Phenotype characterization of human melanoma cells resistant to Dabrafenib

Combined B-RAF and MEK inhibition elicits rapid anti-tumor responses in the most patients with B-RAF (V600E) mutant metastatic melanoma. However, combined treatment is not a sufficient strategy to overcome drug resistance development. We selected in vitro melanoma cells resistant to dabrafenib (B-RAFi, B-RAF inhibitor) alone or combined with trametinib (MEKi, MEK inhibitor) from three different dabrafenib/trametinib-sensitive melanoma cell lines (A375, 397, 624.38). After their characterization, by measuring drug IC50 and examining the drug effect on the cell cycle by FACS analysis, we examined their phenotype. Melanoma cells resistant to dabrafenib alone or combined with trametinib exhibited a spindle shape as compared to the polygonal shape of their sensitive counterparts. Drug-resistant cells showed an increased expression of CD90 with a reduction of CD324 - mesenchymal and epithelial markers, respectively - as well as a higher cell motility and a reorganization of the network of the vimentin mesenchymal marker, mainly localized at perinuclear sites as compared to the drug-sensitive cells. Furthermore, the expression analysis of CD20, CD133, CD44, and Oct4 stem cell-like markers revealed an increased CD20 expression in all drug resistant cells, presenting however different changes in CD133 and CD44 expression levels as well as an Oct4 translocation from cytoplasm to the perinuclear sites and nucleus. No changes in genetic status among the main candidate cancer genes were observed between drug-sensitive and drug-resistant cells. These data support that drug-resistant cells adopt an Epithelial-to-Mesenchymal Transition-like phenotype as well as cell stem-like features to escape the drug effect. Unraveling the molecular changes behind this phenotype will contribute to define novel target(s) for melanoma therapy and to overcome drug resistance development.