TY - JOUR

T1 - Long-Term Vemurafenib Exposure Induced Alterations of Cell Phenotypes in Melanoma

T2 - Increased Cell Migration and Its Association with EGFR Expression

AU - Molnár, Eszter

AU - Garay, Tamás

AU - Donia, Marco

AU - Baranyi, Marcell

AU - Rittler, Dominika

AU - Berger, Walter

AU - Tímár, József

AU - Grusch, Michael

AU - Hegedűs, Balázs

PY - 2019

Y1 - 2019

N2 - Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

AB - Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

KW - Adult

KW - Aged

KW - Cell Line, Tumor

KW - Cell Movement/drug effects

KW - Cell Proliferation/drug effects

KW - Drug Resistance, Neoplasm/drug effects

KW - Epithelial-Mesenchymal Transition/drug effects

KW - ErbB Receptors/metabolism

KW - Erlotinib Hydrochloride/pharmacology

KW - Female

KW - Gene Expression Regulation, Neoplastic/drug effects

KW - Humans

KW - Inhibitory Concentration 50

KW - Male

KW - Melanoma/drug therapy

KW - Middle Aged

KW - Mutation/genetics

KW - Neoplasm Proteins/genetics

KW - Phenotype

KW - Proto-Oncogene Proteins B-raf/genetics

KW - RNA, Messenger/genetics

KW - Signal Transduction/drug effects

KW - Time Factors

KW - Vemurafenib/pharmacology

U2 - 10.3390/ijms20184484

DO - 10.3390/ijms20184484

M3 - Journal article

C2 - 31514305

VL - 20

JO - International Journal of Molecular Sciences (Online)

JF - International Journal of Molecular Sciences (Online)

SN - 1661-6596

IS - 18

M1 - 4484

ER -