Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Dissecting the role of novel oncomiRs and their molecular targets in the evolution of resistance to target therapies in melanoma

AIMS: They have been: a) To characterize the migratory and invasive properties of drug-resistant melanoma cells and to assess the correlation with miR-4443 and miR-4488 overexpression; b) To investigate whether the enforced overexpression of miR-4443 or miR-4488 in drug sensitive melanoma cells is able to phenocopy the biological properties of drug-resistant cells; c) To identify miR-4443 and miR-4488 potential targets and unveil the molecular pathways affected in melanoma. RESULTS: I demonstrated that BRAF-mutant melanoma cells rendered drug resistant to BRAFi have enhanced migrative/invasive properties as compared to their drug sensitive counterparts. Moreover, drug-sensitive melanoma cells transiently transfected with miR-4443/miR-4488 mimics are able to phenocopy the migratory/invasive behaviour of drug-resistant cells. Furthermore, Confocal microscopy analyses showed that MAPKi-resistant A375 cells are increased in size, number of stress fibers and filopodia. In silico predictions of miR-4443/miR-4488 target genes combined to literature studies allowed to hypothesize the intermediated filament protein Nestin, as a promising candidate. Coherently with this, experimental data showed that Nestin is strongly down-regulated in drug-resistant cells in which miR-4443/miR-4488 are in contrast up-regulated. Finally, luciferase assays as well as WB and FACS analyses confirmed the predictions. CONCLUSIONS: I demonstrated that a) drug-resistant cells enhanced migratory and invasive properties as compared to sensitive counterparts, b) this phenotype is influenced by miR-4443/miR-4488, which are upregulated in drug resistant cells and c) I identified a novel oncomiRs-Nestin axis as responsible for the regulation of migrative/invasive properties of metastatic melanoma. Altogether, these evidences unveiled novel non-genetic mechanisms centered around the deregulation of two novel oncomiRs as responsible for the evolution of drug resistance to targeted therapies in BRAF-mutant melanomas; these findings may potential therapeutic implications.