Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

Abstract

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.

Increased copy number of imprinted genes in the chromosomal region 20q11-q13.32 is associated with resistance to antitumor agents in cancer cell lines

“…PHLDA2 expression was associated with resistance to 54 agents with ρ > 0.3, including the strongest associated with the PDK1 inhibitor BX-912 with ρ = 0.402. Consistent with the association of its increased expression with resistance to many agents in our pancancer analysis, PHDLA2 had been previously found to be a part of molecular signatures and pathways overexpressed in melanoma and multiple myeloma cell lines resistant to a BRAF inhibitor/MEK inhibitor combination and proteasome inhibitors, respectively [121,122]. Interestingly, in our analysis PHLDA2 expression was associated with sensitivity to four agents including three MEK inhibitors (trametinib, refametinib, and PD0325901) and an HSP90 inhibitor, tanespimycin (− 0.378 ≤ ρ ≤ − 0.329; Additional file 8: Table S5).…”

Gene Expression Patterns of Osteopontin Isoforms and Integrins in Malignant Melanoma

“…Osteopontin (OPN), a multifunctional protein, has been widely studied as a promising biomarker in various types of tumors for monitoring tumor progression, invasion, metastasis formation and drug resistance (13,25). The association between the aberrant expression of osteopontin and melanoma invasion, metastasis formation, and radio/drug resistance has been recently described (1,3,(25)(26)(27). The biological functions of tumor-associated gene products are extensively regulated via pre-and posttranscriptional modifications, resulting in alternative splicing of OPN.…”

“…Osteopontin (OPN), a multifunctional protein, has been widely studied as a promising biomarker in various types of tumors for monitoring tumor progression, invasion, metastasis formation and drug resistance ( 13 , 25 ). The association between the aberrant expression of osteopontin and melanoma invasion, metastasis formation, and radio/drug resistance has been recently described ( 1 , 3 , 25 – 27 ).…”

Targeting mitochondrial energetics reverses panobinostat‐ and marizomib‐induced resistance in pediatric and adult high‐grade gliomas

“…3I) support a correlation between resistance and enolase‐2 expression, an enzyme involved in the penultimate step of glycolysis that converts 2‐phosphoglycerate to phosphoenolpyruvate (Fig. S2A), and functions as a crucial activator of other oncogenic pathways, leading to cell proliferation and treatment resistance [69–71].…”