Triple inhibition of EGFR, Met, and VEGF suppresses regrowth of HGF-triggered, erlotinib-resistant lung cancer harboring an EGFR mutation

Abstract

Introduction: Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer.

Methods: Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene-transfected PC-9 (PC-9/HGF) cells were examined.

Results: Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment.

Conclusion: These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis.

FIGURE 1.

Combined use of crizotinib or TAS-115 with erlotinib reverses resistance to EGFR-TKI induced by exogenous HGF. A and B, PC-9 cells were incubated with or without erlotinib or crizotinib and TAS-115 in the presence or absence of HGF (20 ng/ml) for 72 hours. Cell viability was determined by MTT assay. Bars show SD. The data shown are representative of five independent experiments with similar results. EGFR, epidermal growth factor receptor; EGFR-TKI, EGFR-tyrosine kinase inhibitor; HGF, hepatocyte growth factor; MTT, Thiazolyl Blue Tetrazolium Bromide.

FIGURE 2.

Combined use of crizotinib or TAS-115 with erlotinib reverses resistance to EGFR-TKI induced by endogenous HGF. A, PC-9/Vec and PC-9/HGF cells were incubated with or without erlotinib for 72 hours. Cell viability was determined by MTT assay. Bars show SD. B and D, PC-9/Vec and PC-9/HGF cells were treated with crizotinib or TAS-115 for 72 hours. C–G, PC-9/Vec and PC-9/HGF cells were incubated with or without erlotinib (0.3 μmol/liter) with or without crizotinib (0.3 μmol/liter) and TAS-115 (1.0 μmol/liter) for 72 hours. The data shown are from three independent experiments with similar results. H and I, PC-9/HGF cells were incubated with TAS-115 (1.0 μmol/liter) or crizotinib (0.3 μmol/liter) and/or erlotinib (0.3 μmol/liter) for 1 hour. Thereafter, cell lysates were harvested, and phosphorylation of the indicated proteins was determined by Western blot analysis. EGFR, epidermal growth factor receptor; EGFR-TKI, EGFR-tyrosine kinase inhibitor; HGF, hepatocyte growth factor; MTT, thiazolyl blue tetrazolium bromide.

FIGURE 3.

Crizotinib and TAS-115 inhibits VEGF production by cancer cells and endothelial proliferation. A and B, Tumor cells were incubated with or without HGF (50 ng/ml) in the presence of different concentrations of crizotinib or TAS-115 for 48 hours. Thereafter, supernatants were harvested, and the number of tumor cells was counted. VEGF concentration in the supernatants was determined by ELISA. VEGF levels corrected by the tumor cell number are shown. C and D, HMVECs were incubated in RPMI-1640 medium with 10% FBS (control) or RPMI-1640 medium with 10% FBS in the presence or absence of VEGF (50 ng/ml) with different concentrations of TAS-115, crizotinib, or bevacizumab for 72 hours. Thereafter, cell viability was determined by MTT assay. Bars show SD. The data shown are from three independent experiments with similar results. VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; ELISA, enzyme-linked immunosorbent assay; HMVECs, human dermal microvascular endothelial cells; FBS, fetal bovine serum; MTT, thiazolyl blue tetrazolium bromide.

FIGURE 4.

Treatment with erlotinib plus TAS-115 inhibits angiogenesis in PC-9/HGF tumors in vivo. Nude mice bearing PC-9/Vec or PC-9/HGF tumors (approximately 100 mm³ in size) were administered erlotinib and/or crizotinib and/or TAS-115 orally, once daily for 4 days, and/or bevacizumab intraperitoneally only once. A, The mice were killed on day 4, and the tumors were harvested. B, Numbers of tumor vessels (mean ± SE) determined by CD31 immunohistochemical staining are shown for groups containing five mice each. Representative graphs are shown. The data shown are representative of two independent experiments with similar results. HGF, hepatocyte growth factor.

FIGURE 5.

Treatment with erlotinib plus TAS-115 inhibits the growth of PC-9/HGF tumors in vivo. A and B, Nude mice bearing PC-9/Vec or PC-9/HGF tumors (approximately 100 mm³ in size) were administered erlotinib and/or crizotinib and/or TAS-115 orally once daily and/or bevacizumab intraperitoneally once a week. Tumor volume was measured using calipers. Mean ± SE tumor volumes on day 39 are shown for groups containing five mice each. The data shown are representative of two independent experiments with similar results. HGF, hepatocyte growth factor.

FIGURE 6.

Treatment with erlotinib plus TAS-115 most strongly prevented the regrowth of PC-9/HGF tumors even after cessation of treatment. A, Nude mice bearing PC-9/HGF tumors were treated as described in Figure 5 until day 39. Thereafter, treatment was terminated, and tumor volume was measured until day 49. B, The mice were killed on day 49, and tumors were harvested. Tumor vessels and apoptotic cells were determined by CD31 immunohistochemical and TUNEL staining, respectively. Asterisk indicates P < 0.01. HGF, hepatocyte growth factor; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling.