Targeted therapies with B-RAF/MEK-inhibitors are a promising therapeutic option for B-RAF mutated melanoma. However, patients frequently acquire resistance during treatment. Potential resistance mechanisms have been described, most of which bypass mutant BRAF by reactivating MAPK and Pl3K/Akt signaling. The aim of this study was to analyze the alterations in protease, ECM and soluble factors expression in drug-resistant melanoma cells that may contribute to alter the microenvironment and drive resistance. By prolonged treatment with Vemurafenib and Trametinib we generated 4 resistant melanoma cells: A375 and Skmel28 carrying the BRAFV600E mutation and BLM and MV3 harboring the NRASQ61R mutation. In all resistant cells MITF was highly upregulated supporting its role in promoting melanoma cell resistance. Morphologically, A375R and SKmel28R, but not BLMR/MV3R cells, acquired a mesenchymal-like phenotype with enhanced vimentin expression. In all resistant cells we found enhanced expression of several MMPs (MMP8,-14,-15,-16) and ADAM10. However, expression of MMP-2 and -9 and further ADAMs was variable. In parallel to altered protease expression, in Skmel28R cells we detected large amounts of secreted tenascin C (TNC). On transcript level, TNC was upregulated in Skmel28R indicating that the detected alterations are the result of transcriptional regulation. TNC was highly secreted by A375, but not majorly altered in A375R and low expressed in MV3R/BLMR cells. Fibronectin secretion was steady in SKmel28R and BLMR as in controls, whereas it was barely expressed in A375 and A375R cells, and downregulated in MV3R. Analysis of proteins secreted by resistant cells identified an increase of proinflammatory proteins such as CCL2, GM-CSF, CCL20 and Osteopontin, an ECM protein and mediator of inflammation. Altogether these factors might contribute to the creation of a proinflammatory environment that might foster the crosstalk between melanoma cells, fibroblasts, macrophages and inflammatory cells and promote, in combination with the ECM and proteolytic enzyme alterations, tumor invasion and metastasis.

Potential mediators of melanoma cell resistance