title: The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma.
abstract: The MYCN oncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy. MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogene MDM2 as a putative target. MDM2 has multiple p53-independent functions modulating cell cycle and transcriptional events. Standard ChIP with MYCN antibodies established the binding of MYCN to a consensus E-box within the human MDM2 promoter. Oligonucleotide pull-down assays further established the capacity of MYCN to bind to this promoter region, confirming the ChIP results. Luciferase reporter assays confirmed the E-box-specific, MYCN-dependent regulation of the MDM2 promoter in MYCN-inducible neuroblastoma cell lines. Real-time quantitative PCR and Western blot analysis demonstrated a rapid increase in endogenous MDM2 mRNA and MDM2 protein upon induction of MYCN. Targeted inhibition of MYCN in a MYCN-amplified neuroblastoma cell line resulted in decreased MDM2 expression levels with concomitant stabilization of p53 and induction of apoptosis. Our finding that MYCN directly modulates baseline MDM2 levels suggests a mechanism contributing to the pathogenesis of neuroblastoma and other MYC-driven malignancies through inhibition of MYC-stimulated apoptosis.
authors.affiliation: Center for Cell and Gene Therapy, Texas Children's Cancer Center, Baylor College of Medicine, 1102 Bates Street, Houston, TX 77030, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Slack
authors.forename: Zaowen
authors.initials: Z
authors.lastname: Chen
authors.forename: Roberto
authors.initials: R
authors.lastname: Tonelli
authors.forename: Martin
authors.initials: M
authors.lastname: Pule
authors.forename: Lisa
authors.initials: L
authors.lastname: Hunt
authors.forename: Andrea
authors.initials: A
authors.lastname: Pession
authors.forename: Jason M
authors.initials: JM
authors.lastname: Shohet
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D024721:E-Box Elements; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000209:etiology*; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D015513:Oncogene Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors*
chemical_list: C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2