Abstract
Simple SummaryMelanoma is the most aggressive, deadliest form of skin cancer. Combined BRAF-MEK inhibitor (BRAFi/MEKi) therapy was a breakthrough in the treatment of melanoma with BRAFV600-mutations. However, many patients frequently develop drug resistance to the combinatory treatment. The aim of our study was to characterize the molecular background behind acquired resistance to BRAFi/MEKi-s. After the successful development of resistant cell lines, we investigated the invasion properties, changes in gene and protein expressions, as well as the effect of the “drug holiday” of the resistant cell lines. Drug-resistant melanoma cells had a higher invasive potential and acquired a spindle-like structure, and many cancer-related proteins were overexpressed in the resistant cells. Furthermore, transcriptome analysis revealed that differentially expressed genes are functionally linked to a variety of biological functions that may lead to resistance to the inhibitors. These results may offer valuable insight into further understanding of BRAFi/MEKi resistance, as well as to the development of therapeutic tools to overcome drug resistance.Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.
Highlights
Despite several advancements in the treatment of patients with malignant melanoma, this disease is still one of the major causes of skin cancer-associated death [1]
Combinational treatment using encorafenib and binimetinib was approved by the US Food and Drug Administration (FDA) for melanoma patients with a BRAFV600E/K mutations in 2018 [6,7]
We investigated the effect of the “drug holiday” on the protein expression changes using the same Proteome Profiler (Proteome Profiler Human XL Oncology Array) as we used before
Summary
Despite several advancements in the treatment of patients with malignant melanoma, this disease is still one of the major causes of skin cancer-associated death [1]. The most frequent mutation is an amino acid change from valine to glutamic acid at the 600-position: BRAFVV600E This mutation enhances the kinase activity of mitogen-activated protein kinase (MAPK) signalling ~500-fold and controls important cellular functions involving cell proliferation, differentiation, migration, survival, and angiogenesis [4]. Combinational treatment using encorafenib and binimetinib was approved by the US Food and Drug Administration (FDA) for melanoma patients with a BRAFV600E/K mutations in 2018 [6,7]. This inhibitor combination showed a high response rate, a favourable toxicity profile, and impressive progression-free survival (approximately 16.9 months compared to the ~9-month BRAFi monotherapy) in melanoma patients with BRAF mutations [6,8]. Studies have shown that several protein alterations, such as those in EGFR, COT, cyclin D1, and PDGFR-β, are associated with BRAFi and MEKi resistance [11]
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