MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells

Abstract

The MYCN gene plays a critical role in determining the clinical behavior of neuroblastoma. Although it is known that genomic amplification occurs in high-risk subsets, it remains unclear how MYCN expression is regulated in the pathogenesis of neuroblastomas. Herein, we report that MYCN expression was regulated by the oncoprotein MDM2 at the post-transcriptional level and was associated with neuroblastoma cell growth. Increasing MDM2 by ectopic overexpression in the cytoplasm enhanced both mRNA and protein expression of MYCN. Mechanistic studies found that the C-terminal RING domain of the MDM2 protein bound to the MYCN mRNA’s AU-rich elements within the 3′-untranslated region (3′UTR) and increased MYCN 3′UTR-mediated mRNA stability and translation. Conversely, MDM2 silencing by specific siRNA rendered the MYCN mRNA unstable and reduced the abundance of MYCN protein in MYCN-amplified neuroblastoma cell lines. Importantly, this MDM2 silencing resulted in a remarkable inhibition of neuroblastoma cell growth and induction of cell death through a p53-independent pathway. Our results indicate that MDM2 plays a p53-independent role in the regulation of both MYCN mRNA stabilization and its translation, suggesting that MDM2-mediated MYCN expression is one mechanism associated with growth of MYCN-associated neuroblastoma and disease progression.

Dual Targeting of Wild-Type and Mutant p53 by Small Molecule RITA Results in the Inhibition of N-Myc and Key Survival Oncogenes and Kills Neuroblastoma Cells In Vivo and In Vitro

“…Aurora A is a negative prognostic factor and a potential therapeutic target in neuroblastoma (46), which, according to our recent study, is a bona fide p53 target (38). In addition, RITA treatment leads to the decrease of MDM2, which upregulates N-Myc (17). Taken together, our data suggest that reactivation of p53 by RITA causes inhibition of N-Myc via induction of its E3 ligase Fbxw7.…”

“…p53 dysfunction in neuroblastoma has been linked to MDM2 amplification and Wip1 activation (5,14), as well as to homozygous deletions of CDKN2A, encoding MDM2 inhibitor p14ARF (15). Moreover, N-Myc inactivates p53 by inducing the expression of MDM2 (16), which in turn upregulates N-Myc (17). p53 mutations occur very seldom in neuroblastoma, but in cell lines established at relapse p53 mutations are more frequent, implicating mutant p53 in the development of therapy-resistant phenotype (8,9).…”

Crosstalk between MYCN and MDM2-p53 signal pathways regulates tumor cell growth and apoptosis in neuroblastoma

“…Furthermore, to confirm that MDM2 and MYCN reciprocally regulate p53, we tested the effect of siMDM2 on p53 expression in SHEP-Tet/21N line with overexpression of MDM2 and with conditional expression of ectopic MYCN that was not regulated by MDM2 due to lack of original 3'UTR. 19,20 We found that the expression of p53 was induced by siMDM2 in both SHEP-Tet/21N in the presence of tetracycline (without MYCN expression) and SHEP-Tet/21N in the absence of tetracycline (with MYCN expression), with higher levels of basic expression and induction of p53 in the latter than in the former (Fig. 3D).…”

“…MDM2, when translocated from the nucleus to the cytoplasm, binds to the AU-rich elements of the MYCN 3'-UTR and regulates MYCN mRNA stabilization and its translation. 19 Because MDM2 and MYCN are not only mutually regulated but also reciprocally regulate p53 in neuroblastoma, we are interested in investigating exactly how that cross talk between MYCN and the MDM2/p53 signaling pathway leads to individual expression of those molecules in neuroblastoma cells, and importantly, what is the impact of that cross talk on neuroblastoma cell growth and induction of apoptosis. In the present study, we selected neuroblastoma cell lines with or without MYCN gene amplification that either have or do not have MDM2 overexpression, and utilized gene transfection and gene knockdown by siRNA to carry out our investigation.…”

The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals

“…Sucrose gradient polysome fractionation was performed as described in Gu et al (2012), with some modifications. ES cells grown in 15-mm plates in 2i medium (5 3 10 7 cells per plate) were incubated with 100 mg/mL cycloheximide (Sigma) for 15 min to arrest ribosome movement on mRNAs.…”