Figure 3. OSM pathway activation protects cells from targeted drug-induced apoptosis and is dependent on an OSMRs/JAK1/STAT3 pathway.

(A) Six NSCLC cell lines were treated with DMSO or a targeted drug: H23 and H2009 (KRAS-mut) with selumetinib (10 μM), H3255 and HCC4006 (EGFR-mut) with erlotinib (100 nM), H3122 (EML4-ALK fusion) and HCC78 (SLC34A2-ROS1 fusion) with crizotinib (500 nM), with or without pretreatment with rhOSM (10 ng/mL) for at least 2 weeks. Cells were stained with crystal violet 7 to 10 days after treatment. Quantification measured by MTT assay (relative to DMSO) is shown at bottom right of each plate.

(B) H2009 cells cultured with or without rhOSM (10 ng/mL) were treated with serial dilutions of selective JAK1 inhibitor filgotinib, JAK1/2 and TBK1 inhibitor momelotinib, or STAT3 inhibitor stattic for 48 hours. Whole-cell proteins were harvested and analyzed by Western blotting. OSMR signals were detected at 180 kD (arrow head).

(C) Six NSCLC cell lines treated or not treated with rhOSM (10 ng/mL) for at least 2 weeks were transfected with siRNA specific for OSMR/LIFR, JAK1, STAT3, or scrambled negative control for 48 hours. The cells then were treated with one of the targeted drugs, as follows, with or without filgotinib: H23 and H2009, selumetinib (10 μM); H3255 and HCC4006, erlotinib (100 nM); H3122 and HCC78, crizotinib (500 nM). After 7 to 10 days of treatment, cell viabilities compared to control cells (without siRNA and drugs) were measured by MTT assay. ^*P <0.05.

(D) Six NSCLC cell lines were treated with the indicated targeted drugs with or without rhOSM for 72 hours, and lysates obtained from cancer cells were examined by Western blotting with the indicated antibodies. Sel, selumetinib; Fil, filgotinib; Erl, erlotinib; Cri, crizotinib.