Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes trough downregulation of intermediate filament nestin

Vittorio Castaldo1, Michele Minopoli2, Francesca Di Modugno3, Andrea Sacconi4, Domenico Liguoro5, Rachele Frigerio6, Arianna Ortolano6, Marta Di Martile7, Luisa Gesualdi5, Gabriele Madonna8, Mariaelena Capone8, Roberto Cirombella9, Angiolina Catizone1, Donatella Del Bufalo7, Andrea Vecchione9, Maria Vincenza Carriero2, Paolo Antonio Ascierto8, Rita Mancini5,9, Luigi Fattore*6, Gennaro Ciliberto10. 1 Department of Anatomy, Histology, Forensic- Medicine and Orthopedics, Sapienza University of Rome, 00161, Rome, Italy. 2 Preclinical Models of Tumor Progression Unit, Istituto Nazionale Tumori IRCCS 'Fondazione G. Pascale', 80131 Naples, Italy. 3 Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Via Chianesi 53, 00144 Rome, Italy. 4 Clinical Trial Center, Biostatistics and Bioinformatics Unit, IRCCS Regina Elena National Cancer Institute, 00144, Rome, Italy. 5 Department of Clinical and Molecular Medicine, Sapienza University of Rome, 00161, Rome, Italy. 6 SAFU Laboratory, Department of Research, Advanced Diagnostics and Technological Innovation, Translational Research Area, IRCCS Regina Elena National Cancer Institute, 00144, Rome, Italy. 7 Preclinical Models and New Therapeutic Agents Unit, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy. 8 Unit of Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, 80131, Naples, Italy 9 Faculty of Medicine and Psychology, Department Clinical and Molecular Medicine, Sant’Andrea Hospital-Sapienza University of Rome, 00118, Rome, Italy 10 Scientific Directorate, IRCSS Regina Elena National Cancer Institute, 00144, Rome, Italy. Abstract Background and rationale: BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. Methods: Invasion and migration have been determined by wound healing, Boyden chamber, transwell invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by immunofluorescence immunohistochemical and flow cytometry analyses. Results: We demonstrate that the two oncomiRs are responsible for the enhanced migration and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we have identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. Conclusions: Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions