Hepatocyte growth factor-induced mesenchymal-epithelial transition factor activation leads to insulin-like growth factor 1 receptor inhibitor unresponsiveness in gastric cancer cells

Abstract

Insulin-like growth factor 1 receptor (IGF-1R) inhibitors have been developed as potential therapeutics for cancer treatment; however, the phase III trials have not produced promising overall survival rates. Therefore, understanding the mechanism underlying intrinsic resistance to IGF-1R-targeted agents is urgently required. A number of studies have revealed that activation of alternative receptor tyrosine kinases can mediate resistance to IGF-1R-targeted therapy. The present study investigated whether activated mesenchymal-epithelial transition factor (MET; also known as c-Met and hepatocyte growth factor receptor) confers resistance to an IGF-1R inhibitor (NVP-AEW541) of gastric cancer (GC) cells. NCI-N87 and MGC-803 cells were treated with varying concentrations and combinations of NVP-AEW541, hepatocyte growth factor (HGF) and MET small interfering (si)-RNA or crizotinib (a MET inhibitor). The effects of these agents on cell proliferation and pro-apoptotic events were assessed by Cell Counting Kit-8 assays and flow cytometry. Receptor activation and the downstream signaling pathway were examined using western blot analysis. Expression and/or activation of MET and IGF-1R in 156 GC specimens were evaluated by immunohistochemistry. The results demonstrated that NVP-AEW541 inhibited cell growth, with dephosphorylation of IGF-1R and protein kinase B (AKT), in NCI-N87 and MGC-803 cells. Application of HGF activated MET and the downstream AKT signaling pathways, decreased apoptotic events and restored cell proliferation, which were reversed by MET inhibition via crizotinib or siRNA knockdown. Furthermore, combination therapy of NVP-AEW541 and crizotinib exhibited an enhanced effectiveness in vitro. In addition, >40% of IGF-1R overexpressed GC specimens showed MET expression and activation. In conclusion, HGF-induced MET activation may represent a novel mechanism conferring unresponsiveness to IGF-1R-targeted agents in GC, and inhibition of MET may improve the efficacy of IGF-1R inhibitors.

Keywords: drug resistance; gastric cancer; hepatocyte growth factor; insulin-like growth factor 1 receptor inhibitor; mesenchymal-epithelial transition factor.

Figure 1.

MET-overexpressed GC cell line displays the most unresponsiveness to NVP-AEW541. (A) Six GC cell lines were treated with increasing concentrations of NVP-AEW541 for 72 h, followed by evaluation of cell proliferation and the IC₅₀. Data points indicate the average of 6 replicates and are presented as the mean ± standard error of the mean. (B) Protein lysates were collected from all 6 GC cell lines and analyzed for baseline IGF-1R and MET expression and activation. β-actin served as a loading control. MET, mesenchymal-epithelial transition; IC₅₀, half-maximal inhibitory concentration; IGF-1R, insulin-like growth factor 1 receptor. GC, gastric cancer.

Figure 2.

NVP-AEW541 sensitivity is abrogated by treatment with HGF in NCI-N87 and MGC-803 cells, which can be restored by MET blocking or silencing in the presence of HGF. (A) NCI-N87 cells were treated with NVP-AEW541 (AEW), NVP-AEW541+HGF 20 ng/ml, NVP-AEW541 + crizotinib 0.1 µM (CRIZO) and NVP-AEW541+HGF+crizotinib 0.1 µM, followed by determination of cell proliferation at 2 weeks. (B) MGC-803 cells were treated with NVP-AEW541 (AEW), NVP-AEW541+HGF 50 ng/ml, NVP-AEW541 + crizotinib 0.5 µM (CRIZO) and NVP-AEW541+HGF+crizotinib 0.5 µM, followed by determination of cell proliferation at 2 weeks. Data points indicate the average of 3 replicates and are presented as the mean ± standard error of the mean. *P<0.001 vs. NVP-AEW541-treated control group. (C) NCI-N87 cells were treated with NVP-AEW541 (AEW)+scrambled siRNA, AEW+HGF (20 ng/ml) +scrambled siRNA and AEW+HGF+MET siRNA, followed by determination of cell proliferation at 1 week. Data points indicate the average of 3 replicates and are presented as the mean ± standard error of the mean. (D) MGC-803 cells were treated with NVP-AEW541 (AEW)+scrambled siRNA, AEW+HGF (50 ng/ml) +scrambled siRNA, and AEW+HGF+MET siRNA, followed by determination of cell proliferation at 1 week. Data points indicate the average of 3 replicates of three and are presented as the mean ± standard error of the mean. (E) NCI-N87 and MGC-803 cells were transfected with MET or scrambled siRNA. The indicated gene expression was measured by western blotting following 3 days of 20 nM siRNA transfection. MET was significantly knocked down by the indicated siRNA. MET, mesenchymal-epithelial transition; HGF, hepatocyte growth factor; siRNA, small interfering RNA.

Figure 3.

HGF decreases the number of apoptotic events in NVP-AEW541-treated NCI-N87 and MGC-803 cells. (A) NCI-N87 cells were treated for 18 h with media, NVP-AEW541 15 µM (AEW), NVP-AEW541+HGF 50 ng/ml and NVP-AEW541+HGF+crizotinib 2 µM (CRIZO), and then analyzed by flow cytometry. (B) MGC-803 cells were treated for 18 h with media, NVP-AEW541 20 µM (AEW), NVP-AEW541+HGF 100 ng/ml and NVP-AEW541+HGF+crizotinib 10 µM (CRIZO), and then analyzed by flow cytometry. **P<0.05. The results are the average of triplicate experiments and data are presented as the mean ± standard deviation. HGF, hepatocyte growth factor.

Figure 4.

HGF-dependent phosphorylation of MET reactivates AKT, which is attenuated by crizotinib via MET inhibition. NCI-N87 cells were treated for 3 h with NVP-AEW541 15 µM, and/or crizotinib 20 µM with or without the addition of HGF 50 ng/ml for the final 30 min. MGC-803 cells were treated for 3 h with NVP-AEW541 15 µM, IGF-1 50 ng/ml and/or crizotinib 10 µM with or without the addition of HGF 10 ng/ml for the final 30 min. Whole cell lysates were analyzed by SDS-PAGE, followed by western blot analysis. HGF-induced MET phosphorylation restored AKT signaling in NVP-AEW541-treated cells; a combination of NVP-AEW541 and crizotinib treatment blocked the activation of AKT and MAPK in HGF-stimulated NCI-N87 cells, and AKT in HGF-stimulated MGC-803 cells. HGF, hepatocyte growth factor; MET, mesenchymal-epithelial transition; AKT, protein kinase B; IGF-1, insulin-like growth factor 1; MAPK, mitogen-activated protein kinase.

Figure 5.

Combination of NVP-AEW541 (AEW) with crizotinib (CRIZO) results in a significant increase in cell growth toxicity. (A) NCI-N87 and (B) MGC-803 cells were treated with increasing concentrations of the indicated drugs for 72 h, followed by evaluation of cell viability by Cell Counting Kit-8. Data points indicate the average of 6 replicates and is presented as the mean ± standard error of the mean.

Figure 6.

Immunohistochemical detection of IGF-1R, MET and p-MET in GC specimens. Typical examples of immunohistochemical staining for IGF-1R, MET and p-MET. Magnification, ×200. GC, gastric cancer; IGF-1, insulin-like growth factor 1 receptor; MET, mesenchymal-epithelial transition; p-, phosphorylated.