Abstract B9: Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation

Abstract

Abstract Introduction: Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behavior, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in neuroblastoma tumors is highly correlated with poor survival of patients. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid, to reduce minimal residual disease, and exposure of a number of NB cell lines to the related compound, all-trans-retinoic acid (ATRA), induces neural differentiation along with the down-regulation of MYCN. We previously determined that miRNA-10a/b plays a critical role in the process of ATRA induced neuroblastoma cell differentiation through direct targeting of nuclear co-repressor receptor 2 (NCOR2) (Foley et al Cell Death & Differentiation 2011: 18:1089). Here, we have profiled three ATRA sensitive (SK-N-BE, LAN5 and SHSY-5Y) and two ATRA resistant (Kelly and SK-N-AS) neuroblastoma cell lines for the expression of 750 miRNA loci at early (3 hours) and late (7 days) time points following ATRA treatment. The purpose of our experiments was to identify critical early miRNA signaling events, as well as miRNAs that might mediate sensitivity versus resistance to ATRA. Methods: All cells were treated with 5 μmol ATRA (Sigma, St. Louis, MO) which was continuously administered by replacing the medium every 24 hrs. MiRNA expression profiling was carried out using TaqMan Low Density Arrays from Applied BioSystems (Carlsbad, CA). Results: miR-10a, -34a, -125b, -28-5p, and -183 were consistently up-regulated and miR-450-5p, -151-3p and -138-2 were consistently down-regulated >2 fold (p < 0.05) in all three ATRA sensitive cell lines at three hours post-ATRA. At 7 days post-ATRA, there were more differentially expressed miRNAs, with 13 miRNAs being up-regulated and 18 down-regulated > 2 fold (p < 0.05) across all three cell lines. Among the miRNAs differentially expressed at 3 hours post-ATRA, miR-10a, miR-125b and miR-34a remained differentially expressed at 7 days post-ATRA. Remarkably, none of the differentially expressed miRNAs in the ATRA sensitive cell lines were differentially expressed in the ATRA resistant cell lines at the three hour time point, and very few displayed altered expression in the ATRA resistant cell lines even after a 7 day exposure to ATRA. qPCR and Western blot analysis of the critical miR-10a target, NCOR2, revealed that this gene is down regulated at both mRNA and protein level at the 3 hour time point. In addition to the targeting of NCOR2 by miR-10a, we noted that two additional up-regulated miRNAs, miR-125b and miR-132 (significantly up-regulated within 7 days post-ATRA) are also predicted to target NCOR2, indicating substantial redundancy in miRNA mediated signaling. Direct targeting of NCOR2 by these additional miRNAs was experimentally validated by luciferase reporter assays. This redundancy in miRNA targeting explains why antagomir inhibition of single miRNAs prior to ATRA treatment only delays, but does not block the induction of differentiation. Finally, we present evidence that an initial early decrease in MYCN levels, which is well known to occur in neuroblastoma cells treated with ATRA, is miRNA mediated. Conclusions: We have identified critical and redundant early miRNA signaling events that take place during the process of ATRA induced neuroblastoma cell differentiation. The absence of these events in ATRA resistant cell lines suggests new avenues of investigation for elucidating potential miRNA mediated mechanisms involved with ATRA therapy failure. Citation Format: Niamh H. Foley, Isabella M. Bray, Raquel Fernandez, Raymond L. Stallings. Critical Early microRNA-Mediated Signalling Events During ATRA Induced Neuroblastoma Cell Differentiation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B9.