Effect of miR-195 overexpression in the miRNA cargo of extracellular vesicles secreted by human melanoma cells and in the regulation of cells sensitivity to targeted-therapy through horizontal transfer of information

Author(s)

Nathalia Leal Santos

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2021-12-06

Examining board members

Roger Chammas; Diana Noronha Nunes; Fátima Solange Pasini; Eduardo Moraes Rego Reis

• Advisor: Roger Chammas; Luciana Nogueira de Sousa Andrade

Abstract

Melanoma is considered the most aggressive form of skin cancer. Combined targeted therapy, with BRAF and MEK inhibitors, considered the standard treatment for BRAF-mutated metastatic melanoma patients, results in 5 years overall survival of 20%. Extracellular Vesicles (EVs)-mediated transfer of information has been reported as a mechanism capable of regulating tumor resistance and progress. The presence of antitumoral molecules within EVs is related to increased response rates and better prognosis for melanoma patients. MiR-195, considered a tumor suppressor miRNA, is down-regulated in melanoma and its overexpression results in decreased cells proliferation and increased sensitivity to chemotherapy with alkylating agents and BRAF inhibitors. We aimed to analyze whether miR-195 overexpression could modulate EVs cargo and the role of these particles in naïve cells response to combined targeted treatment. A375, SKMel-5, SKMel-28 and UACC-62 cells were transfected with miR-195, resulting in increased EVs release and decreased EVs size. Gene expression analysis by RT-qPCR showed that miR-195 mimic induces the expression of GTAPases involved in exosomes biogenesis, while genes involved in microvesicles shedding are down-regulated upon transfection. EVs derived from cells overexpressing miR-195 could transfer its cytostatic effect, inducing the expression of anti-proliferative genes, such as CDKN1A and CDKN3, and inhibiting proproliferative genes, including CCND1 and YAP1, in naïve cells. EVs containing miR-195 also impaired in vivo tumor growth, reducing tumors growth kinetics. Treatment with miR-195 EVs could also sensitize naive cells to combined targeted therapy, resulting in higher expression of BAD, inhibition of BCL2L1 and consequently increased cell death, in addition to decreased Página | 4 clonogenic potential. Additionally, regarding the mechanisms of miR-195 regulation, TCGA data showed higher levels of methylation of this miRNA in melanoma tissues compared to controls, nevertheless, treatment of melanoma cells with Azacitidine only restored miR-195 expression in one out of three cell lines. In silico analysis showed that several lncRNAs capable to sponge miR-195 are overexpressed in melanomas, representing another possible mechanism of down-regulation. In summary, miR-195 overexpression represents a promising strategy to sensitize melanoma cells through horizontal transfer of information (AU)

• FAPESP's process: 19/07278-0 - Effect of miR-195 superexpression in the cargo of microRNAs present in extracellular vesicles secreted by human melanoma cells and in the regulation of sensibility to target-therapy through horizontal transfer of information
• Grantee: Nathalia Leal Santos
• Support Opportunities: Scholarships in Brazil - Master