Phenotype Characterization of Human Melanoma Cells Resistant to Dabrafenib.

In this study, the phenotype of melanoma cells resistant to dabrafenib (B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated from three different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometry analysis, Ion-torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to the sensitive counterparts, accompanied with an increased CD90 (mesenchymal marker) expression and a E-cadherin (epithelial marker) reduction, suggesting an Epithelial-to-Mesenchymal-like phenotypic transition. However, TGF-?1 induced Epithelial-to-Mesenchymal Transition (EMT) melanoma cells, when treated with dabrafenib, were more sensitive to the drug compared to the non-TGF-?1 induced EMT ones, suggesting that TGF-?1-induced EMT was not associated to dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/ vimentin reorganization, as expected in an EMT, all of them showed unvaried E-cadherin mRNA levels, unchanged Snail protein levels, while Twist1 protein expression decreased with exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings supported a distinct active EMT-like program adopted from melanoma cells under drug exposure. Further, dabrafenib-resistant cells exhibited stem-cell features, with an Oct4 translocation from cytoplasm to the peri-nuclear sites and nuclei and an increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib was dependent on a re-activation of MAPK signaling, suggest that it was linked to a distinct active EMT-like program as well as stem cell features adopted from melanoma cells.