Combinatorial treatments targeting MAPK and PI3K/mTOR pathways in metastatic melanoma

Therapeutic targeting of BRAFV600E has shown a significant impact on progression-free and overall survival in advanced melanoma, but only a fraction of patients benefit from these treatments, suggesting that additional signaling pathways involved in melanoma growth/survival need to be identified. In fact MAPK and PI3K/mTOR signaling pathways are constituively activated in most cancers, including melanoma, to sustain the melanoma growth/survival. A large panel of melanoma were characterized for resistance/susceptibility to different inhibitors targeting MAPK and PI3K/mTOR signaling pathways and the synergistic effect of combinatorial treatments affecting both pathways. These effects were evaluated in terms of cell viability (MTT), apoptosis (Annexin V-PI), caspase 3/7 activity and subG1 cell fraction, highlighting a hierarchy in the combination effects. Further, a smaller panel of melanoma cell lines, were treated with inhibitors singularly and in combination to test the effects on the expression of principal proteins involved in these two pathways. Gene expression profile was performed to analyse the gene modulation induced by inhibitors to identify new strategies to fight melanoma resistance. The human melanoma cell line Me13 was established in our laboratory from a surgical specimen. Cells were routinely maintained in RPMI medium 1640 supplemented with 8% FBS and 2 mM glutamine. Cells were maintained at 37°C in a water-saturated atmosphere of 7% CO2 in air. 4.5x10^6 cells were seeded in 75 cm^2 flasks in RPMI medium 1640 supplemented with 8% FBS; after 24h, cells were untreated or treated with MAPK and or PI3K/mTOR inhibitors at different concentrations for 8 hours in RPMI medium 1640 supplemented with 4% FBS. Each treatment or combination was performed in triplicate. The cells were collected and RNA extracted.