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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000170
Original Articles

Triple Inhibition of EGFR, Met, and VEGF Suppresses Regrowth of HGF-Triggered, Erlotinib-Resistant Lung Cancer Harboring an EGFR Mutation

Nakade, Junya MS*; Takeuchi, Shinji MD, PhD*; Nakagawa, Takayuki MS*; Ishikawa, Daisuke MD*; Sano, Takako PhD*; Nanjo, Shigeki MD*; Yamada, Tadaaki MD, PhD*; Ebi, Hiromichi MD, PhD*; Zhao, Lu MS*; Yasumoto, Kazuo MD, PhD*; Matsumoto, Kunio PhD; Yonekura, Kazuhiko PhD; Yano, Seiji MD, PhD*

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Author Information

Divisions of *Medical Oncology and Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; and Tsukuba Research Center, Taiho Pharmaceutical Co., Ltd., Tsukuba, Japan.

Disclosure: Dr. Yano received honoraria from Chugai Pharma and AstraZeneca and research funding from Chugai Pharma. Mr. Nakagawa is an employee of Eisai Co., Ltd. Dr. Yonekura is an employee of Taiho Pharmaceutical Co., Ltd. The remaining authors declare no conflict of interest.

Address for correspondence: Seiji Yano, MD, PhD, Division of Medical Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920–0934, Japan. E-mail:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivitives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.

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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.

Lung cancer is the leading cause of cancer-related deaths worldwide. Recent advances in molecular biology have identified driver oncogenes such as epidermal growth factor receptor (EGFR) mutations or the echinoderm microtubule–associated protein-like 4/anaplastic lymphoma kinase (ALK) fusion gene in non–small-cell lung cancer (NSCLC). In the treatment of NSCLCs harboring these driver oncogenes, the use of EGFR tyrosine kinase inhibitors (TKIs; such as gefitinib and erlotinib) and an ALK inhibitor (such as crizotinib) to block driver oncogene survival signals resulted in marked tumor regression.1–4 Despite these clinical successes, tumors acquire resistance to those agents in almost all cases during the course of therapy.5

Recently, several mechanisms of EGFR-TKI resistance have been identified and classified as follows: (1) alteration of the target EGFR gene (e.g., T790M gatekeeper mutation)6,7; (2) activation of bypass resistance signals (e.g., Met gene amplification,8 hepatocyte growth factor [HGF] overexpression,9 and activation of the nuclear factor-kappa B (NFkB)pathway10 and Gas6-AXL axis)11; and (3) other mechanisms such as transformation to small-cell lung cancer,12–14 epithelial-to-mesenchymal transition,15–17 alteration of microRNA,18 and down-regulation of MED12.19 Previously, we demonstrated that HGF activates, through the Met/PI3K/Akt pathway, bypass signals that trigger resistance; overexpression of HGF was observed more frequently than T790M and Met amplification in tumors from patients with NSCLC who acquired EGFR-TKI resistance in a Japanese cohort.20 These findings indicate that HGF is a clinically relevant target for overcoming EGFR-TKI resistance in EGFR-mutant lung cancer.

Angiogenesis is essential for the progression of various types of solid tumors, including NSCLC. Vascular endothelial growth factor (VEGF) is the most prominent proangiogenic molecule and is considered to be a therapeutic target in NSCLC. We previously reported that overexpressed HGF stimulates VEGF production by means of phosphorylation of Met/Gab1 and promotes tumor growth by stimulating angiogenesis in EGFR-mutant lung cancer models,21 which indicates that HGF is a critical inducer of not only EGFR-TKI resistance but also angiogenesis in EGFR-mutant lung cancer. Therefore, we hypothesized that triple inhibition of the driver signal (EGFR), bypass resistance signal (Met), and angiogenesis (VEGF) may be beneficial for controlling the progression of EGFR-mutant lung cancer with HGF-triggered EGFR-TKI resistance.

EGFR-TKIs, erlotinib, gefitinib, ALK-TKI, crizotinib, and the anti-VEGF antibody bevacizumab have been clinically approved as molecularly targeted drugs in many countries. Crizotinib is known to have activity against Met in addition to ALK and c-ros oncogene 1, receptor tyrosine kinase (ROS1).22,23 In the present study, we investigated the therapeutic effect of triple inhibition against HGF-triggered, EGFR-TKI–resistant lung cancer harboring an EGFR mutation by using clinically available targeted drugs, namely, erlotinib, crizotinib, and bevacizumab. We further assessed the therapeutic potential of erlotinib and TAS-115 (Supplementary Figure 1, Supplementary Digital Content 1,, a novel VEGF receptor 2 (VEGFR-2) inhibitor, which can be orally administered and has Met inhibitory activity, and we compared this doublet treatment with the clinically available triplet. In this study, we demonstrate that the doublet inhibited the progression of HGF-overexpressing EGFR-mutant lung cancer more efficiently than the clinically available triplet treatment. Moreover, TAS-115 combined with erlotinib also controlled tumor growth well and, remarkably, delayed regrowth even after cessation of the treatment.

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Cell Cultures and Reagents

The EGFR-mutant human lung adenocarcinoma cell lines PC-9 (del E746_A750) and HCC827, with deletions in EGFR exon 19, were purchased from Immuno-Biological Laboratories Co. (Gunma, Japan) and from American Type Culture Collection (Manassas, VA) respectively.21 Human HGF-gene transfectant (PC-9/HGF) and vector control (PC-9/Vec) cells were established as previously described.24 These cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. All cells were passaged for less than 3 months before renewal from frozen, early-passage stocks. The human embryonic lung fibroblast cell line MRC-5 was purchased from the Health Science Research Resources Bank (Osaka, Japan). MRC-5 (P30–35) cells were maintained in Dulbecco’s modified Eagle’s medium with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Human dermal microvascular endothelial cells (HMVECs) were incubated in RPMI-1640 medium with 10% FBS (control), RPMI-1640 medium with 10% FBS plus VEGF, or HuMedia-MvG with different concentrations of TAS-115 for 72 hours. Thereafter, cell viability was determined by thiazolyl blue tetrazolium bromide (MTT) assay. Cells were regularly screened for mycoplasma by using MycoAlert Mycoplasma Detection Kits (Lonza, Rockland, ME). The cell lines were authenticated at the laboratory of the National Institute of Biomedical Innovation (Osaka, Japan) by short tandem repeat analysis. TAS-115 was synthesized by Taiho Co., Ltd (Tokyo, Japan). Erlotinib and crizotinib were obtained from Selleck Chemicals (Houston, TX). Bevacizumab was obtained from Chugai Pharma (Tokyo, Japan). Human recombinant HGF was prepared as previously described.24

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Production of HGF and VEGF in Cell Culture Supernatants

Cells (2 × 105) were cultured in a 2 ml of culture medium with 10% FBS for 24 hours, washed with phosphate-buffered saline (PBS), and incubated for 48 hours in the medium supplemented with 10% FBS. In some experiments, HGF was added to the medium. The culture media was harvested and centrifuged, and the supernatants were stored at −80°C until analysis. The concentrations of HGF and VEGF were determined by IMMUNIS HGF EIA (Institute of Immunology, Tokyo, Japan) or Quantikine VEGF enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), respectively, according to the respective manufacturer’s protocol. All samples were run in duplicate. Color intensity was measured at 450 nm by using a spectrophotometric plate reader. Growth factor concentrations were determined by comparison with standard curves. The detection limits for HGF and VEGF were 100 and 31 pg/ml, respectively.

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Cell Viability Assay

Cell growth was measured using the MTT dye reduction method.24 Tumor cells were plated into 96-well plates at a density of 2 × 103 cells/100 ml RPMI-1640 medium with 10% FBS per well. After 24-hour incubation, various reagents were added to each well, and the cells incubated for a further 72 hours, followed by the addition of 50 μl of MTT solution (2 mg/ml; Sigma, St. Louis, MO) to each well and incubation for 2 hours. The media containing MTT solution was removed, and the dark blue crystals were dissolved by adding 100 ml of dimethyl sulfoxide. The absorbance of each well was measured with a microplate reader at test and reference wavelengths of 550 and 630 nm, respectively. The percentage of growth is shown relative to untreated controls. Each reagent concentration was tested at least in triplicate during each experiment, and each experiment was conducted at least three times.

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Antibodies and Western Blotting

Protein aliquots of 25 μg each were resolved by sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad, Hercules, CA) electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad). After washing four times, the membranes were incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 hour at room temperature and overnight at 4°C with primary antibodies to β-actin (13E5), Met (25H2), phospho-Met (Y1234/Y1235;3D7), phospho-EGFR (Y1068), Akt, phospho-Akt (Ser473; 736E11), VEGFR-2 (55B11), phospho-VEGFR-2 (Tyr951;15D2), human EGFR (1 μg/ml), human/mouse/rat Erk1/Erk2 (0.2 μg/ml), and p-Erk1/Erk2 (T202/Y204; 0.1 μg/ml; R&D Systems). After three washes, the membranes were incubated for 1 hour at room temperature with species-specific, horseradish peroxidase–conjugated secondary antibodies. Immunoreactive bands were visualized with Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA) and an enhanced chemiluminescence substrate (Pierce Biotechnology, Rockford, IL). Each experiment was conducted at least three times independently.

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Coculture of Lung Cancer Cells with Fibroblasts or Endothelial Cells

Cells were cocultured in Transwell collagen–coated chambers separated by an 8-mm (BD Biosciences, San Jose, CA) or 3-mm (Corning, Tewksbury, MA) pore size filter. Tumor cells (8 × 103 cells/800 ml) with or without TAS-115 (1.0 μmol/liter) or erlotinib (0.3 μmol/liter) in the lower chamber were cocultured with MRC-5 (1 × 104 cells/300 μl) cells in the upper chamber for 72 hours. The upper chamber was then removed, 200 μl of MTT solution was added to each well, and the cells were incubated for 2 hours at 37°C. The media was removed, and the dark blue crystals in each well were dissolved in 400 μl of dimethyl sulfoxide. Absorbance was measured with an MTP-120 Microplate reader (Corona Electric, Ibaraki, Japan) at test and reference wavelengths of 550 and 630 nm, respectively. The percentage of growth was measured relative to untreated controls. All samples were assayed at least in triplicate, with each experiment conducted three times independently.

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Subcutaneous Xenograft Models

Nude mice (male, 5–6 weeks old) were obtained from Clea (Tokyo, Japan). Cultured tumor cells (PC-9/Vec or PC-9/HGF) were implanted subcutaneously into the flanks of each mouse at 3 × 106 cells/0.1 ml. When tumor volumes reached 100 to 200 mm3, the mice (n = 5 per group) were randomized to the following groups: (1) no treatment (control group), (2) only 50 mg/kg of erlotinib orally, (3) only 25 mg/kg of crizotinib orally, (4) only 100 μg/mouse of bevacizumab intraperitoneally, (5) only 75 mg/kg of TAS-115 orally, (6) erlotinib and crizotinib, (7) crizotinib and bevacizumab, (8) erlotinib and bevacizumab, (9) erlotinib, crizotinib, and bevacizumab, and (10) erlotinib and TAS-115. Each tumor was measured in two dimensions three times a week, and the volume was calculated using the following formula: tumor volume (mm3) = 1/2 (length (mm) × (width (mm))2). All animal experiments complied with the Guidelines for the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center (Approval No. AP-122505).

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Histological Analyses

For detection of endothelial cells (CD31), 5-μm-thick frozen sections of xenograft tumors were fixed with cold acetone and washed with PBS. Then, endogenous peroxidase activity was blocked by incubation in 3% aqueous H2O2 for 10 minutes. After treatment with 5% normal horse serum, the sections were incubated with primary antibodies to mouse CD31 (MEC13.3; BD Biosciences). After probing with species-specific, biotinylated secondary antibodies, the sections were incubated for 30 minutes with avidin–biotinylated peroxidase complex by using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The 3,3′-diaminobenzidine tetrahydrochloride Liquid System (DAKO, Glostrup, Denmark) was used to detect immunostaining. Omission of the primary antibody served as a negative control. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling staining was performed using the Apoptosis Detection System (Promega Corporation, Madison, WI). In brief, 5-μm-thick frozen sections of xenograft tumors were fixed with PBS containing 4% formalin. The slides were washed with PBS and permeabilized with 0.2% Triton X-100. The samples were then equilibrated, and DNA strand breaks were labeled with fluorescein-12-2-deoxy-uridine-5-triphosphate (fluorescein-12-dUTP) by adding the nucleotide mixture and the terminal deoxynucleotidyl transferase enzyme. The reaction was stopped with saline sodium citrate, and the localized green fluorescence of apoptotic cells was detected by fluorescence microscopy (×200). The five areas containing the highest numbers of stained cells within a section were selected for histologic quantitation by light or fluorescent microscopy at a ×400 magnification. All results were independently evaluated by three investigators (JN, TN, and ST).

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Statistical Analysis

Differences were analyzed by one-way analysis of variance. All statistical analyses were carried out using GraphPad Prism Ver. 4.01 (GraphPad Software, Inc., La Jolla, CA). A p value of less than 0.01 was considered statistically significant.

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Effect of Crizotinib and TAS-115 on Bypass Resistance Signals Triggered by Exogenous HGF In Vitro

In the first set of experiments, we examined the effect of crizotinib and TAS-115 on exogenously added HGF-triggered EGFR-TKI resistance in vitro. PC-9 and HCC827 cells are highly sensitive to erlotinib, whereas exogenously added HGF induces resistance to erlotinib in both cell lines. Crizotinib on its own discernibly inhibits the growth of PC-9 cell at high concentrations, consistent with its multikinase activities, and it remarkably sensitizes the cell to erlotinib even in the presence of HGF. TAS-115 does not affect the growth of PC-9 or HCC827 cells at concentrations less than 10 μmol/liter; however, the combined use of TAS-115 with erlotinib reverses HGF-induced resistance in the cell lines in a concentration-dependent manner (Figs. 1A, B and 2A, B, and Supplementary Figure 2, Supplementary Digital Content 2, We previously reported that stromal fibroblasts are a source of exogenous HGF for EGFR-TKI–naive NSCLC and that fibroblast-derived HGF induces resistance to gefitinib and erlotinib in PC-9 and HCC827 cells.25 Crizotinib and TAS-115 reverse the erlotinib resistance of PC-9 cells induced by coculturing with MRC-5 cells (Supplementary Figure 3A, B, Supplementary Digital Content 3, These results indicate that both crizotinib and TAS-115 can reverse the EGFR-TKI resistance induced by exogenous HGF in vitro.

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Figure 1
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Effect of Crizotinib and TAS-115 on Bypass Resistance Signals Triggered by Endogenous HGF

Previously, we showed that HGF is predominantly present in tumor cells of patients with NSCLC with acquired resistance to EGFR-TKIs and that transient HGF-gene transfection into PC-9 cells results in resistance to EGFR-TKIs.20 We, therefore, generated a stable HGF-gene transfectant in PC-9 cells (PC-9/HGF) and assessed the effects against continuously produced endogenous HGF. PC-9/HGF cells secrete high levels of HGF and become resistant to erlotinib, whereas PC-9 or the vector control PC-9/Vec cells do not. Although TAS-115 does not affect the growth of PC-9/HGF cells, crizotinib discernibly inhibits it at high concentrations. The combination of crizotinib or TAS-115 with erlotinib successfully reverses the resistance of PC-9/HGF cells (Fig. 2A–G). Using Western blotting, we examined the effects of crizotinib and TAS-115 on signal transduction in PC-9/Vec and PC-9/HGF cells (Fig. 2H–I). We found that erlotinib inhibits the phosphorylation of EGFR and ErbB3 in PC-9/Vec cells, thereby inhibiting the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2). Met phosphorylation is observed in PC-9/HGF cells but not in PC-9/Vec cells. However, erlotinib fails to inhibit phosphorylation of Akt or Erk1/2 in the presence of HGF. Both crizotinib and TAS-115 suppress the constitutive phosphorylation of Met but not EGFR, ErbB3, or downstream Akt and ERK1/2. HGF stimulates the phosphorylation of Met, but the combined use of crizotinib or TAS-115 with erlotinib inhibits the phosphorylation of Met, Akt, and Erk1/2. These results suggest that crizotinib and TAS-115, when combined with erlotinib, reverse HGF-triggered erlotinib resistance by inhibiting the Met/Gab1/PI3K/Akt pathway.

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Effect of Crizotinib and TAS-115 on Angiogenesis In Vitro and In Vivo

As we reported previously,21 exogenous and endogenous HGF stimulated VEGF production in the PC-9 cancer cell line. Both crizotinib and TAS-115 inhibit VEGF production, presumably because of inhibiting Met activation by HGF (Fig. 3A, B). We also assessed the effect of crizotinib, TAS-115, and bevacizumab on the growth of HMVECs. VEGF promoted HMVEC viability, whereas TAS-115 and bevacizumab, but not crizotinib, inhibit VEGF-stimulated viability of HMVECs in a dose-dependent manner (Fig. 3C, D). We also explored the potential of TAS-115 against VEGFR-2. Western blot analysis indicated that VEGFR-2 is phosphorylated by VEGF stimulation in HMVECs, and TAS-115 and bevacizumab show an inhibitory effect (Supplementary Figure 4, Supplementary Digital Content 4, We next examined the effect on in vivo angiogenesis by using short-term treatment models. Nude mice with established subcutaneous tumors (tumor volume approximately 100 mm3) were treated with erlotinib with or without crizotinib, bevacizumab, and/or TAS-115, and tumor vascularization was determined on day 4 (Fig. 4A, B). In PC-9/Vec tumors, treatment with erlotinib alone, TAS-115 alone, or erlotinib with TAS-115 inhibited vascularization. PC-9/HGF tumors have more vascularization than PC-9/Vec tumors. In PC-9/HGF tumors, treatment with bevacizumab, but not erlotinib or crizotinib, inhibited vascularization. We found that TAS-115 inhibited vascularization more potently than bevacizumab. Under these experimental conditions, treatment with erlotinib plus crizotinib inhibited vascularization. Importantly, erlotinib plus TAS-115 more potently inhibited vascularization, compared with erlotinib plus crizotinib, with or without bevacizumab. These results indicate that TAS-115 has a high potential to inhibit angiogenesis in vivo in EGFR-mutant tumors that produce high levels of HGF. We also confirmed that treatment with crizotinib or TAS-115 inhibits the phosphorylation of EGFR and Met in vivo (Supplementary Figure 5, Supplementary Digital Content 5,

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Figure 4
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Effect of Combined Treatment on Growth of HGF-Overexpressing Tumors In Vivo

Nude mice bearing established subcutaneous tumors (tumor volume approximately 100 mm3) were treated with erlotinib with or without crizotinib, bevacizumab, and/or TAS-115 for 39 days. The treatment was feasible, and no adverse events, including loss of weight, were observed. Tumor volumes on day 39 are shown in Figure 5A and B (tumor growth curves over time are shown in Supplementary Figure 6, Supplementary Digital Content 6, Erlotinib markedly inhibited the growth of PC-9/Vec tumors, but TAS-115 inhibited it only modestly (81.7% and 40%, respectively). In PC-9/HGF tumors, erlotinib alone and crizotinib alone inhibited tumor growth only slightly (30% and 31.9%, respectively). Moreover, bevacizumab alone and TAS-115 alone inhibited tumor growth modestly (67% and 76.6%, respectively). Erlotinib plus crizotinib, with or without bevacizumab, inhibited tumor growth markedly (87.1% and 88.3%, respectively). Importantly, erlotinib plus TAS-115 further inhibited tumor growth significantly (93.7%).

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Effect of Combined Treatment on Regrowth of HGF-Overexpressing Tumors after Cessation of the Treatment

We further evaluated the effect on regrowth of PC-9/HGF tumors after cessation of drug treatment. After 10 days of cessation, tumors treated with erlotinib plus crizotinib with or without bevacizumab regrew to 4.5 and 3.3 times their initial size at the start of cessation, respectively. Tumors treated with erlotinib plus TAS-115 regrew to only 1.7 times their initial size (Fig. 6A). To explore the mechanism of this phenomenon, we again evaluated tumor vascularization on day 49 (10 days after the start of cessation). Consistent with an inhibitory effect against tumor regrowth, vessel density was high (104.6 ± 7.3) and modest (68.6 ± 8.0) in tumors treated with erlotinib plus crizotinib without and with bevacizumab, respectively, whereas vessel density in the tumors treated with erlotinib plus TAS-115 was very low (37.8 ± 3.5; Fig. 6B). However, the number of apoptotic cells was low (1.5 ± 0.6), modest (7.3 ± 5.7), and high (22.7 ± 6.4) in the tumors treated with erlotinib plus crizotinib, crizotinib and bevacizumab, and TAS-115, respectively. These results suggest that erlotinib plus TAS-115 prevents tumor regrowth, even after cessation, by means of sustained inhibition of angiogenesis.

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In the present study, we demonstrated that combined use of erlotinib and TAS-115, a novel angiogenesis inhibitor with Met inhibitory activity, and the use of a triplet of clinically available drugs (such as erlotinib, crizotinib, and bevacizumab) could inhibit the growth of HGF-triggered EGFR-TKI–resistant tumors containing EGFR mutations. Moreover, TAS-115 combined with erlotinib remarkably delayed the regrowth of the HGF-triggered EGFR-TKI–resistant tumors.

Because we reported that HGF is a resistance factor to EGFR-TKI in EGFR-mutant lung cancer,9 HGF has been shown to induce resistance to various molecularly targeted drugs in different types of cancers with driver oncogenes. HGF causes resistance to a selective ALK inhibitor26 and a BRAF inhibitor27 in lung cancer with ALK rearrangement and melanoma with BRAF mutation, respectively, by inducing bypass signals that trigger resistance. Moreover, HGF restores angiogenesis associated with Met expression in tumor vascular endothelial cells and thus induces resistance to sunitinib in various types of cancer.28 These observations indicate that HGF induces resistance to molecularly targeted drugs by multiple mechanisms; therefore, it is an important therapeutic target for circumventing resistance to various molecularly targeted drugs.

HGF and its receptor Met have a close relation with VEGF. Anti-VEGF treatment resulted in a remarkable up-regulation of Met expression in tumors.29 Hypoxia-stimulated expression of VEGF,30 Met,29 and Neuropilin1 (NRP1), a receptor of VEGF, promotes tumor progression.29,31 Furthermore, it was reported that serum levels of HGF and VEGF were inversely correlated with the clinical response to EGFR-TKIs in lung cancer.32–34 In addition, a dual inhibitor of VEGFR-2 and Met (XL-184) was shown to have completely suppressed the invasion and metastasis in a pancreatic cancer model in vivo.29 These studies indicate the rationale for simultaneous inhibition of the HGF-Met and VEGF/VEGFR-2 axes for cancer therapy.

In line with our previous results, we observed that inhibition of both the driver signal (EGFR) and the resistance signal (Met) remarkably suppressed the growth of HGF-triggered EGFR-TKI–resistant tumors in vivo. However, the tumors regrew immediately after the cessation of the dual inhibition, which indicated the presence of cancer cells with proliferating potential that persisted continuously throughout the dual inhibition. Mechanisms of the resistance to dual inhibition should be clarified in the near future.

Additional inhibition of angiogenesis by VEGF neutralization or VEGFR inhibition in addition to dual inhibition (EGFR and Met) could further inhibit growth of HGF-triggered EGFR-TKI–resistant tumor and delay regrowth of the tumors after cessation of the treatment. Bevacizumab in combination with cytotoxic chemotherapy has been shown to prolong progression-free survival in various solid tumors. Our results suggest that the angiogenesis inhibitor in combination with molecularly targeted drugs such as EGFR-TKI and Met-TKI, which directly act on cancer cells, may also delay tumor progression.

It is still controversial whether tumor blood vessels rapidly regrow after cessation of VEGF inhibition. Mancuso et al.35 reported that tumor vasculature regrew within 7 days of cessation of VEGFR inhibitors (given for 7 days) in the RIP-Tag2 pancreatic cancer model and the Lewis lung carcinoma-xenograft model. Bagri et al.36 showed that long-term (7 weeks) treatment with an anti-VEGF antibody prevented the regrowth of tumors compared with control or short-term (2 weeks) treatment, but the effect of the long-term treatment on vasculature regrowth after cessation was not well elucidated. In the present study, we demonstrated that regrowth of tumor vasculature was inhibited even after cessation for 10 days of treatment when, before that, continuous treatment (for 39 days) consisted of bevacizumab plus erlotinib and crizotinib or TAS-115 plus erlotinib; and this inhibition was associated with a high number of apoptotic cells in the tumors and delayed tumor regrowth. These effects were more remarkable with TAS-115 plus erlotinib than with the triplet treatment in our experimental conditions. It is unclear why continuous triple inhibition, especially by TAS-115 plus erlotinib, delayed the recovery of tumor angiogenesis. One possible explanation is that continuous treatment with TAS-115 may impair the function of endothelial progenitor cells expressing VEGFR-2. Further studies with longer follow-up and histochemical analysis will be required to determine the exact mechanisms. On the other hand, VEGFR inhibitory activity may be the disadvantage of TAS-115 for specific cases in which EGFR-TKI resistance caused by only MET amplification. Previous study reported that anti-VEGF therapy elicits malignant progression of tumors to increased local invasion and distant metastasis.37 Therefore, biomarkers for detecting the activities of MET and VEGFRs may be necessary for the optimal use of dual inhibitors for MET and VEGFR.

Inhibition of multiple signaling pathways may cause severe adverse events, especially with continuous admiration of the inhibitors. In our study, 50 mg/kg erlotinib administered daily plus 100 μg/body bevacizumab administered weekly did not show obvious adverse events in nude mice. However, some nude mice treated with daily 50 mg/kg crizotinib plus daily 50 mg/kg erlotinib exhibited severe weight loss and died. Thus, we had to reduce the dose of crizotinib to 25 mg/kg daily when administered along with 50 mg/kg erlotinib. On the other hand, daily administration of 75 mg/kg TAS-115, as expected, inhibited its two targets, Met phosphorylation and angiogenesis, in vivo, and did not show obvious adverse events, including weight loss, even in combination with daily administration of 50 mg/kg erlotinib, suggesting the feasibility of this combined treatment. However, the safety and efficacy of triple inhibition with the triplet of clinically available drugs or with erlotinib plus TAS-115 need to be carefully evaluated in clinical trials.

In conclusion, we demonstrated that triple inhibition of EGFR, Met, and angiogenesis could be achieved by a combination of clinically available drugs (erlotinib, crizotinib, and bevacizumab) or erlotinib and TAS-115 and that the triple inhibition efficiently controlled growth of HGF-triggered, EGFR-TKI–resistant tumors containing EGFR mutations. Clinical trials are warranted to evaluate the efficacy and safety of the triple inhibition in EGFR-mutant lung cancer patients who acquired EGFR-TKI resistance due to HGF.

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This study was supported by Grants-in-Aid for Cancer Research (21390256 to Dr. Yano); Scientific Research on Innovative Areas “Integrative Research on Cancer Microenvironment Network” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (22112010A01 to Dr. Yano); and Taiho Pharmaceutical, Co. Ltd.

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Hepatocyte growth factor; Vascular endothelial growth factor; Epidermal growth factor receptor-tyrosine kinase inhibitor resistance; Lung cancer; Epidermal growth factor receptor mutation

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