RESUMEN
Transcriptional activation of the EVI1 oncogene (3q26) leads to aggressive forms of human acute myeloid leukemia (AML). However, the mechanism of EVI1-mediated leukemogenesis has not been fully elucidated. Previously, by characterizing the EVI1 promoter, we have shown that RUNX1 and ELK1 directly regulate EVI1 transcription. Intriguingly, bioinformatic analysis of the EVI1 promoter region identified the presence of several EVI1 potential binding sites. Thus, we hypothesized that EVI1 could bind to these sites regulating its own transcription. In this study, we show that there is a functional interaction between EVI1 and its promoter, and that the different EVI1 isoforms (EVI1-145kDa, EVI1-Δ324 and MDS1-EVI1) regulate the transcription of EVI1 transcripts through distinct promoter regions. Moreover, we determine that the EVI1-145kDa isoform activates EVI1 transcription, whereas EVI1-Δ324 and MDS1-EVI1 act as repressors. Finally, we demonstrate that these EVI1 isoforms are involved in cell transformation; functional experiments show that EVI1-145kDa prolongs the maintenance of hematopoietic stem and progenitor cells; conversely, MDS1-EVI1 repressed hematopoietic stem and progenitor colony replating capacity. We demonstrate for the first time that EVI1 acts as a regulator of its own expression, highlighting the complex regulation of EVI1, and open new directions to better understand the mechanisms of EVI1 overexpressing leukemias.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Leucemia/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/patología , Humanos , Leucemia/genética , Leucemia/patología , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Proto-Oncogenes/genética , Factores de Transcripción/genéticaRESUMEN
Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 mutations appear to cause loss of function of the mutated allele leading to haploinsufficiency; however, this postulate has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations that are translated into abnormal proteins could affect the transcription of GATA2, triggering GATA2 deficiency. Chromatin immunoprecipitation and luciferase assays showed that the human GATA2 protein activates its own transcription through a specific region located at -2.4 kb, whereas the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation. Accordingly, GATA2 expression was decreased to â¼58% in a patient with p.Arg396Gln, compared with controls. p.Arg396Gln is the second most common mutation in these syndromes, and no previous functional analyses have been performed. We therefore analyzed p.Arg396Gln. Our data show that p.Arg396Gln is a loss-of-function mutation affecting DNA-binding ability and, as a consequence, it fails to maintain the immature characteristics of hematopoietic stem and progenitor cells, which could result in defects in this cell compartment. In conclusion, we show that human GATA2 binds to its own promoter, activating its transcription, and that the aforementioned mutations impair the transcription of GATA2. Our results indicate that they can affect other GATA2 target genes, which could partially explain the variability of symptoms in these diseases. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, which is unable to retain the progenitor phenotype in cells where it is expressed.
Asunto(s)
Factor de Transcripción GATA2/genética , Mutación de Línea Germinal , Síndromes de Inmunodeficiencia/inmunología , Infección por Mycobacterium avium-intracellulare/inmunología , Transcripción Genética , Alelos , Sitios de Unión , Línea Celular Tumoral , Femenino , Factor de Transcripción GATA2/inmunología , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Haploinsuficiencia , Humanos , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Persona de Mediana Edad , Modelos Moleculares , Complejo Mycobacterium avium/inmunología , Infección por Mycobacterium avium-intracellulare/complicaciones , Infección por Mycobacterium avium-intracellulare/genética , Infección por Mycobacterium avium-intracellulare/patología , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de SeñalRESUMEN
Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24-RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24-RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24-RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24-RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24-RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24-RGD for this devastating disease.
Asunto(s)
Adenoviridae/fisiología , Cisplatino/uso terapéutico , Oligopéptidos/uso terapéutico , Virus Oncolíticos/fisiología , Osteosarcoma/terapia , Adolescente , Animales , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Niño , Cisplatino/farmacología , Terapia Combinada , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones Desnudos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/ultraestructura , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We undertook this study to understand how the transcription factor Sox2 contributes to the malignant phenotype of glioblastoma multiforme (GBM), the most aggressive primary brain tumor. We initially looked for unbalanced genomic rearrangements in the Sox2 locus in 42 GBM samples and found that Sox2 was amplified in 11.5% and overexpressed in all the samples. These results prompted us to further investigate the mechanisms involved in Sox2 overexpression in GBM. We analyzed the methylation status of the Sox2 promoter because high CpG density promoters are associated with key developmental genes. The Sox2 promoter presented a CpG island that was hypomethylated in all the patient samples when compared to normal cell lines. Treatment of Sox2-negative glioma cell lines with 5-azacitidine resulted in the re-expression of Sox2 and in a change in the methylation status of the Sox2 promoter. We further confirmed these results by analyzing data from GBM cases generated by The Cancer Genome Atlas project. We observed Sox2 overexpression (86%; Nâ=â414), Sox2 gene amplification (8.5%; Nâ=â492), and Sox 2 promoter hypomethylation (100%; Nâ=â258), suggesting the relevance of this factor in the malignant phenotype of GBMs. To further explore the role of Sox2, we performed in vitro analysis with brain tumor stem cells (BTSCs) and established glioma cell lines. Downmodulation of Sox2 in BTSCs resulted in the loss of their self-renewal properties. Surprisingly, ectopic expression of Sox2 in established glioma cells was not sufficient to support self-renewal, suggesting that additional factors are required. Furthermore, we observed that ectopic Sox2 expression was sufficient to induce invasion and migration of glioma cells, and knockdown experiments demonstrated that Sox2 was essential for maintaining these properties. Altogether, our data underscore the importance of a pleiotropic role of Sox2 and suggest that it could be used as a therapeutic target in GBM.