Development of a melanoma B16F1 cell line that overexpress human B-RAFV600E

COLLI C; RODRIGUEZ Y; ZARATE JM; ALVAREZ SE

Congreso; XXXV Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2017

Mutations in B-RAF protein are encountered in about 6 percent of all cancers. Remarkably, approximately 50% of melanoma patients exhibit the V600E mutation in the B-RAF protein (B-RAFV600E) increasing its activity and promoting cell proliferation. Although the development of B-RAF inhibitors such as vemurafenib have increased the survival of patients with melanoma, the durability of response is limited and tumors become quickly resistant. To better understand the molecular mechanism driving this resistance we decided to generate B16F1 murine melanoma cells that express B-RAFV600E. To this end, B16F1 cells were transfected with pBABE-B-RAFV600E or pBABE-B-RAF1-636 by using polyethylenimine (PEI) reagent. Selection of transfected cells was performed with the antibiotic puromycin. We compared the expression levels of B-RAFV600E in both cell lines by RT-PCR by using specific human oligonucleotides. Melanoma cells overexpressing B-RAFV600E displayed increased ERK phosphorylation levels as compared to cells transfected with p.CDNA3-GFP control cells, suggesting that the protein is fully functional. Moreover, subcutaneous implantation of B16F1 melanoma cells expressing B-RAFV600E in mice induced larger tumors than cells expressing the truncated and non-functional B-RAF1-636 protein. These results suggest that B-RAFV600E enhances melanoma tumor growth. In addition, this tool will be useful to evaluate mechanisms of resistance to chemotherapeutic agents, as well as the melanoma immune response in vivo. Supported by PICT-2014-2378 and PIP2015-2017- 112215-0100603CO