Comparing the effect of different growth factors on melanoma cell signalling pathways

Chan XY, Osman N and Piva TJ

School of Health & Biomedical Sciences, RMIT University, Bundoora, Vic 3083.

Vemurafenib (PLX4032) is often used to treat melanomas that possess the BRAFV600E mutation. While the tumours are shown initially to regress, they become resistant to the drug and the patient relapsed and eventually dies. It has been shown that the growth factors secreted by adjacent cells activated signalling pathways in these melanoma cells. Using specific signalling pathway inhibitors, we investigated the growth factor-activation of intracellular signalling pathways in four melanoma cell lines. MM418-C1 (1° tumour) and C32 (2° tumour) cells harbour the BRAFV600E mutation, while MM329 (1° tumour) and D24 (2° tumour) cells do not. Growth factors (HGF or TGFα) were added to cells that had been serum starved for 24 h in low serum (0.5% FBS) containing media, and the expression of p-BRAF, p-Akt, p-ERK1/2, p-MEK, p-p38, p-JNK1/2 were quantified using Western blots. From preliminary data, we observed that HGF signalled through BRAFV600E in MM418-C1 cells, but TGFα signalled through BRAFWT in D24 cells. However, expression of other downstream signalling intermediates (p-ERK1/2, p-p38 and p-JNK1/2) of BRAF was increased when either HGF or TGFα was added to the cell lines. We also observed the activation of p-Akt (Ser473 and Thr308) by both growth factors which suggests that the PI3K-AKT-mTOR pathway is active in these cell lines. The effect of these growth factors and/or specific signalling pathway inhibitors on these signaling intermediates and cell migration were examined and the significance of the results will be discussed.