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Soluble c-Met receptors inhibit phosphorylation of c-Met and growth of hepatocyte growth factor: c-Met–dependent tumors in animal models

Angela Coxon, Karen Rex, Susanne Meyer, Jianling Sun, Jilin Sun, Qing Chen, Robert Radinsky, Richard Kendall and Teresa L. Burgess
DOI: 10.1158/1535-7163.MCT-08-1032 Published May 2009

Abstract

c-Met is a receptor tyrosine kinase frequently overexpressed or amplified in many types of human cancers. Hepatocyte growth factor (HGF, also known as scatter factor) is the only known ligand for c-Met. In this study, soluble human and murine c-Met receptor-Fc fusion proteins were generated and were shown to bind to human and murine HGF as measured by fluorescence-activated cell sorting and surface plasmon resonance (Biacore) assays. Also, both human and murine c-Met-Fc showed activity in functional cell assays, inhibiting HGF-induced c-Met phosphorylation in PC3 and 4T1 cells, respectively, and inhibiting HGF-driven cellular invasion in a dose-dependent manner. Pharmacokinetic analysis showed that both reagents were suitable for in vivo testing. Systemic administration of human c-Met-Fc significantly inhibited tumor growth in the human HGF-dependent U-87 MG xenograft model at daily doses of 30 or 100 μg (P < 0.0001). Similarly, murine c-Met-Fc, at 100 μg daily, significantly inhibited tumor growth in the murine HGF–dependent CT-26 syngeneic tumor model (P < 0.002). Human and murine c-Met-Fc seemed to be well-tolerated in animals. In conclusion, both mouse and human versions of c-Met-Fc effectively block HGF-induced activation of c-Met and inhibit growth of tumor xenografts, providing further evidence that c-Met is an important target for oncology therapeutics.[Mol Cancer Ther 2009;8(5):1119–25]

  • c-Met
  • hepatocyte growth factor/scatter factor
  • tumor xenograft

Introduction

c-Met, the receptor for hepatocyte growth factor (HGF, also known as scatter factor), is a well-characterized receptor tyrosine kinase that mediates a variety of normal cell functions including proliferation, survival, motility, migration, angiogenesis, and morphogenesis. c-Met is expressed in epithelial tissues, and HGF is expressed in mesenchymal tissues, and together they play an essential role in embryonic development (1).

Extensive evidence suggests that dysregulation of the HGF:c-Met pathway plays an important role in a variety of human malignancies, and that HGF activation of c-Met occurs through both paracrine and autocrine pathways.1 Activating point mutations in the c-Met receptor have been reported in some human malignancies including renal carcinoma, non–small cell lung cancer, and small cell lung cancer (2–7). Expression of recombinant mutant c-Met in cell lines has been shown to lead to constitutive phosphorylation of the receptor and activation of downstream signaling events (6, 8–12), and in some cases, transformation with mutant c-Met was shown to be ligand dependent (13). Some of these recombinant c-Met mutants have been shown to cause cancer in animal models (10, 14). Coexpression of HGF and c-Met in cells leads to activation through an autocrine loop, a mechanism that has been described in gliomas, osteosarcomas, breast cancer, and prostate cancer (15, 16). In addition, paracrine HGF expression was shown to drive growth of c-Met–expressing human tumor xenografts in a transgenic mouse model (17).

Recent preclinical data have shown that the HGF:c-Met pathway can be inhibited by fragments of HGF (18, 19), decoy receptors and soluble c-Met domains that prevent receptor dimerization (19–23), small-molecule tyrosine kinase inhibitors (24–28), and antibodies directed against c-Met or HGF (29–37). Several of these agents are currently being tested in clinical trials (36–43).

In this study, we generated soluble forms of both human and murine c-Met receptor–Fc fusion proteins and evaluated their binding to either human or murine HGF. We also evaluated their effect on HGF-induced c-Met phosphorylation in cells, HGF-driven cellular invasion, and HGF-dependent tumor growth in mouse xenograft models.

Materials and Methods

Cells and Culture

Chinese hamster ovary cells, 293T cells, the PC3 human prostate carcinoma cell line, the 4T1 mouse breast cancer cell line, the HGF-expressing U-87 MG human glioblastoma cell line, the HGF-expressing CT-26 murine colon carcinoma cell line, and the NMuMG normal mouse mammary epithelial cell line were obtained from the American Type Culture Collection. Cells were grown as monolayers using standard cell-culture conditions.

Generation of Human and Murine c-Met-Fc Fusion Proteins and Human and Mouse Avidin-HGF Proteins

The human c-Met-Fc construct was generated by fusing the extracellular domain of human c-Met (amino acid residues 1–914) to the 226-amino acid Fc fragment of human IgG1. Similarly, the murine c-Met-Fc construct was generated by fusing the extracellular domain of murine c-Met (amino acid residues 1–929) to the 226-amino acid Fc fragment of human IgG1. The resulting constructs were inserted into a mammalian expression vector and transfected into Chinese hamster ovary cells, and c-Met-Fc fusion proteins expressed and purified using standard methods.

Human and murine HGF were subcloned into a mammalian expression vector (pCep4avidin-N) containing a cDNA sequence encoding recombinant chicken avidin to create avidin-HGF fusion/chimeric proteins as already described (29). The resulting constructs were transiently expressed in 293T cells using the Lipofectamine transfection method following the manufacturer's instructions (Invitrogen).

Binding of c-Met-Fc proteins to HGF

Fluorescence-Activated Cell Sorting Assay

Conditioned media from 293T cells expressing the avidin-HGF fusion proteins was collected and applied to biotin-coated polystyrene beads according to the manufacturer's instructions (Spherotech, Inc.). Purified c-Met-Fc was applied to the bead complex and bound c-Met-Fc was detected using a phycoerythrin-labeled goat anti-human F(ab")2 antibody according to the manufacturer's instructions (Southern Biotech Association). The bead complexes were then subjected to fluorescence-activated cell sorting analysis using a FACScan (Becton Dickinson) flow cytometer.

Biacore Assay

A surface plasmon resonance Biacore 3000 (Biacore, Inc.) affinity assay was used to analyze whether human or murine c-Met-Fc disrupted binding of human or murine HGF to c-Met-Fc. The assay was done according to the manufacturer's instructions. Human or murine c-Met-Fc was immobilized on a CM5 chip to form the receptor surfaces. Then, human or murine HGF (1 nmol/L) was incubated with various concentrations (10−13–10−7 M) of c-Met-Fc before injection over the immobilized receptor surfaces, and the signal for HGF binding to c-Met-Fc on the chip detected. Maximum (100%) HGF binding signal was determined in the absence of c-Met-Fc in solution. Because only free HGF molecules bound to c-Met-Fc surfaces, decreased binding with increasing concentrations of c-Met-Fc indicated binding of HGF to c-Met-Fc in solution. EC50 values were determined using the one-site competition model in GraphPad Prism software (GraphPad).

Functional Cell Assays

c-Met Autophosphorylation Assay

The effect of human or murine c-Met-Fc on HGF-mediated c-Met autophosphorylation was evaluated using a quantitative electrochemiluminescent immunoassay as described (25). Briefly, PC3 or 4T1 cells were plated in 96-well plates in growth media for 24 h then serum starved for 18 to 20 h. Human or murine HGF (100 ng/mL) was preincubated with various concentrations (0–1,000 nmol/L) of c-Met-Fc or anti-HGF monoclonal antibody (R&D Mab 294) at 4°C for 16 h. PC3 or 4T1 cells were treated with the human HGF or murine HGF mixture, respectively, at 37°C for 10 min. Cells were washed once with PBS and lysed [1% Triton X-100, 50 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, 300 μmol/L Na3OV4, and protease inhibitors]. Cell lysates were incubated with a biotin-labeled goat-anti-c-Met antibody (BAF358, for human c-Met; BAF527, for murine c-Met; R&D Systems) for capture, followed by a mouse anti-phosphotyrosine antibody (4G10; Upstate) and a BV-tag–labeled anti-mouse IgG (BioVeris, Inc.) as the detection antibody. Levels of c-Met phosphorylation were then measured on a BioVeris M-Series instrument.

NMuMG Matrigel Invasion Assay

In this study, we used the normal mouse mammary epithelial cell line (NMuMG) Matrigel invasion assay, with Matrigel-coated FluoroBlok Invasion System inserts (BD Bioscience, Inc.), to evaluate the effect of c-Met-Fc fusion proteins on cell migration and invasion. The assay was done as described (29), using cell-attractant medium containing 50 ng/mL 293T-derived HGF. c-Met-Fc was diluted in attractant media, and duplicate samples for each condition were evaluated. The cell suspension was added to the upper chamber and incubated at 37°C for 18 h. Cells were then stained with Cell Tracker Green (Molecular Probes) to detect migrated cells on the bottom of the chamber filter. Fluorescent intensities of migrated cells were quantified with a Victor 2 model 1420 Multilabel Counter plate reader (Wallac, PerkinElmer) using the bottom-reading mode.

Animals

CD1 athymic nude (nu/nu) and BALB/c female mice (Charles River Laboratories) were used in pharmacokinetic and efficacy experiments. Animals were housed in sterilized cages on a 12-h light/dark cycle with food and water provided ad libitum in compliance with all Association for Assessment and Accreditation of Laboratory Animal Care specifications. All procedures were conducted in accordance with federal animal care guidelines and were preapproved by the Amgen Institutional Animal Care and Use Committee.

Pharmacokinetic Analysis

Eight-week-old female CD1 nu/nu mice or female BALB/c mice were injected either i.p. or s.c. with 50 μg per mouse human c-Met-Fc or 100 μg per mouse murine c-Met-Fc, respectively. At various time points after injection, mice were sacrificed (3 animals per time point), blood was collected, and plasma was prepared. Levels of intact c-Met-Fc were determined by ELISA. Delphia assay plates (Wallac) were coated with 5 mg/mL human Fc polyclonal antibody (Pierce). Both human and murine c-Met-Fc were used as standards. Plasma samples were added to Fc-coated plates, and c-Met binding to the plate was detected with biotinylated anti-human or anti-murine c-Met antibodies (R&D Systems) and Eu-streptavidin (Wallac). Signal enhancement and time-resolved fluorescence detection were done using the Victor Multilabel Counter plate reader (Wallac). The concentrations of c-Met were determined using the linear equation (Excel Version 2.0.6; Microsoft).

Tumor Xenograft Models

Tumor Growth and Measurement

To evaluate in vivo activity of human c-Met-Fc, 5- to 6-wk-old female CD1 nu/nu mice (10 animals per group) were injected with 5 × 106 U-87 MG human glioblastoma cells in 0.2 mL DMEM, s.c. in the right flank. Mice were treated with human c-Met-Fc (30 or 100 μg per mouse) or human Fc control (300 μg per mouse), i.p. daily, starting on day 2. To evaluate in vivo activity of murine c-Met-Fc, 5 to 7-week-old female BALB/c mice (10 animals per group) were injected with 3 × 105 CT-26 mouse colon carcinoma cells in 0.2 mL DMEM, s.c. in the right flank. Mice were treated with murine c-Met-Fc (100 μg per mouse) or human Fc control (100 μg per mouse), s.c. daily, starting on day 2. Tumor size and body weights were determined twice a week. Tumors were measured using Pro-Max Digital Ultra Calipers (Fred Fowler Co.) and tumor volumes calculated as length × width × height in mm3.

Statistical analysis

Tumor volumes are expressed as means ± SE. Data were analyzed by repeated-measures Analysis of Variance followed by Scheffé's post hoc test to determine P values. All data analyses were done using StatView software v5.0.1 (SAS Institute, Inc.).

Results

Generation and Biochemical Characterization of c-Met-Fc Fusion Proteins

We generated human and murine c-Met-Fc fusion proteins in Chinese hamster ovary cells and human and murine HGF in 293T cells, and then evaluated binding of c-Met-Fc to HGF by fluorescence-activated cell sorting analysis (Fig. 1A and B). Both human and murine HGF bound to either human or murine c-Met-Fc. Next, we used a Biacore solid-phase assay to assess whether human or murine c-Met-Fc disrupted binding of human or murine HGF to immobilized c-Met-Fc receptors. Preincubation of either human or murine HGF with increasing concentrations of human c-Met-Fc resulted in decreased interaction of either human HGF with immobilized c-Met-Fc receptors (EC50, 0.56 nmol/L) or murine HGF with immobilized human c-Met-Fc receptors (EC50, 0.51 nmol/L; Fig. 1C). Similar results were observed for murine c-Met-Fc disruption of either human HGF interaction with immobilized murine c-Met-Fc receptors (EC50, 0.42 nmol/L) or murine HGF interaction with immobilized murine c-Met-Fc receptors (EC50, 0.47 nmol/L; Fig. 1D). Thus, both human and murine HGF bound equally well to either human or murine c-Met-Fc.

Figure 1.
Figure 1.

Human and murine c-Met-Fc bind to both human and murine HGF. A and B, fluorescence-activated cell sorting analysis. Conditioned media from 293T cells containing either human or murine HGF was collected and applied to biotin-coated polystyrene beads. Either human or murine c-Met-Fc was applied to the bead mixture and binding detected using a phycoerythrin-labeled goat anti-human IgG by fluorescence-activated cell sorting analysis. C and D, Biacore measurements. Inhibition of HGF:c-Met binding was measured over different concentrations of either human or murine c-Met-Fc using a Biacore assay. Hu HGF, human HGF; mu HGF, murine HGF.

Human and Murine c-Met-Fc Neutralize HGF-Mediated Cellular Activities and Inhibit HGF-mediated c-Met Phosphorylation

Next, we tested the effects of the c-Met-Fc fusion proteins on c-Met phosphorylation in cells. Neither PC3 human prostate carcinoma nor 4T1 mouse breast cancer cells express HGF; therefore, the assay mimicked a paracrine model of ligand-mediated receptor activation. We used PC3 cells to measure human c-Met-Fc inhibition of HGF-mediated c-Met phosphorylation and 4T1 cells to measure murine c-Met-Fc inhibition of HGF-mediated c-Met phosphorylation. Preincubation of HGF with either human or murine c-Met-Fc led to dose-dependent inhibition of human HGF-mediated c-Met phosphorylation in PC3 cells (Fig. 2A) and murine HGF-mediated c-Met phosphorylation in 4T1 cells (Fig. 2B). In contrast, neither human Fc nor mouse Fc inhibited HGF-mediated phosphorylation of c-Met, whereas a monoclonal anti-HGF antibody inhibited c-Met phosphorylation in this assay (data not shown; Fig. 2B).

Figure 2.
Figure 2.

Human and murine c-Met-Fc inhibit HGF-mediated c-Met phosphorylation in cells. A, inhibition of human HGF-induced c-Met phophorylation in PC3 human prostate carcinoma cells. B, inhibition of murine HGF-induced c-Met phosphorylation in 4T1 mouse breast cancer cells. Inhibition of HGF-mediated phosphorylation in PC3 or 4T1 cells was determined using a bead-based-ECL immunoassay. Columns, mean; bars, SD. Human Fc control, Fc fragment of human IgG1; murine Fc control, Fc fragment of mouse IgG1.

Invasion Assay

HGF induces c-Met–dependent migration and invasion in a variety of epithelial-derived tumor cell lines. Several studies have suggested that this cellular activity is responsible for the invasion of tumor cells in breast cancer (39, 43). We used the quantitative NMuMG cell–based Matrigel invasion assay to evaluate the effect of the c-Met-Fc fusion proteins on cell invasion. Both human and murine c-Met-Fc blocked both human and murine HGF–induced cell invasion (Fig. 3). Incubation of 50 ng/mL human HGF with 11, 33, or 100 μg/mL human c-Met-Fc led to inhibition of cellular invasion by 12%, 8%, and 55%, respectively; and incubation of 50 ng/mL human HGF with 11, 33, or 100 μg/mL murine c-Met-Fc led to inhibition of cellular invasion by 54%, 44%, and 64%, respectively. Similarly, incubation of 50 ng/mL murine HGF with 11, 33, or 100 μg/mL human c-Met-Fc led to inhibition of cellular invasion by 0%, 12%, and 68%, respectively; and incubation of 50 ng/mL murine HGF with 11, 33, or 100 μg/mL murine c-Met-Fc led to inhibition of cellular invasion by 0%, 23%, and 44%, respectively. Neither human nor murine Fc had an effect on cell invasion in this assay (data not shown).

Figure 3.
Figure 3.

Human and murine c-Met-Fc block human and murine HGF-induced invasion of NMuMG cells. A, human HGF incubated with human c-Met-Fc; B, human HGF incubated with murine c-Met-Fc; C, murine HGF incubated with human c-Met-Fc; D, murine HGF incubated with murine c-Met-Fc. Inhibition of NMuMG cells invading through a Matrigel-coated filter was measured in the presence of 50 ng/mL HGF with or without different concentrations of either human c-Met-Fc or murine c-Met-Fc in the cell attractant. Migrated cells were stained with Cell Tracker Green and quantified by fluorescence. Duplicate samples were evaluated for each condition.

Pharmacokinetic Profile of Human and Murine c-Met-Fc in Mice

A single dose of human c-Met-Fc (50 μg) was administered to CD1 nu/nu mice by either i.p. or s.c. injection. Mice were sacrificed at various time points for determination of plasma levels of human c-Met-Fc. I.p. injection resulted in greater exposure compared with s.c. injection, as shown by the human c-Met-Fc concentration versus time curves (Fig. 4A and B). The half-life (t1/2) for i.p. and s.c. administration was 27 and 37 h, respectively. Similar results were observed after administration of murine c-Met-Fc (100 μg per mouse) to BALB/c mice by either i.p. or s.c. injection (Fig. 4C and D), with t1/2 of 45 and 32 hours for i.p. and s.c. administration, respectively. These results were used for determining the dosing schedule used in the mouse xenograft studies.

Figure 4.
Figure 4.

Pharmacokinetic profiles of c-Met-Fc proteins in mice. Plasma levels of human c-Met-Fc after administration of a single dose to athymic nude mice by i.p. (A) or s.c. (B) injection. Plasma levels of murine c-Met-Fc after administration of a single dose to BALB/c mice by i.p. (C) or s.c. (D) injection. Mice were injected with 100 μg per mouse of either human (nude mice) or murine (BALB/c mice) c-Met-Fc fusion protein in 200 μL PBS. At various time points after injection, mice were sacrificed and plasma collected (n = 3 per time point). Plasma levels of intact c-Met-Fc were determined by ELISA. Points, mean for each group; bars, SE. Hu, human; mu, murine.

Human and Murine c-Met-Fc Inhibit Autocrine HGF:c-Met–Driven Tumor Growth in Xenograft Models

U-87 MG human glioblastoma and CT-26 mouse colon carcinoma cells express the respective forms of both HGF and c-Met, and proliferation and survival of these cells in culture are at least partially driven by a c-Met–dependent autocrine-signaling loop. To evaluate the effect of human and murine c-Met-Fc on tumor growth, we used a minimum disease model where c-Met-Fc treatment began 2 d after s.c. implantation of U-87 MG human glioblastoma cells or CT-26 mouse colon carcinoma cells. Both human and murine c-Met-Fc significantly inhibited growth of tumors in mice compared with Fc control [60% and 100% inhibition, P < 0.0001 for 30 and 100 μg human c-Met-Fc, respectively (Fig. 5A); 59% inhibition, P < 0.002 for 100 μg murine c-Met-Fc (Fig. 5B)].

Figure 5.
Figure 5.

Human and murine c-Met-Fc inhibit autocrine HGF:c-Met–driven tumor growth in mouse xenograft models. A, U-87 MG human glioblastoma cells were injected s.c. into female athymic nude mice. Treatment with human c-Met-Fc was initiated on day 2 at 30 or 100 μg/daily by i.p. injection, and tumor volumes were measured twice per week. B, CT-26 mouse colon carcinoma cells were injected s.c. into female BALB/c mice. Treatment with murine c-Met-Fc was initiated on day 2 at 100 μg per mouse daily by s.c. injection, and tumor volumes were measured twice per week. Points, mean (n = 10); bars, SE. *, P < 0.0001; **, P < 0.002.

Based on body weights, no evidence of overt toxicity was observed in animals treated with either human or murine c-Met-Fc (data not shown).

Discussion

In this study, we generated and characterized human and murine c-Met-Fc fusion proteins. Both these fusion proteins bind equally well to either human or murine HGF (Fig. 1), and this binding leads to inhibition of HGF-induced responses in cells, including inhibition of c-Met phosphorylation and cell invasion (Figs. 2 and 3). Systemic administration of the c-Met-Fc fusion proteins enabled determination of pharmacokinetic parameters in mice. Both human and murine c-Met-Fc exhibited long half-lives after i.p. or s.c. dosing in mice, ranging from 27 to 45 h (Fig. 4). Human c-Met-Fc, at daily doses of 30 or 100 μg, significantly inhibited tumor growth in an autocrine HGF:c-Met–driven minimal disease model of U-87 MG glioblastoma (Fig. 5A). Similarly, murine c-Met-Fc, at a dose of 100 μg daily, significantly inhibited tumor growth in a minimal disease model of CT-26 mouse colon carcinoma tumors (Fig. 5B). CT-26 cells are known to express HGF; however, our results are the first demonstration that growth of CT-26 tumors in mice is sensitive to inhibition of HGF, although CT-26 tumors seem to be less sensitive compared with U-87 MG tumors. Both human and murine c-Met-Fc seemed to be well-tolerated in animals.

The in vitro and in vivo effects of c-Met-Fc fusion proteins we observed in this study are similar to results previously observed with fully human IgG2 monoclonal antibodies to HGF (29). In that study, we showed that these antibodies bind to human HGF and block HGF binding to c-Met. The antibodies inhibited HGF-driven c-Met phosphorylation and cell invasion, and also inhibited tumor growth in autocrine HGF:c-Met–driven xenograft models of U-87 MG human glioblastoma. Currently, one of these antibodies, AMG 102, is being developed as a potential antitumor therapeutic (34–37). One of the major distinctions between anti-HGF antibodies such as AMG 102 and the c-Met-Fc fusion proteins described in the present report is that the antibodies do not bind to murine HGF. Therefore, preclinical assessment of the efficacy of HGF antibodies has been limited to autocrine xenograft models that express human HGF. The c-Met-Fc fusion proteins will permit the evaluation of HGF inhibition in paracrine settings; and might prove especially useful in syngeneic or orthopic models of cancer in which tumors develop in a native microenvironment in the context of a normal immune system.

Several different strategies are being pursued to interdict the HGF:c-Met pathway in cancer to validate the pathway clinically. The data presented here complement and expand the existing data suggesting that c-Met is a critical target for therapeutic intervention. Furthermore, the reagents generated here provide a means of selectively inhibiting the pathway in preclinical rodent models. The soluble c-Met receptors exhibit favorable pharmacologic properties in vivo and bind to both human and mouse HGF. These properties will allow for the expanded evaluation of HGF inhibition in multiple preclinical tumor models, likely deepening our understanding of the role of the HGF:c-Met pathway in cancer growth and metastasis; especially in a paracrine setting.

In conclusion, we have developed c-Met soluble receptors that effectively block HGF-induced activation of c-Met and inhibit growth of tumor xenografts, further demonstrating the potential of the HGF:c-Met axis as an oncology target.

Disclosure of Potential Conflicts of Interest

All authors are past or present employees of and stockholders in Amgen, Inc.

Acknowledgments

We thank Joanna Ho, Trace Tsuruda, Nayima Houston, Liu Jiang, and Lisa Renshaw-Gegg from the Protein Science group at Amgen, and Donald Bottaro at the National Cancer Institute for their contributions to this study. At Amgen, we also thank Martha Mutomba for writing assistance, and Paula Kaplan-Lefko and Isabelle Dussault for providing comments on the manuscript.

Footnotes

  • Grant support: Supported by Amgen Inc.

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1VAI. Hepatocyte growth factor/scatter factor, Met and cancer references. Available at: http://www.vai.org/met/. Updated September 16, 2008. Accessed October 9, 2008.

    • Received November 3, 2008.
    • Revision received February 24, 2009.
    • Accepted March 1, 2009.

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Molecular Cancer Therapeutics: 8 (5)
May 2009
Volume 8, Issue 5

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Soluble c-Met receptors inhibit phosphorylation of c-Met and growth of hepatocyte growth factor: c-Met–dependent tumors in animal models
Angela Coxon, Karen Rex, Susanne Meyer, Jianling Sun, Jilin Sun, Qing Chen, Robert Radinsky, Richard Kendall and Teresa L. Burgess
Mol Cancer Ther May 1 2009 (8) (5) 1119-1125; DOI: 10.1158/1535-7163.MCT-08-1032
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