Elucidating the role of metastasis suppressor microRNA-335 in neuroblastoma disease pathogenesis.

Neuroblastoma is a highly metastatic paediatric cancer in which advanced stage disease is associated with a high incidence of mortality despite the intensive multimodal nature of its treatment regimens. Amplification of MYCN is the single most important genetic indicator of unfavourable, metastatic disease. As a transcription factor, MYCN can regulate a large network of protein-coding genes and non-coding RNA transcripts, such as miRNAs, to manipulate cellular functioning and promote malignant transformation. In this study, we provide a comprehensive outline of the function of the MYCN regulated tumour suppressor miRNA, miR-335, in neuroblastoma. It was previously determined that miR-335 expression is significantly down-regulated in the MYCN amplified subtype of tumour, indicative of a tumour suppressor function. Furthermore, we established that low expression of miR-335 is significantly associated with poor overall survival among neuroblastoma patients. The aim of this study was to identify the phenotypic function of miR-335 in neuroblastoma tumorigenesis, to characterise its mechanism of action and the mechanism of regulation of the miRNA itself. We provide conclusive evidence that miR-335 functions as a potent suppressor of neuroblastoma cell motile and invasive capacity. Enhancing the expression of miR-335 significantly diminishes the cells’ motile and invasive capacity. Moreover, we demonstrate that modification in cell migratory potential mediated by miR-335 is accompanied by rearrangements in the actin cytoskeleton network. The actin cytoskeleton constitutes the cells’ migratory machinery and dynamic actin rearrangements provide the forces necessary for cell motility. We illustrate that miR-335 regulates cancer cell migration and invasion by directly targeting a plethora of genes that effectively control the migratory machinery of the cell. Firstly, we validate that miR-335 directly targets multiple members of the oncogenic non-canonical TGF-P pathway that functions to enhance tumour cell migration, invasion and metastasis. In particular, ROCK1 and MAPK1 belong to two independent branches of the non-canonical pathway and are directly repressed by miR-335. In addition, miR-335 directly suppresses the expression of the putative TGF-|3 pathway member LRG1. Secondly, we identify the formin family of actin nucleators as important targets of miR-335. By targeting the formin family, miR-335 gains direct access to the actin assembly and disassembly machinery of the cell, manipulating its activity to modify cancer cell migratory and invasive capacity. In this study, we also address the mechanism of miR-335 regulation in neuroblastoma tumorigenesis. It is determined that the miR-335 promoter is subject to direct binding and repression of its transcript by the oncogenic transcription factor MYCN. Thus, we conclude that in MYCN amplified tumours, miR-335 expression is directly repressed leading to the increased expression of its metastasis enhancing target genes, non-canonical TGF-p members and the formin homology family, ultimately accelerating the development of advanced stage disease and contributing to the poor survival phenotype of MYCN amplified tumours