588. MYCN Silencing Using RNA Interference Causes Apoptosis and Differentiation in MYCN Amplified Neuroblastoma Cell Lines

Abstract

Neuroblastoma (NB) is the most common malignant solid tumour in childhood and accounts for 15% of childhood cancer deaths. High risk NB is strongly correlated with MYCN amplification and the survival rate is very low at approximately 30-50%. MYCN, a member of the MYC proto-oncogene family, is an evolutionarily conserved bHLHZip transcription factor. MYCN controls various target genes that regulate essential cellular processes such as proliferation, cell growth, apoptosis and differentiation in certain tissues in the embryo, while it is low or absent in adults. However, overexpressed MYCN in MYCN-amplified tumours induces proliferation and cell growth and suppresses apoptosis and differentiation pathways in NB tumour cells. Therefore, we hypothesise that MYCN is a promising target gene for NB therapy as its suppression may lead to apoptotic cell death, retarded proliferation or differentiation resulting in their terminal differentiation, and eventual senescence. Transcription factors are generally difficult targets to design drugs and thus we aim to silence MYCN in NB cells using RNA interference (RNAi). Our hypothesis is that MYCN silencing by RNAi triggers apoptosis and differentiation, and affects up/down-regulation of the genes downstream of MYCN, including p53. To observe the silencing efficiency and the biological downstream effects following the knocking down, we performed short interfering RNA (siRNA) transfections at 4different concentrations (50-5nM) using a commercial transfection reagent Lipofectamine RNAiMAX. These transfections were performed in different MYCN-amplified NB cell lines, Kelly and SK-N-BE(2), and the silencing efficiency measured at the mRNA and protein levels. SK-N-BE(2) cells have non-functional p53 and have shown resistance against cytotoxic therapy and conventional chemo/radio therapies and therefore, these cells were used as a NB model, that scarcely apoptose even when MYCN level goes down. Following transfections in both cell lines, approximately 40-50% silencing was obtained at the mRNA level. MYCN protein reduction was demonstrated in a dose dependent manner up to a maximum of 70% at a dose of 50nM in Kelly cells. Knockdown in protein levels were higher than that at the mRNA level. SK-N-BE(2) cells following MYCN siRNA silencing differentiated significantly after 6 days, and differed in their cell morphology when compared with control siRNA-transfected cells. Both, the number of cells with longer neurites and the total number of longer neurites, increased in 5 nM MYCN siRNA-treated cells when compared to control siRNA-transfected cells by approximately 21-fold and 2.4-fold, respectively. In conclusion, MYCN siRNA leads to the knockdown of MYCN mRNA levels, and that in turn induces downstream biological effects leading to apoptosis and differentiation. This suggests that MYCN silencing by RNAi might be applied as a therapy of NB with or without functional p53, in tumours that have resistance against chemotherapeutics. MYCN silencing by siRNA could be a promising therapy for a wide range of NB types with MYCN amplification.