RESUMEN
In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target.
Asunto(s)
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteína 1 Relacionada con Twist/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Genes myc , Humanos , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Transfección , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
BACKGROUND: The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNDelta1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. METHODS: Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNDelta1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. RESULTS: Both are translated, but higher levels of protein were seen with MYCNDelta1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNDelta1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNDelta1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCNDelta1b mRNA. CONCLUSIONS: Existence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.
Asunto(s)
Empalme Alternativo/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , Adulto , Secuencia de Bases , Supervivencia Celular/genética , Células Cultivadas , Exones/genética , Feto/metabolismo , Dosificación de Gen/fisiología , Expresión Génica/fisiología , Humanos , Proteína Proto-Oncogénica N-Myc , Sistemas de Lectura Abierta/fisiología , Isoformas de Proteínas/genética , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Recent progress has enlightened the involvement of Hox genes in organogenesis. Several Hox genes are expressed in normal and neoplastic mammary glands. Using Hoxa5 mutant mice, we showed that Hoxa5-/- females present nursing defects. Characterization of the Hoxa5-/- mammary gland phenotype reveals changes in proliferation and differentiation of the epithelium of nulliparous and pregnant Hoxa5-/- females that precede the abnormal secretory activity at parturition. These defects likely underlie the incapacity of Hoxa5-/- dams to properly feed their pups. Hoxa5 expression is restricted to the mammary stroma at specific stages of mammary gland development. The loss of Hoxa5 function causes accelerated lobuloalveolar epithelium development, a phenotype that can be rescued upon grafting of mutant mammary epithelium into wild-type fat pads. Conversely, reciprocal grafting experiments demonstrate that Hoxa5-/- stroma cannot support normal proliferation of wild-type mammary epithelium. These data establish the essential contribution of Hoxa5 to mammary epithelium instruction by means of mesenchymal-epithelial crosstalk.
Asunto(s)
Células Epiteliales/citología , Proteínas de Homeodominio/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Fosfoproteínas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Ciclo Estral , Femenino , Proteínas de Homeodominio/genética , Lactancia , Glándulas Mamarias Animales/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Mutantes , Fosfoproteínas/genética , Embarazo , Receptores de Esteroides/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de TranscripciónRESUMEN
N-Myc oncogene amplification is a frequent event in neuroblastoma and is strongly correlated with advanced disease stage and treatment failure. Similarly to c-Myc oncogenic activation, N-Myc deregulation promotes both cell proliferation and p53-dependent apoptosis by sensitizing cells to a variety of insults. Intriguingly, p53 mutations are uncommon in neuroblastomas, strongly suggesting that an alternative cooperating event circumvents this safeguard against oncogene-driven neoplasia. By performing a pangenomic cDNA microarray analysis, we demonstrate that human Twist is constantly overexpressed in N-Myc-amplified neuroblastomas. H-Twist overexpression is responsible for the inhibition of the ARF/p53 pathway involved in the Myc-dependent apoptotic response. This oncogenic cooperation of two key regulators of embryogenesis causes cell transformation and malignant outgrowth.