RESUMEN
The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.
Asunto(s)
Carcinogénesis/genética , ARN Largo no Codificante/metabolismo , Proteínas Ribosómicas/metabolismo , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Factor de Transcripción E2F1/metabolismo , Femenino , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Pronóstico , Biosíntesis de Proteínas , Estabilidad Proteica , ARN Largo no Codificante/genética , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
Neuroblastoma is the most common solid tumor in early childhood. Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN, MYCN antisense NCYM and lncUSMycN genes show poor prognosis. BET bromodomain inhibitors show anticancer efficacy against neuroblastoma partly by reducing MYCN gene transcription and N-Myc mRNA and protein expression. We have previously shown that the long nocoding RNA lncUSMycN upregulates N-Myc mRNA expression by binding to the RNA-binding protein NonO. In this study, we found that lncUSMycN upregulated NCYM expression, and knocking-down lncUSMycN reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the NCYM gene promoter. NCYM upregulated N-Myc mRNA expression, NCYM RNA formed a complex with NonO protein, and knocking down NCYM expression reduced neuroblastoma cell proliferation. Importantly, treatment with BET bromodomain inhibitors reduced NCYM expression. In human neuroblastoma patients, high levels of NCYM expression in tumor tissues correlated with high levels of N-Myc, NonO and lncUSMycN expression as well as poor patient prognosis. Taken together, our findings suggest that lncUSMycN upregulates NCYM expression by activating its gene transcription, and that NCYM RNA upregulates N-Myc mRNA expression by binding to NonO. Our findings also provide further evidence for the application of BET bromodomain inhibitors for the therapy of neuroblastoma characterized by MYCN/NCYM gene locus amplification.