Only inhibitors of the RAS/RAF/MEK Selonsertib pathway (including the MEK inhibitors PD098059 and U0126 and the Erk2 inhibitor 5-iodotubercidin) showed promising antitumor activity in terms of reduced cell viability, as measured Smad inhibitor by MTT assay. The other drugs, except for the broadly toxic compound staurosporin used as positive control, were nearly unable to reduce cell viability/proliferation, although all compounds were used at doses higher than the described IC50 in order to enhance their activity. A similar drug response was observed for the different samples (Figure 2C shows
a representative one). In line with the melanosphere sensitivity to compounds targeting the MAPK pathways, we observed the activation of this PHA-848125 datasheet signaling pathway with high levels of phosphorylation of Erk and downstream S6 (Figure 2D). We also found high levels of Cyclin-D and undetectable p16 (Figure 2D). These results are in agreement with the frequent alteration of the RAS/RAF/MEK pathway and cell cycle deregulation
found in melanomas. Next, we analyzed DNA sequences of genes whose alterations may contribute to the abnormal pathway activation. As reported in the Additional file 3: Table S1, NRAS was never mutated in the analyzed samples. Instead, despite the ubiquitous Erk phosphorylation found in melanospheres, the BRAF-V600E mutation was detected in samples 1, 2 and 4, BRAF-V600K mutation was found in samples 5 and 8, while samples 3, 6 and 7 displayed wild type BRAF. All samples displayed wild type PTEN. Finally, sequence analysis
of the exon 4 and 5 of GNAQ gene, whose mutations have been associated with wild type BRAF and NRAS melanomas, revealed wild type status in all samples (Additional file 3: Table S1 and Additional file 4) [45]. Treatment with MEK inhibitor PD0325901 results Rapamycin chemical structure in strong antitumor activity against melanospheres The encouraging activity of the MEK inhibitors used in the pathway inhibitor screening (see above) prompted us to study the antitumor effect of the MEK inhibitor PD0325901 on the melanospheres, based on its antitumor activity described in clinical studies [16]. Following 3 day-exposure to PD0325901, at doses comparable with those achieved in vivo, both wild type and mutated-BRAF cells displayed decreased proliferation/viability, with mutated-BRAF samples being more sensitive to the drug (Figure 3A). In order to distinguish the cytostatic from the cytotoxic effect and to unravel the molecular mechanisms of PD0325901 antitumor activity against malenospheres, we first performed cell cycle analysis of control and treated samples. After short exposure (2 days), PD0325901 greatly affected cell cycle progression by determining accumulation of cells in the G1 phase, both in the wild type and mutated-BRAF samples (Figure 3B).