Figure 5. Targeting cMET reverses HGF-induced resistance to trametinib in UM cells.

(A) UM001 and UM003 cells were treated with crizotinib for 4 hr, followed by 10 ng/ml of HGF stimulation for 15 min. Phosphorylation of cMET was evaluated by Western blotting of cell lysates with p-cMET antibody. Actin was used for loading. (B) Exponentially growing UM001 and UM003 cells were treated with DMSO or 100 nM trametinib, in combination with 10 ng/ml HGF and/or crizotinib for 3 days (UM001) and 5 days (UM003). Cell viability was determined by AlamarBlue® staining. **P<0.01 based on two-tail Student's t-test assuming unequal variance. (C) Exponentially growing UM001 and UM003 cells were treated 100 nM trametinib, in combination with HGF and/or crizotinib for 3 days (UM001) and 5 days (UM003). Culture medium was changed and new drug and growth factors were added on day 3). Cells were washed and stained with crystal violet. Images were taken (×200 magnification). (D) UM001 cells were treated with DMSO or 100 nM of trametinib, in combination with 10 ng/ml NRG1 (left) or 10 ng/ml HGF (right) for a total of 3 days. In some conditions, 2 ug/ml MK2206 was also added. Cell growth was determined by crystal violet staining. Images were taken at ×200 magnification. (E) UM001 cells were pretreated with vehicle, 100nM trametinib or 2 μM MK2206 overnight. Cells were then stimulated with 10 ng/ml NRG1 (left) or HGF (right) for 1 hr, as indicated. Activation of AKT, ERK1/2, and TSC2 were analyzed by Western blotting.