Melanoma plasticity creates a plethora of opportunities for cancer cells to escape treatment. Thus, therapies must target all cancer cell subpopulations bearing the potential to contribute to disease. The role of the differentiation/pigmentation program in intrinsic and acquired drug resistance is largely uncharacterized. MITF level and expression of MITF-dependent pigmentation-related genes, MLANA, PMEL, TYR, and DCT, in drug-naïve and vemurafenib- or trametinib-treated patient-derived melanoma cell lines and their drug-resistant counterparts were analysed and referred to genomic alterations. Variability in execution of pigmentation/differentiation program was detected in patient-derived melanoma cell lines. Acute treatment with vemurafenib or trametinib enhanced expression of pigmentation-related genes in MITF-Mhigh melanoma cells, partially as the consequence of transcriptional reprograming. During development of resistance, changes in pigmentation program were not unidirectional, but also not universal as expression of different pigmentation-related genes was diversely affected. In selected resistant cell lines, differentiation/pigmentation was promoted and might be considered as one of drug-tolerant phenotypes. In other resistant lines, dedifferentiation was induced. Upon drug withdrawal (“drug holiday”), the dedifferentiation process in resistant cells either was enhanced but reversed by drug reexposure suggesting involvement of epigenetic mechanisms or was irreversible. The irreversible dedifferentiation might be connected with homozygous loss-of-function mutation in MC1R, as MC1RR151C  +/+ variant was found exclusively in drug-naïve MITF-Mlow dedifferentiated cells and drug-resistant cells derived from MITFhigh/MC1RWT cells undergoing irreversible dedifferentiation. MC1RR151C  +/+ variant might be further investigated as a parameter potentially impacting melanoma patient stratification and aiding in treatment decision.

Clinical evidence has revealed that while RAS/RAF/MEK/ERK pathway is a crucial component of melanomagenesis, other signaling pathways can also contribute to the malignant growth and development of resistance to targeted therapies. We explored the response of BRAF melanoma cells derived from surgical specimens and grown in stem cell medium to vemurafenib and trametinib, drugs targeting the activity ofBRAF and MEK1/2, respectively. Cell growth and apoptosis were monitored by real-time imaging system, immunophenotype and cell cycle by flow cytometry, gene expression by quantitative real-time PCR, immunoblotting and enzyme-linked immunosorbent assay. The BRAF melanoma cell populations were diverse. Differences in morphology, pigmentation, cell cycle profiles, and immunophenotype were observed. At the molecular level, melanoma cells differed in the phosphorylation of ERK1/2, NF-κB, and β-catenin, and expression of several relevant genes, including MITF-M, DKK1, CCND1, BRAF, CXCL8, and CTGF. Despite having different characteristics, melanoma cells responded similarly to vemurafenib and trametinib. Both drugs reduced ERK1/2 phosphorylation and percentages of cells expressing Ki-67 at high level, inhibited expression of CCND1 and induced cell cycle arrest in the G/G phase. These expected cytostatic effects were accompanied by increased CD271 expression, a marker of stem-like cells. NF-κB activity was reduced by both drugs, however, not completely abolished, whereas the level of active β-catenin was increased by drugs in three out of six cell populations. Interestingly, expression of IL-8 and CTGF was significantly reduced by treatment with vemurafenib and trametinib. Simultaneous inhibition of NF-κB activity and induction of ERK1/2 phosphorylation revealed that CTGF expression depends on ERK1/2 activity but not on NF-κB activity. Both, the positive effects of treatment with vemurafenib and trametinib such as the newly identified CTGF suppression and undesired effects such as increased CD271 expression suggesting selection of melanoma stem-like cells should be considered in the development of combination treatment for melanoma patients.

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. Electronic supplementary material The online version of this article (10.1007/s10495-019-01542-y) contains supplementary material, which is available to authorized users.

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It has been shown that the response of V600EBRAF melanoma cells to targeted therapeutics is affected by growth factors. We have investigated the influence of three different growth factors, bFGF, EGF and HGF used either alone or in combination, on the response of V600EBRAF melanoma cell populations established from surgical specimens to vemurafenib and trametinib, targeting V600EBRAF and MEK1/2, respectively.We report that proliferation and phenotype of V600EBRAF melanoma cell populations were not detectably influenced by exogenous growth factors. Neither cell distribution in cell cycle and CCND1 expression nor activity of signaling pathways crucial for melanoma development and maintenance, including the RAF/MEK/ERK pathway, WNT/β-catenin pathway and NF-κB signaling, were affected by the presence of different growth factors. We furthermore show that vemurafenib and trametinib abrogated the activity of ERK1/2, arrested cells in G0/G1 cell cycle phase, triggered apoptosis, induced changes in the expression of CXCL8, CCND1 and CTGF and the frequency of Ki-67high and CD271high cells. These effects were, however, similar in the presence of different growth factors. Interestingly, comparable results were also obtained for melanoma cells grown without exogenous growth factors bFGF, EGF and HGF for a period as long as 4 months prior the drug treatment.We conclude that the composition or lack of exogenous growth factors bFGF, EGF and HGF do not markedly influence viability and phenotype of V600EBRAF melanoma cells and their response to vemurafenib and trametinib in vitro. Our results question the necessity of these growth factors in the medium that is used for culturing V600EBRAF melanoma cells.