Critical role of miR-10b in BRaf^V600E dependent anchorage-independent growth and invasion of melanoma cells

Abstract

26Recent high-throughput-sequencing of cancer genomes has identified oncogenic 27 mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. 28 BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) 29 and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, 30 the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf 31 gain-of-function mutations in melanoma frequently lead to unrestrained growth, 32 enhanced cell invasion and increased viability of cancer cells. Although it is clear that 33 the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on 34 BRaf-MEK-ERK activation, the downstream effector targets that are required for 35 oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have 36 regulatory functions towards the expression of genes that are important in 37 carcinogenesis. We observed that miR-10b expression correlates with the presence of 38 the oncogenic BRaf (BRaf V600E ) mutation in melanoma cells. While expression of miR-39 10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-40 10b silencing decreases BRaf V600E cancer cell invasion in vitro. Importantly, the 41 expression of miR-10b is required for BRaf V600E -mediated anchorage-independent 42 growth and invasion of melanoma cells in vitro. Taken together our results suggest that 43 miR-10b is an important mediator of oncogenic BRaf V600E activity in melanoma.44 45 46 3 47 48 49 Melanoma is the most aggressive of all the skin cancers. BRaf is a 50 serine/threonine protein kinase that activates the MEK/ERK-signaling pathway. About 51 25-70% of malignant melanomas harbor gain-of-function mutations in the oncogene 52 BRaf (1). Among several BRaf gain-of-function mutations, BRaf V600E is the most 53 common mutation and accounts for nearly 80% of them (1). Not only does BRaf V600E 54 cause a sustained activation of ERK signaling pathway in melanoma, it is also critical for 55 the malignant process and is one of the few identified driver mutations essential for 56 melanoma proliferation and survival. The transformation of melanocytes to melanoma 57 by BRaf V600E requires activation of the MEK-ERK kinases cascade with multiple 58 downstream components (2-4). The mechanism that integrates the diverse components 59 into a coordinated response to the BRaf V600E mutation remains undefined. 60 61 MicroRNAs (miRNAs) are small, non-protein coding RNA molecules and they 62 regulate gene expression through a combination of translational repression and mRNA 63 destabilization (5). Each miRNA targets ~200 mRNA molecules (6). Because of their 64 pleiotropic potentials, miRNAs are attractive candidates as master regulators of the 65 BRaf V600E oncogenic transformation program. In this study, we identified for the first 66 time, significant positive correlation between BRaf V600E mutation and microRNA-10b 67 (miR-10b) expression. Furthermore, we show that miR-10b is a novel ...