Phenotype characterization of human melanoma cells resistant to dabrafenib

F. G. Cordaro, A. L. De Presbiteris, R. Camerlingo, N. Mozzillo, G. Pirozzi, E. Cavalcanti, A. Manca, G. Palmieri, A. Cossu, G. Ciliberto, P. A. Ascierto, S. Travali, E. J. Patriarca, E. Caputo

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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-beta1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGFbeta1induced EMT melanoma cells, suggesting that TGF-beta1-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.
Original languageEnglish
Pages (from-to)2741-2751
Number of pages11
JournalOncology Reports
Volume38
Issue number5
DOIs
Publication statusPublished - Nov 1 2017

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Melanoma
Epithelial-Mesenchymal Transition
Phenotype
Cadherins
Transforming Growth Factor beta1
Stem Cells
dabrafenib
Snails
Vimentin
Biochemistry
Cell Movement
Fluorescent Antibody Technique
Cytoplasm
Proteins
Ions
Technology
Cell Line
Messenger RNA
Pharmaceutical Preparations

Cite this

Cordaro, F. G., Presbiteris, A. L. D., Camerlingo, R., Mozzillo, N., Pirozzi, G., Cavalcanti, E., ... Caputo, E. (2017). Phenotype characterization of human melanoma cells resistant to dabrafenib. Oncology Reports, 38(5), 2741-2751. https://doi.org/10.3892/or.2017.5963 [doi]

Phenotype characterization of human melanoma cells resistant to dabrafenib. / Cordaro, F. G.; Presbiteris, A. L. De; Camerlingo, R.; Mozzillo, N.; Pirozzi, G.; Cavalcanti, E.; Manca, A.; Palmieri, G.; Cossu, A.; Ciliberto, G.; Ascierto, P. A.; Travali, S.; Patriarca, E. J.; Caputo, E.

In: Oncology Reports, Vol. 38, No. 5, 01.11.2017, p. 2741-2751.

Research output: Contribution to journalArticle

Cordaro, FG, Presbiteris, ALD, Camerlingo, R, Mozzillo, N, Pirozzi, G, Cavalcanti, E, Manca, A, Palmieri, G, Cossu, A, Ciliberto, G, Ascierto, PA, Travali, S, Patriarca, EJ & Caputo, E 2017, 'Phenotype characterization of human melanoma cells resistant to dabrafenib', Oncology Reports, vol. 38, no. 5, pp. 2741-2751. https://doi.org/10.3892/or.2017.5963 [doi]
Cordaro FG, Presbiteris ALD, Camerlingo R, Mozzillo N, Pirozzi G, Cavalcanti E et al. Phenotype characterization of human melanoma cells resistant to dabrafenib. Oncology Reports. 2017 Nov 1;38(5):2741-2751. https://doi.org/10.3892/or.2017.5963 [doi]
Cordaro, F. G. ; Presbiteris, A. L. De ; Camerlingo, R. ; Mozzillo, N. ; Pirozzi, G. ; Cavalcanti, E. ; Manca, A. ; Palmieri, G. ; Cossu, A. ; Ciliberto, G. ; Ascierto, P. A. ; Travali, S. ; Patriarca, E. J. ; Caputo, E. / Phenotype characterization of human melanoma cells resistant to dabrafenib. In: Oncology Reports. 2017 ; Vol. 38, No. 5. pp. 2741-2751.
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AU - Pirozzi, G.

AU - Cavalcanti, E.

AU - Manca, A.

AU - Palmieri, G.

AU - Cossu, A.

AU - Ciliberto, G.

AU - Ascierto, P. A.

AU - Travali, S.

AU - Patriarca, E. J.

AU - Caputo, E.

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N2 - 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-beta1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGFbeta1induced EMT melanoma cells, suggesting that TGF-beta1-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.

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