Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells

Abstract

There is a pressing need to identify new drug targets and novel approaches for treatment of non-small-cell lung carcinoma (NSCLC). Members of the epidermal growth factor receptor (EGFR) and Met receptor families have been identified as important molecular targets for NSCLC. Two EGFR tyrosine kinase inhibitors (TKIs; erlotinib and gefitinib) are in current clinical use, but a majority of patients do not respond to these targeted therapies. We used receptor TK (RTK) capture arrays to identify receptors active in NSCLC cell lines. As Met and ErbBs were active, we explored the potential therapeutic advantage of combined targeting of Met with ErbB receptor family inhibitors for treatment of NSCLC. We found that Met physically interacts with both EGFR and Her2 in a NSCLC cell line with overexpression/overactivation of Met. Combined use of a dual EGFR/Her2 inhibitor with a Met inhibitor yields maximal growth inhibition compared with the use of EGFR and/or Met inhibitors. This suggests that simultaneous inhibition of multiple RTKs may be needed to effectively abrogate tumour cell growth. Phosphoproteomic analysis by RTK capture arrays may be a valuable tool for identifying the subset of tumours with functional receptor activation, regardless of mechanism.

Figure 1

Activation of Met and response to GW2974 in H441 cells. (A) Multiple RTKs are activated in H441 and H1666 cells in full serum conditions. Whole cell extracts (200 μg) were incubated with RTK capture array membranes. RTK activation was determined by probing with phosphotyrosine antibody conjugated to horse-radish peroxidase. Paired spots correspond to 1: EGFR; 2: Her2; 3: Her3; 4: Her4; 5: Mer; 6: Met; 7: MSPR; 8: Flt3; 9: Ret; and 10: Dtk. The four sets of duplicate spots at each corner of the RTK array membrane serve to orient and align the membrane to identify and correlate the positive set of spots to individual RTKs. (B) Sensitivity of cell lines to GW2974. Cells were treated with indicated concentrations of GW2974 for 5 days, and cell proliferation was measured using a WST-1 colorimetric assay. The GI₅₀ of H1666 is 0.1 μM compared with 8.6 μM for H441 cells. Error bars represent s.d. of three data sets. Representative data are shown from multiple experiments. (C) Dose-dependent inhibition of EGFR-dependent signalling in H1666 cells and Met-dependent signalling in H441 cells by GW2974. Whole cell extracts were made from cells treated with GW2974 at indicated concentrations for 5 days as in panel B, and subjected to immunoblotting with the indicated antibodies. Arrows indicate respective protein bands. β-Tubulin was used as a loading control. GW2974 concentration in lanes 1: 1 μM; 2: 0.5 μM; 3: 0.25 μM; 4: 0.125 μM; 5: 0.0625 μM; 6: 0 μM; 7: 25 μM; 8: 12.5 μM; 9: 6.25 μM; 10: 3.125 μM; 11: 1.56 μM and 12: 0 μM.

Figure 2

GW2974 inhibits constitutively activated Met, Her2 and Her3 in H441 cells. (A) GW2974 abrogates the activation of multiple receptors in H441 cells. Cells were starved for 24 h and then treated for 2 h with GW2974 before incubation with EGF (100 ng ml⁻¹) for 10 min. Whole cell extracts (500 μg) were analysed on each RTK array membrane, and activation status of receptors was assessed using antiphosphotyrosine antibody and numbered as in Figure 1A. 1: EGFR; 2: Her2; 3: Her3; 6: Met; 9: Ret; and 11: VEGFR-2. (B and C) GW2974 inhibits Met signalling and EGF-activated signalling. The extracts analysed in panel A were analysed by immunoblotting with indicated antibodies. M and numbers in panel B indicate molecular weight marker for proteins. β-Tubulin was used as a loading control.

Figure 3

Cross-talk between Met and EGFR family receptors. GW2974 inhibits Met activation in a dose-dependent manner in H441 cells (A), but not in the absence of EGFR family receptors in 32D/Met cells (B). (A) GW2974 inhibits Met activation in the presence or absence of HGF. Cells were serum starved for 24 h followed by treatment with either vehicle (DMSO) or GW2974 or a Met inhibitor (PHA) for 2 h before activation with HGF (50 ng ml⁻¹) for 30 min. Whole cell extracts were made and subjected to western blot with indicated antibodies. (B) GW2974 and gefitinib do not inhibit HGF-dependent activation of Met in stably transfected 32D cells with Met (32D/Met). Cells were treated as in panel A. β-Tubulin was used as a loading control.

Figure 4

Met interacts with both EGFR and Her2, and the interaction is inhibited by GW2974. (A and B) Whole cell extracts were made from H441 cells treated with GW2974 for 2 h or DMSO vehicle control. Extracts (1 mg) were subjected to immunoprecipitation with anti-Her2 (A) or with anti-EGFR (B) antibodies followed by immunoblotting with indicated antibodies. Immunoprecipitation with IgG was used as a negative control in each experiment. Numbers on the side of panels indicate molecular weight marker. The middle panels of A and B are from the ErbB receptor region of the blot. (C) Whole cell extracts were immunoblotted with anti-Met antibody to indicate equal amounts of Met in each treatment. β-Tubulin was used as a loading control.

Figure 5

Met shRNA downregulates ErbB family receptor activation. Whole cell extracts were made from control (H441-pLKO.1) and Met shRNA (H441-Met shRNA) cells. (A) Whole cell extracts (200 μg) were analysed on each RTK array membrane, and activation status of receptors was assessed using antiphosphotyrosine antibody and numbered as in Figure 1A. 1: EGFR; 2: Her2; 3: Her3; 6: Met; 7: MSRP; and 9: Ret. (B) The cell extracts analysed in panel A were analysed by immunoblotting with indicated antibodies. β-Tubulin was used as a loading control. (C) Cell extracts (500 μg) used in panel A were subjected to immunoprecipitation using either anti-Her2 or anti-Her3 antibodies followed by immunoblotting with indicated antibodies. IgG was used as a negative control.

Figure 6

Synergistic inhibition of cell proliferation by combination of a Met inhibitor (PHA) with either gefitinib or GW2974 in H441 cells. Cells were treated with indicated concentrations of gefitinib and PHA (Ai–iii) or GW2974 and PHA (Bi–iii) either alone or in combination at 0.125–3 μM concentration range for 5 days. For combination of drugs, a fixed ratio of 1 was used. Cell proliferation was assessed as in Figure 1B. Data were plotted as dose–response regression curves using Xlfit program. Each error bar represents the s.d. from three data sets. Ai–Bi: H441 cells; Aii–Bii: H441-pLKO.1 cells; and Aiii–Biii: H441-Met shRNA cells. (C) Whole cell extracts were made from cells treated with drugs for 5 days as indicated, and were subjected to western blot analysis with indicated antibodies. β-tubulin was used as a loading control.