RESUMEN
Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/fisiopatología , Proteínas de la Ataxia Telangiectasia Mutada , Autofagia , Beclina-1 , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Humanos , Immunoblotting , Estimación de Kaplan-Meier , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mitocondrias/genética , Mitocondrias/fisiología , Consumo de Oxígeno , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/metabolismo , Timocitos/ultraestructura , Proteínas Supresoras de Tumor/genéticaRESUMEN
Mdm4 is a critical inhibitor of the p53 tumor suppressor. Mdm4 null mice die early during embryogenesis due to increased p53 activity. In this study, we explore the role that Mdm4 plays in the intestinal epithelium by crossing mice carrying the Mdm4 floxed allele to mice with the Villin Cre transgene. Our data show that loss of Mdm4 (Mdm4intDelta) in this tissue resulted in viable animals with no obvious morphological abnormalities. However, these mutants displayed increased p53 levels and apoptosis exclusively in the proliferative compartment of the intestinal epithelium. This phenotype was completely rescued in a p53 null background. Notably, the observed compartmentalized apoptosis in proliferative intestinal epithelial cells was not due to restricted Mdm4 expression in this region. Thus, in this specific cellular context, p53 is negatively regulated by Mdm4 exclusively in highly proliferative cells.
Asunto(s)
Apoptosis/genética , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Bromodesoxiuridina/metabolismo , Cruzamientos Genéticos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Transgenes , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/análisis , beta-Galactosidasa/metabolismoRESUMEN
The p53 tumor suppressor is mutated in most human tumors. MDM2, a well-known inhibitor of p53, is overexpressed in a large number of tumors, suggesting that increased levels of MDM2 also contribute to tumorigenesis. A novel p53 inhibitor, MDM4, was more recently identified. The role of MDM4 in cancer development is not well understood. We set out to examine the levels of MDM4 by immunohistochemistry in head and neck squamous carcinomas (HNSC) to ask whether high MDM4 levels could contribute to its development and progression. In addition, MDM2 and p53 levels were examined to identify overlapping expression patterns. MDM4 is present at high levels in 50% of HNSC. In addition, overexpression of MDM2 was detected in 80% of tumors, many of which were also positive for MDM4. A subset of tumors displayed high levels of all 3 proteins. Sequencing of the p53 gene revealed that tumors with positive immunoreactivity for MDM2 or MDM4, some of which also had high levels of p53, did not carry mutations in this gene. Thus, the detection of p53 by immunohistochemistry was not synonymous with the presence of p53 mutations. Expression of both MDM2 and MDM4 in tumors without p53 mutations strongly suggests that MDM2 and MDM4 inhibit the activity of this tumor suppressor in HNSC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular , Análisis Mutacional de ADN , Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis.
Asunto(s)
Neoplasias Cerebelosas/genética , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Meduloblastoma/genética , Mutación/genética , Carcinogénesis , Células HEK293 , Células HeLa , Humanos , Meduloblastoma/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Análisis de la Célula IndividualRESUMEN
The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.
Asunto(s)
Autofagia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/deficiencia , Proteínas de Unión al ADN/deficiencia , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
High levels of the critical p53 inhibitor Mdm4 is common in tumors that retain a wild-type p53 allele, suggesting that Mdm4 overexpression is an important mechanism for p53 inactivation during tumorigenesis. To test this hypothesis in vivo, we generated transgenic mice with widespread expression of Mdm4. Two independent lines of transgenic mice, Mdm4(Tg1) and Mdm4(Tg15), developed spontaneous tumors, the most prevalent of which were sarcomas. To determine whether overexpression of Mdm4 also cooperated with p53 heterozygosity to induce tumorigenesis, we generated Mdm4(Tg1) p53(+/-) mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53(+/-) or Mdm4(Tg1) mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4(Tg1) p53(+/-) tumors. Mdm4 is thus a bona fide oncogene in vivo and cooperates with p53 heterozygosity to drive tumorigenesis. These Mdm4 mice will be invaluable for in vivo drug studies of Mdm4 inhibitors.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Experimentales/genética , Proteínas Proto-Oncogénicas/genética , Proteína p53 Supresora de Tumor/deficiencia , Ubiquitina-Proteína Ligasas/genética , Actinas/genética , Animales , Transformación Celular Neoplásica/metabolismo , Pollos , Daño del ADN , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/biosíntesis , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/biosíntesisRESUMEN
Human homolog of murine double minute 2 (HDM2) and HDM4 (or HDMX) are negative regulators of p53. HDM4 has not been assessed in precursor B (pre-B) lymphoblastic leukemia (ALL). We examined bone marrow samples obtained at time of diagnosis from 55 adults with pre-B ALL. A tissue microarray composed of 2 cores per specimen was constructed and immunohistochemical techniques were used to assess HDM4, HDM2, p53, and p21. HDM4 was expressed in 39 of 49 (80%) cases. HDM2 was expressed in 14 of 54 (26%). All HDM2-positive cases were also positive for HDM4 (P<0.05). We confirmed expression of HDM4 and HDM4 variants by Western blotting and sequencing of reverse transcription-polymerase chain reaction products in a subset of ALL tumors. Results were correlated with the presence of the Philadelphia chromosome (Ph). p53 (P<0.05) and p21 (P<0.001) were expressed significantly more often in Ph+ pre-B ALL. HDM4 and HDM2 showed no correlation with Ph status. HDM4 expression in most cases of adult pre-B ALL suggests that HDM4 is a potential therapeutic target.
Asunto(s)
Biomarcadores de Tumor/análisis , Médula Ósea/patología , Proteínas Nucleares/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas/análisis , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Médula Ósea/química , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/análisis , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Mutación , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-mdm2/análisis , ARN Mensajero/análisis , Factores de Tiempo , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.