1. Phenotype characterization of human melanoma cells resistant to dabrafenib.
- Author
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Cordaro FG, De Presbiteris AL, Camerlingo R, Mozzillo N, Pirozzi G, Cavalcanti E, Manca A, Palmieri G, Cossu A, Ciliberto G, Ascierto PA, Travali S, Patriarca EJ, and Caputo E
- Subjects
- Antigens, CD, Biomarkers, Tumor genetics, Cadherins genetics, Cadherins metabolism, Cell Line, Tumor, Humans, Imidazoles, MAP Kinase Signaling System, Melanoma genetics, Mutation, Octamer Transcription Factor-3 genetics, Octamer Transcription Factor-3 metabolism, Oximes, Phenotype, Proto-Oncogene Proteins B-raf genetics, Thy-1 Antigens genetics, Thy-1 Antigens metabolism, Vimentin genetics, Vimentin metabolism, Biomarkers, Tumor metabolism, Drug Resistance, Neoplasm, Epithelial-Mesenchymal Transition, Melanoma metabolism
- Abstract
In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric 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 their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF‑β1‑induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.
- Published
- 2017
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