Abstract 130: MiR-204 acts as a tumor suppressor in neuroblastoma through down-regulation of the neurotrophic receptor TrkB

Abstract

Abstract Objectives: The purpose of this study was to elucidate the functional effects of miR-204 in neuroblastoma. Background: Neuroblastoma (NB) is a leading cause of cancer linked mortality in children. Previously, we identified numerous microRNAs, differentially expressed in favourable versus unfavourable tumor subtypes, that are significantly associated with patient overall survival (OS) and event free survival (EFS). In particular, lower miR-204 expression at diagnosis in our NB tumor cohort (n=147) was significantly associated with poor EFS and OS (P < 0.0001), indicating a potential tumor suppressive role. Here we present our findings from the study of functional effects of exogenous miR-204 expression in NB cell lines, and we identify NTRK2/TrkB as a direct target of miR-204. Results: In order to elucidate the mechanism of miR-204 tumor suppression, mature miR-204 mimics were transiently transfected into three NB cell lines (Kelly, SKNAS, SHSY5Y). Ectopic over-expression had no significant effect on cell viability (MTS and Cyquant assays) or apoptosis (Annexin V and Caspase 3/7 assays). In addition, transfection of miR-204 into either NB1691 or SKNAS cells prior to retroperitoneal implantation of the cells into immune deficient mice had no detectable effect on tumor establishment and growth (n=4). Completely counter-intuitive to expectations, ectopic up-regulation of miR-204 in the MYCN amplified (MNA) Kelly cells resulted in a statistically significant increase in motility, invasion, and colony formation (P < 0.05). Based on these observations, we hypothesised that an important target of miR-204 in primary tumors might not be expressed in NB cell lines. Computationally predicted targets included NTRK2/TRKB and its ligand, BDNF, both of which are expressed at high levels in unfavourable NB, and promote cell survival and chemotherapy resistance through the P13K/AKT pathway. TrkB is detectable in 30-40% of primary NBs (∼70% of MNA tumours) but rarely found in NB cell lines. By artificially inducing NB cell lines to express full length TrkB, we determined that miR-204 expression resulted in decreased TrkB protein levels. Direct targeting of miR-204 with the 3’UTR of TrkB (but not BDNF) was validated by a luciferase reporter assay. Consistent with expectations, decreased expression of TrkB by miR-204 targeting resulted in increased sensitivity to cisplatin treatment. Conclusion: We conclude that miR-204 associated survival in NB patients can be attributed in part to TrkB targeting. Our results demonstrate that miR-204 is a candidate prognostic marker and natural occurring regulator of TrkB, which could be exploited to improve response to treatment in NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2011-130