Figure 5.

Reactivation of MAPK signaling by suppression of DUSP6 promotes ALK-inhibitor resistance in EML4-ALK lung adenocarcinoma. (a) Immunoblot analysis of DUSP6 expression in H3122 cells and CAR sub-lines. Data shown represent three independent experiments. (b,c) Effects of DUSP6 re-expression in H3122 CAR sub-lines on ALK-inhibitor sensitivity, assessed by crystal violet cell-growth assays (b), and on ALK signaling, assessed by immunoblot analysis (c). Data shown represent three independent experiments. (d) Growth-inhibition response (as in Fig. 1g) to 72 h of treatment with crizotinib in H3122 cells expressing scrambled shRNA or either of two independent shRNAs targeting DUSP6. *P < 0.05, at each concentration (unpaired t-test). (e) Left panel, level of knockdown (by immunoblot analysis), and right panel, IC₅₀ values, of cells in d. (f) Immunoblot analysis in H3122 cells treated with shRNA as in d and treated with 250 nm crizotinib for 30 min. (g,h) Immunohistochemistry analysis (IHC) of DUSP6 expression in pre-treatment (Pre-TKI) and post-resistance (Post-TKI) lung adenocarcinoma tumor biopsies from patients treated with ALK inhibitor (n = 25). TKI, tyrosine kinase inhibitor. Each symbol represents an individual patient tumor; small horizontal lines indicate the mean (±s.d.); dotted lines link results for six individual patients with corresponding paired pre-treatment and after-ALK-inhibitor-resistance tumor samples for analysis. P = 0.0026 between each group (pre-treatment versus post-resistance) (unpaired t-test). Representative images of those used to obtain the immunohistochemistry scores are shown in Supplementary Figure 10d. Red arrows indicate tumor cells. Scale bars indicate 200 μm and 400 μm.