Abstract B43: MYCN-amplified neuroblastoma maintain an undifferentiated phenotype through interference with estrogen and NGF signaling

Abstract

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. It is a highly heterogenic cancer of the sympathetic nervous system and features clinical phenotypes ranging from spontaneous differentiation and regression to aggressive growth and metastasis. Amplification of the MYCN gene is found in approximately 20-30% of all neuroblastoma cases and is linked to an undifferentiated phenotype and a poor prognosis. MYCN is a member of the MYC network of transcription factors, which are key players in the regulation of various fundamental cellular processes, e.g., cell growth, apoptosis and differentiation. We have previously demonstrated that miR-18a from the MYCN-regulated miR-17~92 microRNA cluster interferes with neuronal differentiation through repression of estrogen receptor alpha (ERα) levels. This study aims to analyze the effect of ERα overexpression on the morphology and functional behavior of MYCN-amplified neuroblastoma cells. Real time PCR, Western Blot and immunofluorescence analysis were performed to analyze mRNA levels and the amount, activation and localization of proteins. The morphological phenotype after activation of estrogen and/or NGF signaling was studied using bright field microscopy. Anchorage independent growth, WST-1 cell viability and transwell migration and invasion assays were conducted to study the effect of ERα overexpression and activation of NGF signaling on the functional phenotype of NB cells. Here we show that overexpression of ERα or interference with miR-18a is sufficient to increase mRNA and protein levels of the nerve growth factor (NGF) receptors TrkA and p75NTR in the MYCN-amplified neuroblastoma cell line SK-N-BE(2). Both NGF receptors negatively correlate with MYCN-amplification and are strongly linked to an improved prognosis in neuroblastoma patients. Importantly, neuronal differentiation was enhanced upon activation of NGF signaling in neuroblastoma cells with increased ERα levels, suggesting a crosstalk between estrogen and NGF signaling. Furthermore, overexpression of ERα resulted in a robust interference with processes linked to tumorigenesis, including cell survival, proliferation, migration, and anchorage independent growth. Together our results suggest a new mechanism by which MYCN-amplified neuroblastoma maintain an undifferentiated phenotype: MYCN-induced miR-18a targets ERα and thereby interferes with activation of NGF signaling and induction of differentiation. Conversely, reintroduction of ERα potentiates NGF signaling, facilitates neuronal differentiation and inhibits processes linked to tumorigenesis. Our data propose that restauration of ERα expression and activity, e.g., by interference with miR-18a, could be a promising strategy for the development of new therapies for neuroblastoma. Citation Format: Johanna Dzieran, Ulrica K. Westermark, Marie Arsenian Henriksson. MYCN-amplified neuroblastoma maintain an undifferentiated phenotype through interference with estrogen and NGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B43.