Abstract 474: Alterations of micro-RNAs are associated with melanoma resistance to BRAF inhibitors: Role of miR-126
Authors: Caporali Simona , Levati Lauretta , Alvino Ester , Amaro Adriana , Lacal Pedro Miguel , Bonmassar Laura , Bassi Cristian , Lupini Laura , Cappellini Gian Carlo Antonini , Pfeffer Ulrich , Negrini Massimo , Felli Nadia , Carè Alessandra , Russo Giandomenico , D'Atri Stefania 
Published: August 2018

Abstract
Altered expression of miRNAs has been demonstrated in tumor tissue and plasma/serum of cancer patients. miRNAs have been shown to have both diagnostic and prognostic significance and to potentially constitute novel targets and therapeutic agents for cancer treatment. Experimental evidences also support an involvement of miRNAs in tumor cell response to therapy. In this study, we explored the role of miRNAs in melanoma resistance to BRAF inhibitors (BRAFi). The melanoma cell line A375 and its dabrafenib-resistant subline A375R, which displays increased invasiveness and VEGF secretion, were analyzed for miRNA expression using Affymetrix GeneChip® miRNA 3.1 microarrays. Differential expression of selected miRNAs was confirmed in the two cell lines using qRT-PCR. miR-126, previously shown to act as a tumor suppressor gene in melanoma, was chosen for additional studies that were performed also in a second pair of matched melanoma cell lines, sensitive or resistant to dabrafenib (i.e SK-Mel28 and SK-Mel28R). The resistant sublines were transfected with 50 nM Pre-miR hsa-miR-126 miRNA Precursor (pre-miRNA-126) or Pre-miR miRNA Precursor Negative Control#1 (Ambion®) and analyzed for proliferation 6 days later using the MTT assay. Seventy-two hours after transfection, the cells were also assayed for invasion of the extracellular matrix and VEGF-A secretion using Boyden Chamber and ELISA assays, respectively. In 33 melanoma patients treated with BRAFi, alone or in combination with MEKi, plasma samples were collected before the beginning of therapy (T0), and two months later (T2) and subjected to miRNA expression profiling by small RNA-seq. A375R cells displayed 13 up-regulated miRNAs and 32 down-regulated miRNAs with respect to A375 cells (SAM analysis; fold change ≥ 2). Down-regulation of miR-126 was confirmed by qRT-PCR analysis in both A375R and SK-Mel28R cells. Restoration of miR-126 expression in A375R and SK-Mel28R cells by pre-miRNA-126 transfection impaired proliferation, invasion and secretion of VEGF-A, a validated target of miR-126. Fifthy and 28 circulating miRNAs were differentially expressed at T0 and at T2, respectively, between patients who responded to therapy (n=28) and patients who did not (n=5). Our results demonstrate that down-regulation of miR-126 expression is associated with acquired resistance to dabrafenib in melanoma cells and suggest that restoration of this miRNA in dabrafenib-resistant melanomas might restrain tumor growth and metastasis. They also show that in melanoma patients with primary resistance to BRAFi, a set of circulating miRNAs is differentially expressed with respect to patients responding to therapy, suggesting a potential role of circulating miRNAs as biomarkers for early prediction of drug response. Supported by the Italian Ministry of Health, grant 5PerMille-2010 and AIRC, IGP 17585
Citation Format: Simona Caporali, Lauretta Levati, Ester Alvino, Adriana Amaro, Pedro Miguel Lacal, Laura Bonmassar, Cristian Bassi, Laura Lupini, Gian Carlo Antonini Cappellini, Ulrich Pfeffer, Massimo Negrini, Nadia Felli, Alessandra Carè, Giandomenico Russo, Stefania D'Atri. Alterations of micro-RNAs are associated with melanoma resistance to BRAF inhibitors: Role of miR-126 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 474.