB loop of OSM determine species-specific activities. These mutations might reflect a key step in the evolutionary process of this cytokine, in which receptor promiscuity gives way to ligand-receptor specialization.
Asunto(s)
Oncostatina M/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/genética , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/metabolismo , Ratones , Oncostatina M/genética , Subunidad beta del Receptor de Oncostatina M/genética , Subunidad beta del Receptor de Oncostatina M/metabolismo , Multimerización de Proteína/genética , Estructura Secundaria de Proteína , Especificidad de la EspecieRESUMEN
Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , ADN Helicasas/genética , Músculo Esquelético/fisiología , Regeneración , Células Madre/citología , Células Madre/metabolismo , Animales , Biología Computacional , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Modelos Biológicos , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
Somatic stem cells expand massively during tissue regeneration, which might require control of cell fitness, allowing elimination of non-competitive, potentially harmful cells. How or if such cells are removed to restore organ function is not fully understood. Here, we show that a substantial fraction of muscle stem cells (MuSCs) undergo necroptosis because of epigenetic rewiring during chronic skeletal muscle regeneration, which is required for efficient regeneration of dystrophic muscles. Inhibition of necroptosis strongly enhances suppression of MuSC expansion in a non-cell-autonomous manner. Prevention of necroptosis in MuSCs of healthy muscles is mediated by the chromatin remodeler CHD4, which directly represses the necroptotic effector Ripk3, while CHD4-dependent Ripk3 repression is dramatically attenuated in dystrophic muscles. Loss of Ripk3 repression by inactivation of Chd4 causes massive necroptosis of MuSCs, abolishing regeneration. Our study demonstrates how programmed cell death in MuSCs is tightly controlled to achieve optimal tissue regeneration.
Asunto(s)
Epigénesis Genética/genética , Músculo Esquelético/metabolismo , Necroptosis/genética , HumanosRESUMEN
The identity of tumor-initiating cells in many cancer types is unknown. Tumors often express genes associated with embryonic development, although the contributions of zygotic programs to tumor initiation and formation are poorly understood. Here, we show that regeneration-induced loss of quiescence in p53-deficient muscle stem cells (MuSCs) results in rhabdomyosarcoma formation with 100% penetrance. Genomic analyses of purified tumor cells revealed spontaneous and discrete oncogenic amplifications in MuSCs that drive tumorigenesis, including, but not limited to, the amplification of the cleavage-stage Dux transcription factor (TF) Duxbl. We further found that Dux factors drive an early embryonic gene signature that defines a molecular subtype across a broad range of human cancers. Duxbl initiates tumorigenesis by enforcing a mesenchymal-to-epithelial transition, and targeted inactivation of Duxbl specifically in Duxbl-expressing tumor cells abolishes their expansion. These findings reveal how regeneration and genomic instability can interact to activate zygotic genes that drive tumor initiation and growth.
Asunto(s)
Autorrenovación de las Células , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Neoplasias/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Cigoto/metabolismo , Animales , Células Cultivadas , Inestabilidad Genómica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Músculo Esquelético/patología , Mioblastos/patología , Neoplasias/metabolismo , Neoplasias/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The hallmark of stem cells is their capability to either self-renew or to differentiate into a different cell type. Adult skeletal muscle contains a resident muscle stem cell population (MuSCs) known as satellite cells, which enables regeneration of damaged muscle tissue throughout most of adult life. During skeletal muscle regeneration, few MuSCs self-renew to maintain the muscle stem cell pool while others expand rapidly and subsequently undergo myogenic differentiation to form new myofibers. However, like for other stem cell types, the molecular networks that govern self-renewal and/or differentiation of MuSCs remain largely elusive. We recently reported a method to isolate sufficient amounts of purified MuSCs from skeletal muscle which enables us to study their cell autonomous properties. Here, we describe a lentiviral, image-based loss-of function screening pipeline on primary MuSCs that enables systematic identification of genes that regulate muscle stem cell function.
Asunto(s)
Diferenciación Celular/genética , Autorrenovación de las Células/genética , Perfilación de la Expresión Génica/métodos , Músculo Esquelético/citología , Células Madre/citología , Células Madre/metabolismo , Biomarcadores , Citometría de Flujo , Biblioteca de Genes , Vectores Genéticos/genética , Células HEK293 , Humanos , Inmunofenotipificación , Lentivirus/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción GenéticaRESUMEN
Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Homeostasis , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Músculo Estriado/metabolismo , Envejecimiento/patología , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Diferenciación Celular/genética , Islas de CpG/genética , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Ratones Transgénicos , Mitocondrias Cardíacas/metabolismo , Músculo Estriado/embriología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Regiones Promotoras Genéticas/genética , Unión ProteicaRESUMEN
The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKß-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which â¼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKß substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKß on serine 298. We provide evidence that IKKß-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.