RESUMEN
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Asunto(s)
Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares , Proteínas Oncogénicas , Proteínas de Unión al ARN , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas , Línea Celular Tumoral , Neuroblastoma/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Exosomas/metabolismo , Exosomas/genética , Intrones , Unión Proteica , Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación Neoplásica de la Expresión Génica , ARN/metabolismo , ARN/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proliferación CelularRESUMEN
The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
Asunto(s)
Núcleo Celular/enzimología , Proliferación Celular , Replicación del ADN , Exosomas/enzimología , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/enzimología , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Roturas del ADN de Doble Cadena , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Exosomas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Células 3T3 NIH , Neuroblastoma/genética , Neuroblastoma/patología , Regiones Promotoras Genéticas , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Polimerasa II/genética , Terminación de la Transcripción GenéticaRESUMEN
Inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts (n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain-of-function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re-expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow-moving cells. An impressive increase of acetylated alpha-tubulin was observed in ITIH5-positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis-inhibiting properties of ITIH5 in pancreatic cancer.
Asunto(s)
Adhesión Celular , Movimiento Celular , Neoplasias Pancreáticas , Transducción de Señal , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Movimiento Celular/genética , Adhesión Celular/genética , Línea Celular Tumoral , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Adhesiones Focales/metabolismo , Adhesiones Focales/genética , Metilación de ADN/genética , Regiones Promotoras Genéticas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Inhibidoras de Proteinasas SecretorasRESUMEN
MYC oncoproteins are key drivers of tumorigenesis. As transcription factors, MYC proteins regulate transcription by all three nuclear polymerases and gene expression. Accumulating evidence shows that MYC proteins are also crucial for enhancing the stress resilience of transcription. MYC proteins relieve torsional stress caused by active transcription, prevent collisions between the transcription and replication machineries, resolve R-loops, and repair DNA damage by participating in a range of protein complexes and forming multimeric structures at sites of genomic instability. We review the key complexes and multimerization properties of MYC proteins that allow them to mitigate transcription-associated DNA damage, and propose that the oncogenic functions of MYC extend beyond the modulation of gene expression.