Cancer cell phenotype largely depends on oxygen availability. The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue. Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O₂), normoxia (6% O₂) and hypoxia (1% O₂). A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines. Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner. Increased protein stability might be responsible for high PGC1α level in MITF^low melanoma cells. Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner. In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy. Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAF^V600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis. Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered. This might result from an inframe deletion in EIF2AK3 leading to a PERK^L21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRAS^Q61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1^P187S). In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAF^V600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone. As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAF^V600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation. Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.

Background: Despite the progress made in the clinical management of metastatic melanoma, a patient’s response to treatment cannot be fully predicted, and intrinsic or acquired resistance that is developed in most melanoma patients warrants further research efforts. In addition to genetic factors, microenvironmental input should be considered to explain the diversity of response to treatment among melanoma patients. In this study, we evaluated the impact of insulin on patient-derived BRAF^V600E melanoma cells, either untreated or treated with vemurafenib or trametinib, inhibitors of BRAF^V600 and MEK1/2, respectively. Methods: Cells were cultured in serum-free conditions, either with or without insulin. The activity of the MAPK/ERK and PI3K/AKT pathways was assessed by Western blotting, cell viability, and percentages of Ki-67- and NGFR-positive cells by flow cytometry. Transcript levels were analyzed using qRT-PCR, and γ-H2AX levels by immunoblotting and confocal microscopy. A luminescence-based assay was used to measure glutathione content. Results: While insulin did not influence the MAPK/ERK pathway activity, it had a strong influence on melanoma cells, in which this pathway was suppressed by either vemurafenib or trametinib. In the presence of insulin, both drugs were much less efficient in 1) inhibiting proliferation and reducing the percentage of Ki-67-positive cells, and 2) inducing apoptosis and phosphorylation of histone H2AX in melanoma cells. Changes induced by vemurafenib and trametinib in glutathione homeostasis and DNA repair gene expression were also attenuated by insulin. Moreover, insulin impaired the combined effects of targeted drugs and doxorubicin in melanoma cells. In addition to insulin-induced PI3K/AKT activity, which was either transient or sustainable depending on the cell line, an insulin-triggered increase in the percentage of cells expressing NGFR, a marker of neural crest stem-like cells, may contribute to the reduced drug efficacy. Conclusion: Our results demonstrate the role of insulin in reducing the efficacy of vemurafenib and trametinib. This needs clinical assessment.