Abstract 2290: MiR-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-beta signalling pathway
Authors: Lynch Jennifer , Fay Joanna , Meehan Maria , Bryan Kenneth , Watters Karen M. , Murphy Derek M. , Stallings Raymond L. 
Published: August 2012

Abstract
MicroRNAs are post-transcriptional gene regulators that play a ubiquitous role in cellular functioning. Aberrant expression of miRNAs has been correlated with many cancer types, including neuroblastoma. Neuroblastoma is an often fatal pediatric cancer derived from precursor cells of the sympathetic nervous system. The single most important indicator of unfavourable metastatic disease is amplification of the MYCN oncogenic transcription factor. MiR-335 is significantly down-regulated in MYCN amplified tumors (Bray et al Plos One 2009; 4:e7850) suggesting a possible tumor suppressive function. This study aims to determine if miR-335 functions as a tumor suppressor in neuroblastoma and to elucidate the mechanism by which it elicits this effect. Ectopic up-regulation of miR-335 significantly reduced the motile and invasive capacity of neuroblastoma cell lines. Inversely, down-regulation of miR-335 significantly increased their migratory and invasive potential. mRNA expression profiling arrays in conjunction with the miRNA target prediction algorithm TargetScan identified potential targets of miR-335. Three of these candidate genes, ROCK1, MAPK1 and LRG1, were selected for further investigation because of their known or predicted involvement in cell migration and invasion. Down-regulation of these genes by miR-335 was validated at mRNA and protein levels and direct targeting by miR-335 was confirmed by luciferase reporter assays. siRNA-mediated inhibition of each of these three genes recapitulated the diminished cell migration and invasion associated with miR-335 up-regulation. ROCK1 and MAPK1 are members of two of the major branches of the oncogenic non-canonical TGF-beta signalling pathway, whose signals converge to tightly regulate the phosphorylation or activation status of the motor protein myosin light chain (MLC). LRG1 is a putative TGF-beta pathway member that has been proposed to bind to TGF-beta receptor II. We demonstrate that siRNA mediated inhibition of ROCK1, MAPK1 and LRG1 leads to a significant reduction in phosphorylated levels of MLC protein. Furthermore, we demonstrate that up-regulation of miR-335 inhibits phosphorylation or activation of MLC through targeting of these three genes. Finally, we demonstrate using a MYCN repressible cell line and MYCN ChIP-chip experiments that miR-335 is directly repressed by the oncogenic transcription factor MYCN. Therefore, we conclude that miR-335 is directly repressed by MYCN in MYCN amplified neuroblastoma tumors leading to the increased expression of the oncogenic non-canonical TGF-beta pathway members ROCK1, MAPK1 and LRG1 which function to enhance the metastatic propensity of the tumor cells.
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 2290. doi:1538-7445.AM2012-2290