Fig. 1
MEK1 and AKT1/2 drive osimertinib resistance. A. Method to establish osimertinib resistant cell lines. B. Verification of osimertinib sensitivity in parental cells and resistant cells by MTT assay. Both parental cells and resistant cells of PC-9, HCC827 and H1975 were exposed to osimertinib at 0, 0.001, 0.01, 0.1, 1, 2.5,5 µM concentrations for 48 h. Normalized cell viability is shown on the Y axis. IC50 values were calculated using GraphPad Prism 7.0. C. Enriched phosphoproteins in resistant cells were analyzed by KEGG. D. Cell lysates from resistant cells and parental cells were loaded to compare phosphorylation of MEK1/2 and downstream ERK as well as AKT/ GSK3β. E. Cell sensitivity to osimertinib after knockdown of MEK1 and AKT1/2 or dual knockdown of MEK1 and AKT1/2 in PC9-Osi and HCC827-Osi cells. After knockdown, cells were treated with osimertinib at 0, 0.001, 0.01, 0.1, 1, 2.5 µM concentrations for 48 h and cell viability were measured by MTT assay. F. Changes in MEK1, ERK and RSK expression by 200 nM osimertinib treatment at the indicated times in PC-9 and HCC827 cells. G. Phosphorylation of AKT and GSK3β in HCC827 and H1975 cells treated with 200 nM osimertinib for 1-48 h. H. Over-expression of MEK1 and AKT1/2 in PC-9, HCC827 and H1975 cells. pUSE-CA-MEK1 and pUSE -CA-AKT1/2 were transfected into PC-9, HCC827 and H1975 cells; after 24 h, cell lysates were collected to detect expression level of MEK1 and AKT by Western blotting. I. Osimertinib sensitivity in control cells and in cells over-expressing MEK1 or AKT1/2. Twenty-four hours after transfection with pUSE-CA-MEK1 and pUSE -CA-AKT1/2, cells were seeded and treated with various concentration of osimertinib for another 48 h. Cell viability was then measured by MTT assay. Quantitative analysis of western blotting bands was performed by Image J software in (D, E, F, G and H). Bars indicate the mean ± SD from 3 independent experiments in (B, E and I). One-way ANOVA with a multiple comparisons test and unpaired t-test were used in (E and I), ns P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001