Abstract
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.
Highlights
We further explored the influence of growth conditions on melanoma cells and cell response to treatment with vemurafenib (Zelboraf) targeting V600EBRAF and trametinib (Mekinist) targeting mitogenactivated kinase/ERK kinase 1/2 (MEK1/2)
hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF) were used in the culture medium for 10 days, either alone or in combination
Cell proliferation was investigated in 3 melanoma cell lines established from surgery specimens, DMBC11, DMBC12 and DMBC21 using IncuCyte (Fig 1a)
Summary
The present study on the influence of growth factors on melanoma cell phenotype and response to vemurafenib (Zelboraf) and trametinib (Mekinist) was justified by recently. BFGF, EGF and HGF do not influence V600EBRAF melanoma and response to vemurafenib and trametinib published reports. It has been demonstrated that untreated V600EBRAF melanoma cells exhibit high levels of feedback inhibition in the RAF/MEK/ERK signaling pathway that diminishes the ability of receptor tyrosine kinase signaling to activate ERK [1]. This feedback inhibition becomes deregulated in vemurafenib-treated cells, which increases cell responsiveness to growth factors, such as bFGF, HGF and EGF [1]. Vemurafenib treatment increased the fibroblast-mediated release of HGF [3], and HGF secretion by stroma cells evoked resistance to RAF inhibitors [2,4,5] or as suggested by Filitis et al, just induced HGF-mediated mitogenesis [6]
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