Article Figures & Data
Figures
- Figure 1.
ERBB3 mediates feedback activation of AKT and ERK phosphorylation following trametinib treatment in ERBB3-expressing cell lines.A, NCI-H358 and NCI-H1792 cells were treated with either 50 nmol/L trametinib or 500 nmol/L selumetinib for indicated times, and lysates were probed with the indicated antibodies. B, NCI-H358 cells were treated with DMSO or 50 nmol/L trametinib for 72 hours, and cell lysates were analyzed for levels of phosphorylated RTKs using phospho-RTK arrays. Key RTKs are indicated. C, NCI-H358 and NCI-H1573 cells were treated with 50 nmol/L trametinib for the indicated times, and lysates were probed with the indicated antibodies. D, cells were treated as in B and cell lysates were immunoprecipitated with an antibody to p85. Interaction between p85 and ERBB3 was determined by immunoblotting. In parallel, whole-cell extracts (WCE) were immunoblotted to detect the indicated proteins. E, cells were treated with 1 μmol/L of the pan-EGFR inhibitor afatinib, 50 nmol/L trametinib, or the combination of these two drugs for 48 hours, and lysates were probed with the indicated antibodies. F, NCI-H358 xenografts were treated with the vehicle (control), afatinib 7.5 mg/kg, trametinib 0.6 mg/kg, or the combination at the same doses. Drugs were administered once daily by oral gavage. Tumor volumes were plotted over time from the start of treatment (mean ± SEM). *, P < 0.05 by the Student t test. G, NCI-H1792 and LU99 cells were treated with indicated drug and drug combination as in E. These cells were absent expression of ERBB3. Lysate from NCI-H358 was used as a positive control for ERBB3 expression. All immunoblots are representative of three independent experiments.
- Figure 2.
FGFR1 is dominantly expressed in mesenchymal-like KRAS-mutant lung cancer cell lines. A, expression of ERBB3, E-cadherin, and vimentin protein were analyzed by Western blotting of lysates from KRAS-mutant lung cancer cell lines. Actin is a loading control. Independent experiments were performed three times, and a representative result is shown. B and C, NCI-H358 cells were treated with TGFβ1 (4 ng/mL) or PBS for 14 days in order to induce EMT. B, RNA was extracted from each cell and gene expression profiles were compared. The list of 30 genes most upregulated following TGFβ1 treatment is shown. C, lysates were extracted from each cell and immunoblotted with antibodies against indicated RTKs and EMT markers. Actin was used as a loading control. Independent experiments were performed three times, and a representative result is shown. D, unsupervised hierarchical clustering of 39 KRAS-mutant NSCLC cell lines from the CCLE database. A total of 2,635 genes were analyzed which show more than 5-fold change among cell lines with a median expression value of 6 or more. E and F, scattered plot analysis showing the relationship between ERBB3 and CDH1 (E) and an inverse relationship between FGFR1 and CDH1 (F). P < 0.001, both by linear regression analysis.
- Figure 3.
Trametinib induces feedback activation of FRS2 phosphorylation via FGFR1. A, expression of FGFR1, vimentin, and E-cadherin protein was analyzed by Western blotting of lysates from KRAS-mutant lung cancer cell lines. Actin is the loading control. Independent experiments were performed three times, and a representative result is shown. B, NCI-H358 cells were treated with TGFβ1 (4 ng/mL) for 14 days in order to induce EMT. Then, EMT-induced cells (H358–TGFβ) were transfected with two different siRNAs targeting ZEB1 or scramble siRNA and cultured for 72 hours. Lysates were probed with the indicated antibodies. Independent experiments were performed twice, and a representative result is shown. C, long-term MEK inhibition resulted in strong upregulation of FRS2 phosphorylation. NCI-H1792 and LU99 cells were treated with 50 nmol/L trametinib for the indicated times, and lysates were probed with the indicated antibodies. Immunoblots are representative of three independent experiments. D, FGFR1 mediates FRS2 phosphorylation following trametinib treatment. Cells were transfected with two different siRNAs targeting FGFR1 or scramble siRNA and cultured for 48 hours. Then, media were replaced with or without 50 nmol/L trametinib and cells were treated for an additional 48 hours. Lysates were probed with the indicated antibodies. Independent experiments were performed three times, and a representative result is shown.
- Figure 4.
Downregulation of SPRY4 expression is associated with FGFR1–FRS2 activation. A, trametinib suppresses expression of SPRY4 and DUSP6. NCI-H1792 and LU99 cells were treated with 50 nmol/L trametinib for the indicated times, and lysates were probed with the indicated antibodies. Independent experiments were performed three times, and a representative result is shown. B, FRS2 phosphorylation is not induced by DUSP6 knockdown; however, it is induced by SPRY4 knockdown. LU99 cells were transfected with two different siRNAs against SPRY4, DUSP6, or scramble siRNA for 72 hours. Lysates were probed with the indicated antibodies. Lysate from LU99 cells treated with trametinib at 50 nmol/L for 48 hours was used as a positive control for FRS2 activation. Independent experiments were performed three times, and a representative result is shown. C and D, SPRY4 overexpression negated feedback activation of FGFR signaling. LU99 cells infected with a GFP control or SPRY4 expressing lentiviral plasmid were treated with 50 nmol/L trametinib for 48 hours. Lysates were probed with indicated antibodies (C) or cells were analyzed by FACS to quantify annexin-positive cells (D). The average amount of apoptosis ± SD of 3 independent experiments is shown (P < 0.05 by the Student t test). E, trametinib does not activate FRS2 phosphorylation in epithelial-like KRAS-mutant cancer cells with low FGFR1 expression. NCI-H358 and NCI-H1573 cells were treated with 50 nmol/L trametinib for the indicated times, and lysates were probed with the indicated antibodies. Lysate from LU99 was used as a positive control for FRS2 activation following trametinib treatment. Please note that trametinib downregulated SPRY4 expression in both LU99 and epithelial-like cells. Independent experiments were performed three times, and a representative result is shown. F, H358–TGFβ cells were transfected with two different siRNAs targeting ZEB1 or scramble siRNA and cultured for 48 hours. Then, media were replaced with or without 50 nmol/L trametinib, and cells were treated for an additional 48 hours. Lysates were probed with the indicated antibodies. Independent experiments were performed twice, and a representative result is shown.
- Figure 5.
Combination of trametinib with FGFR inhibition effectively leads to cell death in mesenchymal-like KRAS-mutant lung cancer. A, mesenchymal-like KRAS-mutant NCI-H1792 and LU99 cells were treated with 1 μmol/L of the pan-FGFR inhibitor NVP-BGJ398, 50 nmol/L trametinib, or the combination of these two drugs for 48 hours, and lysates were probed with the indicated antibodies. B and C, cell lines were treated with DMSO, 1 μmol/L afatinib, 1 μmol/L NVP-BGJ398, with or without 50 nmol/L trametinib, and drug was replenished every 72 hours for 6 days. Plates were then stained with crystal violet and imaged. A representative plate of 2 independent experiments is shown. D and E, NCI-H1792 (D) or LU99 (E) cells were treated with drug and drug combinations as in A for 72 hours and analyzed by FACS to quantify annexin-positive cells. The average amount of apoptosis ± SD of 3 independent experiments is shown (P < 0.05 by the Student t test). F, epithelial-like KRAS-mutant NCI-H358 and NCI-H1573 cells were treated as in A. Lysates from LU99 were used as a positive control for the induction of FRS2 phosphorylation following trametinib treatment. Independent experiments were performed three times, and a representative result is shown. G, the levels of phosphorylated ERK after treatment with trametinib or trametinib with NVP-BGJ398 were quantified for 11 mesenchymal-like KRAS-mutant cancer cell lines examined (raw data shown in A and Supplementary Fig. S8A). A paired Student t test was used for comparisons. H, induction of apoptosis by trametinib or the combination of trametinib with NVP-BGJ398 in mesenchymal-like KRAS-mutant lung cancer cell lines. Raw data are shown in D, E, and Supplementary Fig. S8D. A paired Student t test was used for comparisons.
- Figure 6.
The combination of an FGFR inhibitor and a MEK inhibitor leads to tumor regressions in mesenchymal-like KRAS-mutant lung cancer in vivo.A, LU99 xenografts were treated with the vehicle (control), NVP-BGJ398 15 mg/kg, trametinib 0.6 mg/kg, or the combination at the same doses. Drugs were administered once daily by oral gavage. Tumor volumes were plotted over time from the start of treatment (mean ± SEM). *, P < 0.05 by the Student t test.B, waterfall plot showing the percentage change in tumor volume (relative to initial volume) for individual LU99 tumors following 25 days of treatment. Note that data for control group were taken on day 11 due to their growth. C, LU99-derived xenograft tumors from mice treated as indicated were lysed and immunoblotted with the indicated antibodies. D, NCI-H23 xenografts were treated the same way as in A. Tumor volumes were plotted over time from the start of treatment (mean ± SEM). *, P < 0.05 by the Student t test. E and F, tumor regression in a PDX by FGFR and MEK inhibition. E, PDX tumors implanted into NSG mice were lysed and immunoblotted with the indicated antibodies. Lysates from NCI-H358 and LU99 were used as control for epithelial and mesenchymal cells, respectively. F, PDXs were untreated (control), or treated with NVP-BGJ398 15 mg/kg, or trametinib 0.6 mg/kg (n = 3 in each cohort). Once the average of the tumors became more than 1,000 mm3 in the trametinib cohort, the combination of trametinib and NVP-BGJ398 was started at the same doses as the single-agent cohorts. Drugs were administered once daily by oral gavage. Tumor volumes were plotted over time from the start of treatment (mean ± SEM).
- Figure 7.
Expression of mesenchymal markers is associated with FGFR1 expression in patients with KRAS-mutant lung adenocarcinoma. A, unsupervised hierarchical clustering of 75 KRAS-mutant adenocarcinoma extracted from the TCGA dataset is shown using 28 genes listed as EMT-related genes by Kalluri and Weinberg (35). Expression of FGFR1 and ERBB3 in each tumor is also shown at the bottom. FGFR1 expression was significantly higher in mesenchymal-like tumors compared to epithelial-like tumors (P < 0.001 by the Student t test). B, proposed treatment strategies for the treatment of KRAS-mutant lung cancer based on EMT status.
Additional Files
Supplementary Data
- Supplementary Figures S1 - S14, Tables S1 - S5 - Supplementary Figure S1. Feedback activation of ERK signaling and upregulation of AKT signaling following MEK inhibitor treatment in KRAS mutant lung cancers. Supplementary Figure S2. Scattered plot analysis showing inverse relationship between ERBB3 and Vimentin and positive correlation between FGFR1 and Vimentin. p < 0.01, both by linear regression analysis. Supplementary Figure S3. MEK inhibition promotes activation of FRS2 in mesenchymal-like KRAS mutant lung cancer cells. Supplementary Figure S4. FGFR inhibition has minimal effect on downstream signaling in mesenchymal-like KRAS mutant cancer cell lines. Supplementary Figure S5. FGF modestly induces trametinib resistance in mesenchymal-like KRAS mutant lung cancer cell lines. Supplementary Figure S6. Results of microarray analysis showing strong suppression of DUSP6 and SPRY4 mRNA levels following trametinib treatment. Supplementary Figure S7. FGFR1-FRS2 pathway is involved in feedback activation of MAPK signaling in an EMT induced NCI-H358 epithelial-like KRAS mutant cancer cells. Supplementary Figure S8. Combination of MEK inhibition with FGFR inhibition leads to further ERK suppression and apoptosis in mesenchymal-like KRAS mutant lung cancer cell lines. Supplementary Figure S9. Magnitude of apoptosis induced by FGFR and MEK inhibition is related to that which is induced by PI3K and MEK inhibition in mesenchymal-like KRAS mutant lung cancer cell lines. Supplementary Figure S10. Suppression of both MAPK and PI3K signal is not enough to induce cell death in cells resistant to MEK inhibitor with FGFR inhibitor. Supplementary Figure S11. Combination of trametinib with NVP-BGJ398 was tolerable in a mouse xenograft model. Supplementary Figure S12. No relationship between sensitivity to combination of FGFR inhibitor with trametinib and mutations in p53 and/or LKB1 in mesenchymal-like KRAS mutant cancers. Supplementary Figure S13. No relationship between EMT and subgroups of KRAS mutant lung adenocarcinoma defined by a previous study. Supplementary Figure S14. No relationship between sensitivity to combination of FGFR inhibitor with trametinib and amino acid substitutions in KRAS in mesenchymal-like KRAS mutant cancers. Supplementary Table S1. Differentially expressed genes following EMT in the NCI-H358 cell line. Supplementary Table S2. No significant alterations in the expression of FGF ligands and receptors after trametinib treatment in NCI-H1792 cells. Supplementary Table S3. Differentially expressed genes by microarray analysis between control and trametinib-treated NCI-H1792 cells. Supplementary Table S4. Mutational status of cell lines used in this study. Supplementary Table S5. Details of the antibodies.
Volume 6, Issue 7
