Abstract 816: Functional analysis of miRNA in chemotherapy resistant neuroblastoma
Authors: Harvey Harry M. , Bray Isabella M. , Stallings Raymond L. 
Published: August 2012

Abstract
Neuroblastoma is a paediatric cancer of the sympathetic nervous system, accounting for 15 % of childhood cancer related deaths. Treatment of neuroblastoma (NB) patients involves intensive rounds of multimodal chemotherapy, with initial responses often being positive, but with a high likelihood of relapse and development of therapy resistance. The aim of this project is to identify miRNAs associated with resistance to chemotherapeutics in NB, and elucidate the molecular mechanisms involved. In order to understand miRNA involvement in NB drug resistance, two MYCN amplified (KellyCis83, CHP-212Cis100) and one MYCN single copy cell line (SK-N-ASCis24) resistant to cisplatin (CDDP) were developed using ‘drug pulse selection’. An increase in resistance to CDDP was confirmed by examination of IC50s before and after selection. Expression of 770 miRNAs was analyzed using Taqman arrays and drug resistant profiles were compared to those of parental lines. 41, 51 and 72 miRNAs were differentially expressed in KellyCis83, SKNASCis24 and CHP-212Cis100 respectively. A total of 8 miRNA exhibited the same pattern of differential expression in both KellyCis83 and SKNASCis24, 6 miRNA in KellyCis83 and CHP-212Cis100, and 8 miRNA in SKNASCis24 and CHP-212Cis100. 2 miRNA were commonly differentially expressed across all three cell lines. Assessment of cellular proliferation revealed resistant variants KellyCis83 and CHP-212Cis100 grown in the presence of low-dose CDDP consistently outgrew cells grown in normal media. This increase in growth is not observed in the parental cell lines. MiRNA expression profiles of cells challenged with 0.2 μM CDDP were obtained and 31 miRNAs differentially expressed in KellyCis83 and CHP-212Cis100 identified. Many miRNAs identified have been previously associated with drug resistance, confirming the efficacy of this approach. For example, in the panel of 8 miRNA common to KellyCis83 and SK-N-ASCis24, all miRNA are predicted to target at least one of two key copper transporters involved in CDDP transport; hCtr1 and ATP7A. Each cell line was also analyzed using aCGH to identify DNA copy number alterations. A region of focal gain containing NAIP (NLR family, apoptosis inhibitory protein) was identified in SKNASCis24, and a 15-fold increase of NAIP mRNA was confirmed using qPCR. NAIP inhibits caspases-3, 7 and 9 resulting in increased cell survival. This discovery was of interest because 13 of the 33 miRNAs down-regulated in SK-N-ASCis24 are predicted to target NAIP. SiRNA depletion of NAIP mRNA led to increased sensitivity to etoposide, but not CDDP in SK-N-ASCis24. However, both drugs have different mechanisms of action and it is possible that NAIP has a greater influence over the function of etoposide in our model. In conclusion, we have identified miRNA associated with drug resistance in NB, and are now working to elucidate the mechanism of miRNA involvement in the process of multidrug resistance.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 816. doi:1538-7445.AM2012-816