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 MITFlow 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.
Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma.,Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi.,miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR.,The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA.,ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting.,Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines.,Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development.,VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA.,miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity.,VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines.,ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance.,At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not.,In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2.,Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity.,Circulating VEGF-A is a promising biomarker for predicting patients’ response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.,The online version of this article (10.1186/s13046-019-1238-4) contains supplementary material, which is available to authorized users.