Melanoma-derived extracellular vesicles: Signaling through their miRNA content

Abstract

Melanoma is the deadliest type of skin cancer. Early diagnosis is of great importance for patient outcome. Currently, genetic testing is required to determine the mutational background. Promising research on extracellular vesicles (EVs) could offer a potential new method to diagnose melanoma. Melanoma cells are known to secrete these EVs as an oncogenic signaling mechanism. One of the components packaged in EVs are miRNAs. It was hypothesized that melanoma mutants BRAF, NRAS, and double mutant distinctively regulate the miRNA composition in secreted EVs. Identifying this mutant-specific miRNA profile in the blood of patients could be used to diagnose melanoma and the mutation. To test whether EV-enriched miRNAs are melanoma-mutant dependent, and whether these signature miRNAs also alter gene expression in recipient cells, we compare the miRNA profiles of EVs and their cellular counterparts for different melanoma mutant cell lines (BRAF, NRAS, double mutant). Target prediction and in vitro target validation is performed to elucidate the regulatory role of these candidate signature miRNAs. We show that EV miRNA profiles are distinct from their matched cellular profiles. The signature miRNA identified for BRAF mutant melanoma cell lines, hsa-mir-6728-3p, enhances TGF-beta signaling via ITCH and Smad2/3 phosphorylation. TGF-beta signaling is known to promote tumorigenesis, drug resistance, and immune surveillance in melanoma. In addition to target validation, we aimed to shed light on miRNA sorting mechanisms in EVs. The existing hypothesis of selective miRNA sorting is based on the specific recognition of motifs within the miRNA sequence by sorting proteins. In our miRNA dataset, such sorting motifs could be identified in the EV-enriched miRNAs and cell-retained miRNAs. Although sorting motifs are highly cell line dependent, some of the identified motifs were shared between the melanoma cell lines and also between previously identified motifs by others, suggesting a conserved nature. Interestingly, these motifs are located primarily at the 3’ end of the miRNA sequences. Altogether, we identified a signature miRNA for BRAF and double mutant melanoma cell lines. For the BRAF signature miRNA we were able to show its capability to regulate the TGF-beta pathway, underlying the importance of EV-miRNAs in tumorigenesis. Sorting of miRNAs into EVs is an extremely complex mechanism that is highly cell line dependent. Our data suggest, in line with previous literature, the existence of sorting motifs within the miRNA sequences.