Therapeutic targeting of human hepatocyte growth factor with a single neutralizing monoclonal antibody reduces lung tumorigenesis

Abstract

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody (mAb), demonstrated profound inhibition of human HGF-induced P-MAPK induction, wound healing and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were utilized to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), over 4 weeks. Beginning at week 3, intraperitoneal (i.p.) treatment with 100μg L2G7 or isotyped matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7 treated group was significantly lower than in the control group (1.58 versus 3.19, P=0.0005). Proliferative index was decreased by 48% (P=0.013) in tumors from L2G7 treated mice versus 5G8 treated mice while extent of apoptosis was increased in these same tumors by 5–fold (P=0.0013). P-MAPK expression was also significantly decreased by 84% in tumors from L2G7 treated mice versus 5G8 treated mice (P=0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results demonstrate that blocking the HGF/c-Met interaction with a single mAb delivered systemically can have profound inhibitory effects on development of lung tumors.

Introduction

Lung cancer is the leading cause of cancer death in the United States and the 5-year survival rate is only 16% (1). Lung cancer patients have few therapeutic options and therefore new targeted strategies are essential to make an impact on this disease. The hepatocyte growth factor (HGF)/c-Met signaling pathway plays a key role in the development and progression of many human cancers, including lung cancer, and represents an attractive targeted pathway for therapeutic intervention (2).

c-Met is a receptor tyrosine kinase (RTK) and is the only known receptor for HGF, also known as scatter factor (3, 4). Under normal physiological conditions, the HGF/c-Met pathway plays a role in development and wound healing and is not required for normal tissue homeostasis in the adult. Thus few adverse effects may result from therapy targeting this pathway. In many types of human cancer, including lung, HGF and/or c-Met is commonly overexpressed compared to normal tissue (5, 6). Furthermore, this correlation has been associated with disease state and clinical outcome (7). An important property of HGF is the ability to induce cell movement. Increased HGF levels within the tumor at time of resection may be an indicator of prior occult migration of malignant cells to other sites, thus increasing the probability of disease recurrence. A correlation between poor outcome and c-Met overexpression has also been observed (8), as well as with co-expression of both c-Met and HGF in lung tumors (9, 10). Other well-characterized biological effects induced by HGF including proliferation, invasion, angiogenesis and anti-apoptotic activity may also explain why overexpression of this pathway could lead to enhanced tumor development and progression. The HGF/c-Met pathway is a point of convergence for heterogeneous interacting signaling networks, thus a drug targeting this pathway could interfere with many different tumorigenic pathways to increase clinical benefit.

HGF has a unique structure composed of an α-chain containing the N-terminal domain and four kringle domains covalently disulfide linked to a serine protease like β-chain. Kim et al. and Burgess et al. have independently developed single potent neutralizing anti-HGF monoclonal antibodies (mAbs) that can inhibit the various HGF-induced biological activities attributable to both the α and β subunits in vitro (11, 12). These antibodies have been demonstrated to be highly specific to human HGF with no cross-reactivity to mouse HGF. Using human glioblastoma xenograft models, which express both HGF and c-Met in an autocrine manner, both antibodies were able to inhibit tumor growth and regression in nude mice. Additionally, histological analysis revealed that tumors from animals treated with the HGF mAb, L2G7, demonstrated decreased cell proliferation and blood vessel area with increased apoptosis (11).

HGF/c-Met signaling in the lung is primarily through a paracrine mechanism whereby the tumors do not express HGF but rather the surrounding stromal tissue expresses and secretes HGF which then acts on neighboring tumor cells expressing the c-Met receptor (13). This paracrine action of HGF in the lung renders testing these novel HGF mAbs difficult in conventional lung tumor xenografts, since murine HGF produced by the tumor stroma will be unaffected. We recently described a novel transgenic mouse model that overexpresses human HGF in the airways under control of the Clara cell secretory protein promoter and showed that these mice express significantly higher HGF levels in the airway luminal space and have a significantly increased susceptibility to carcinogen-induced lung adenocarcinoma (14). These mice develop lung tumors that mimic aggressive human lung adenocarcinoma with high HGF levels. This model provides a powerful preclinical system to evaluate anti-tumor agents that target the HGF/c-Met pathway, specifically agents developed against human HGF.

We utilized this animal model to test the therapeutic potential of anti-human HGF antibody, L2G7. The HGF transgenic mouse model is unique for studying effects of an anti-human HGF neutralizing antibody, since the HGF being overexpressed is human, and there is little evidence of murine HGF in the lungs of these animals. We show for the first time that the L2G7 neutralizing human HGF antibody can significantly decrease carcinogen-induced lung carcinogenesis in human HGF transgenic mice and inhibit downstream cancer-related signaling pathways within the tumors. The L2G7 single antibody may have potential as a therapeutic agent in NSCLC.

Materials and Methods

Reagents

L2G7 anti-HGF mAb and 5G8 isotype control were obtained under a Material Transfer Agreement with Galaxy Biotech (Mt. View, CA). Nitrosoamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) was from Toronto Research Chemicals (North York, ON, Canada). Recombinant human and mouse HGF was purchased from R&D Systems (Minneapolis, MN). NSCLC cell line, 201T, was established in our laboratory from primary tissue specimen as described previously (15). These cells do not harbor a K-ras mutation (16).

Protein Extraction and Western Analysis

Cells were grown to 75% confluency in T75 flasks. Cells were serum-deprived for 48 h followed by addition of recombinant human or mouse HGF (rhHGF or rmHGF) (10 ng/ml), recombinant human EGF (rhEGF) (10 ng/ml), L2G7 (0-300 ng/ml) or 5G8 (0-300 ng/ml) to the cells and protein was harvested at 10 min after HGF or EGF addition to examine phospho-MAPK expression. Cells were washed one time with ice-cold PBS. Protein was extracted by adding 300 μl ice-cold RIPA buffer (1X PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS containing 1 protease inhibitor cocktail/10ml buffer (Roche Diagnostics, Indianapolis, IN)). Protein concentration in the supernatant was measured using the BCA-200 Protein Assay Kit (Pierce, Rockford, IL).

Equal amounts of protein (25 μg) were loaded on a 10% Tricine-SDS gel for phospho-p44/p42 MAPK detection. Protein was transferred to nitrocellulose membrane followed by blocking with 5% milk, 1 X TBS-T. Primary antibody was a 1:1000 dilution of phospho-MAPK (Cell Signaling Technology, Danvers, MA) in 5% milk, 1 X TBS-T at 4°C overnight. Secondary antibody was horseradish peroxidase conjugated IgG at a 1:2000 dilution. West Pico chemiluminescent detection was used followed by autoradiography. Immunoreactive bands were quantitated by densitometry and ImageQuant analysis. Blots were stripped and reprobed with actin antibody (Chemicon/Millipore, Billerica, MA), at a 1:10,000 dilution. Separate gels were run for total MAPK detection using primary antibody at a 1:1000 dilution (Cell Signaling Technology) and secondary antibody at a 1:2000 dilution.

Cell Wound Healing Assay

For wound healing assays, cells were grown to confluency in 6-well plates. Cells were serum starved for 24 h, wounded with a pipette tip, and treated with HGF (10 ng/ml) alone or in combination with L2G7 (300 ng/ml) or 5G8 (300 ng/ml). Cells were examined by light microscopy prior to addition of experimental treatments (0 h) and at 72 h after treatment. The wound width was measured at each time point and the percent closure at 72 h versus 0 h was calculated. Three wells per experimental treatment and three wounds per well were examined. Results reported are the mean ± SE.

Invasion Assay

In vitro invasion assays were carried out in Matrigel-coated Transwell chambers (BD Biosciences, San Jose, CA). Briefly, serum deprived 201T NSCLC cells (1 X 10⁴ per well) were plated in a 24-well Biocoat Matrigel Transwell chamber. HGF (10 ng/ml) was added to the media in the lower chamber as indicated in the figure. L2G7 (300 ng/ml) or 5G8 (300 ng/ml) were added the top and lower chambers as indicated. Cells were incubated at 37°C with 5% CO₂ for 48 hours. Cells that invaded the Transwell chamber were fixed and stained using the Diff-Quik staining solutions according to the manufacturers' instructions (VWR International, West Chester, PA). The membranes were placed on microscope slides and the number of invading cells were scored on a microscope by counting 5 fields per membrane under 40 X magnification. Results reported are the mean ± SE.

Mouse Model

All mice used for experiments were HGF transgenic (FVB/N strain) and heterozygous for the transgene with high copy number. Only HGF transgenic mice were used since they carry the human HGF transgene. Wildtype mice produce only murine HGF which is not inhibited by L2G7. Breeding and identification of transgenic mice were as described previously (14). Equal numbers of males and females were in each experimental group. Mice were given a total of eight i.p. injections of 3 mg NNK (15μg/μl) over 4 weeks. Beginning at week 3, i.p. treatment with 100μg L2G7 or isotyped matched antibody control, 5G8, was initiated and continued through week 15. At week 15, all animals were sacrificed, the lungs were inflated with 10% buffered formalin under 25 cm intra-alveolar pressure and removed. Tumors were counted under a dissecting microscope and tumor size was measured using Motic Images 2000 software. Animal care was in strict compliance with the institutional guidelines established by the University of Pittsburgh.

Immunohistochemistry

Lungs were fixed in 10% buffered formalin. Lung samples were paraffin embedded, sliced and mounted on slides. Paraffin was removed from the slides with xylenes, and slides were stained according to standard procedures. Primary antibody was anti-phospho-p44/p42 MAPK (Cell Signaling Technology) or anti-Ki67 (DakoCytomation, Carpinteria, CA) at a 1:100 dilution. The secondary antibody was a biotinylated IgG specific for the primary antibody. Brown staining was considered positive. Negative control staining was done without the addition of primary antibody. For P-MAPK and Ki67 quantitation, tumors from 5 tumors or preneoplastic lesions per experimental treatment were read and scored for the number of positive cells per five high-powered fields. Results are reported as the mean ± SE.

For the apoptosis assay, the number of apoptotic cells was determined using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Intergen Company, Purchase, NY). Sections were deparafinized as above, incubated with proteinase K for 15 min and washed two times with water. Slides were incubated in 3% H₂O₂ in PBS for 5 min at room temperature and washed two times with PBS. The slides were then incubated for 15 min at 37°C with a terminal transferase enzyme that catalyzes the addition of digoxigenin-labeled nucleotides to the 3′-OH ends of the fragmented DNA. After color development and counterstain, the specimens were mounted. Slides were read and scored for the number of positive cells in either tumor or preneoplastic lesions per five high-powered fields. Results are reported as the mean ± SE.

Laser Capture Microdissection of Tumors and K-ras Mutation Analysis

For isolation of tumors from the lung, 5-micron tissue sections were prepared from each paraffin-embedded lung tissue block. Each tissue section was transferred to a membrane slide, stained with H&E, and examined by an experienced pathologist to identify and histologically analyze each tumor. Once identified, the areas of interest were taken from the membrane slide, by using a fluorescent laser capture microdissection Leica microscope (Application Solution Laser MicroDissection, McHenry, IL). Upon the laser action, the dissected area was dropped into the cap of the microcentrifuge tube containing cell lysis buffer and DNA was extracted by proteinase digestion, followed by phenol-chloroform extraction and ethanol precipitation. The DNA was dissolved in 10 mM Tris-HCl, pH 8.5, 1 mM EDTA and stored at −20°C until use.

K-ras mutations were analyzed as described previously (17) by using the combination of nested-PCR to amplify K-ras exon 1 that contains codon 12/13 and denaturing gradient gel electrophoresis (DGGE) to separate mutant from wild type alleles. Experimentally, for the first round PCR, an aliquot containing an equivalent of 20 – 50 cells were taken from each DNA sample and used for PCR amplification in a final 25 μl volume containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 100 μM each dNTP, 0.3 μM each of the pair of primers (sense: 5′-GACATGTTCTAATTTAGTTG-3′; anti-sense: 5′- AGGCGCTCTTGCCTACGGCA-3′) and 1.0 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchburg, NJ). The mixture was heated at 95°C for 9 minutes then subjected to 40 cycles (94°C/1 min, 53°C/1 min, 72°C/1 min). For the second round PCR, one μl of each of the first round PCR products was then diluted into a final 25 μl reaction mixture containing the same buffer composition as above except that 0.25 μl of [α-³²P]-dATP (3000 Ci/mmol, Perkin Elmer, Boston, MA) was added and that primers (antisense) 5′- AGGCGCTCTTGCCTACGGCA-3′; (sense)5′-GCCGCCTGCAGCCCGCGCCCCCCGTGCCCCCGCCCCGCCGCCGGCCCGGCGCCCTATTGTAAGGCCTGCTGAAAAT-3′ were used to amplify exon 1. Each reaction mixture was heated at 95°C for 9 min then subjected to 25 PCR cycles (94°C/1 min, 60°C/1 min, 72°C/1 min). The resulting PCR products were separated by gel electrophoresis and autoradiographed. Bands containing the expected 126 bp exon 1 fragment were excised from the gel. DNA was eluted and analyzed by DGGE (Bio-Rad, CA) under the conditions described previously (17) and mutant alleles were further characterized by sequencing.

Statistical Analysis

The number of tumors per group in the NNK carcinogenesis animal study was compared with Poisson regression. The tumor sizes per group (after log-transformation) were compared with linear mixed-effects models fitted by the maximum likelihood method. Data from two experiments were combined, and the analysis was adjusted for sex. Significance tests were performed with two-sided significance level 0.05. Tukey one way ANOVA with post-test was used for analysis in Figure 2. For immunohistochemical analysis in Figures 4 and 5, an unpaired Student's t-test was used. For K-ras mutation analysis in Table I, a Fisher's Exact test was used.

Figure 2.

L2G7 blocks HGF-induced wound healing and cell invasion. A) 201T cells were serum deprived followed by treatment with HGF (10ng/ml), L2G7 (300ng/ml) or 5G8 (300ng/ml) as indicated for 72 h. Cell wound healing assays were performed and the mean of 6 samples per treatment group is expressed compared to control, untreated wells. Bars, SE. ***, P<0.001, ANOVA. B) Representative photographs of cells at 72 hr following experimental treatments as indicated. C) 201T cells were serum deprived prior to plating in invasion chamber wells. Cells were treated with HGF (10ng/ml), L2G7 (300ng/ml) or 5G8 (300ng/ml) as indicated in the figure. 48 h following treatment, invading cells were fixed and stained. Positively stained cells in 5 high-powered fields per slide were counted at 40X magnification. Mean of 4 samples per treatment group. Bars, SE. **, P<0.01, ***P<0.001, ANOVA. All comparisons were respective to control untreated cells.

Figure 4.

Representative tumor sections from 5G8 and L2G7 treated animals showing immunohistochemical staining for A) Ki67 B) phospho-MAPK and C) apoptotic cells. Inset is a 2-fold high-powered view. Quantitation for each marker was performed and results are presented as the mean ± SE number of positive cells for each marker from 5 high-powered fields from 5 tumors per experimental treatment. *, P<0.05, **, P<0.005, ***, P<0.0005, Student's t-test.

Figure 5.

Representative preneoplastic sections from 5G8 and L2G7 treated animals showing immunohistochemical staining for A) Ki67 B) phospho-MAPK and C) apoptotic cells. Inset is a 2-fold high-powered view. Quantitation for each marker was performed and results are presented as the mean ± SE number of positive cells for each marker from 5 high-powered fields from 5 tumors per experimental treatment. *, P<0.05, **, P<0.005, Student's t-test.

Table 1.

K-ras mutations in tumors from L2G7 and 5G8 treated mice.

K-ras status	L2G7	5G8
G12D	25 (73.5%)	12 (44.4%)
WT	9 (26.5%)	15 (55.6%)
Total	34 (100%)	27 (100%)

Results

L2G7 specifically inhibits HGF-induced biological activities in vitro

We first examined the effect of L2G7 on biological activities of HGF on a lung cancer cell line in vitro. We chose the 201T lung adenocarcinoma cell line for these assays because it has been demonstrated to overexpress c-Met and respond to HGF, and we previously documented these cells do not secrete HGF (18, 19). We first examined the inhibition of HGF- induced phospho-MAPK in response to increasing concentrations of L2G7. Phospho-MAPK is downstream of c-Met activation, and MAPK induction has been shown to be a highly consistent indicator of HGF activity (19). 201T lung adenocarcinoma cells were treated with L2G7 (0-300 ng/ml) and rhHGF (10 ng/ml). rhHGF induced phospho-MAPK expression by 6-fold compared to control. L2G7 decreased the rhHGF-induced phospho-MAPK expression in a concentration-dependent manner (Fig. 1A). A maximum decrease of 76% was observed for phospho-MAPK expression using 300 ng/ml L2G7. No additional benefit was observed with up to 500 ng/ml L2G7 (data not shown). These experiments were repeated three times with similar results. L2G7 also inhibited HGF-induced phospho-c-Met to a similar extent in vitro.

Figure 1.

L2G7 inhibits human HGF-induced phospho-MAPK expression. A) 201T were serum deprived for 48 h followed by treatment with 0-300ng/ml L2G7 and the addition of A) 10ng/ml rhHGF B) 10ng/ml rhEGF or C) 10ng/ml rmHGF for 10 min. Cell lysates were prepared and analyzed for P-MAPK expression. The blots were stripped and reprobed for β-actin. Relative quantitation is shown beneath each blot. Separate gels were run for total MAPK expression. D) 201T cells were cultured as described above. Following serum deprivation, cells were treated with 5G8 control antibody (0-300ng/ml) in the presence or absence of rhHGF (10ng/ml). P-MAPK, total MAPK and β-actin expression were analyzed as in panels A-C.

We next demonstrated that L2G7 was specific to inhibition of human HGF-induced biological activities. In this regard, rhEGF (10 ng/ml) stimulated phospho-MAPK by almost 7–fold over control and L2G7 (up to 300 ng/ml) showed no ability to inhibit rhEGF-induced phospho-MAPK (Fig. 1B). Similarly, L2G7 could not block murine HGF; rmHGF- induced phospho-MAPK expression was unaffected in the presence of L2G7 (Fig. 1C) consistent with previous results (12). L2G7 alone had no effect on basal phospho-MAPK, and the isotyped matched control antibody, 5G8, also had no effect either in the presence or absence of HGF (Fig. 1A & D).

We next analyzed the effect of the neutralizing antibody on HGF-induced wound healing (Figs. 2A and B). Serum-starved 201T cells were treated with or without 10 ng/ml HGF in the presence or absence of 300 ng/ml L2G7 or 5G8. HGF treatment alone was able to close a pipette generated wound 80.53% compared to the no treatment wound closure of only 1.67% after 72 h (P<0.001). The percent wound closure in the presence of L2G7 and HGF was only 13.67% (greater than 80% inhibition of HGF-induced wound healing, P<0.001, HGF vs. L2G7 plus HGF) while 5G8 plus HGF wound closure was not significantly different from HGF treatment alone wound closure (P>0.05, HGF vs. 5G8 plus HGF). L2G7 or 5G8 alone had no significant effect on wound healing and was similar to control untreated cells.

Another well-documented biological activity of HGF is cell invasion. We used movement through an artificial extracellular Matrigel matrix as a measure of relative invasion of 201T cells to assess whether L2G7 could inhibit HGF-induced invasion (Fig.2C). HGF induced invasion by 1.3-fold in this assay (P<0.001). This stimulation was inhibited below basal levels in the presence of L2G7 treatment (P<0.001, HGF vs. HGF plus L2G7). 5G8 in the presence of HGF had no effect on HGF-induced cell invasion (P>0.05, HGF vs. 5G8 plus HGF). L2G7 or 5G8 alone had no significant effect on invasion and were similar to control untreated cells. Similar effects of L2G7 were observed on HGF-induced cell proliferation (data not shown). Thus, several distinct biological responses to HGF were found to be specifically inhibited by L2G7 in human lung tumor cells in vitro. This established a rationale to test L2G7 in an in vivo preclinical model.

Inhibition of lung tumor formation by L2G7 treatment

We utilized a transgenic mouse model which overexpresses human HGF in the airways to test the efficacy of L2G7 in an in vivo model of lung adenocarcinoma tumorigenesis. We previously documented that these animals express high levels of human HGF in the lungs (14). Since the L2G7 antibody does not recognize murine HGF, this transgenic mouse is a unique system to study this mAb in vivo against lung tumors that normally show only paracrine HGF secretion. All mice were treated with a tobacco carcinogen, NNK, for 2 weeks prior to the initiation of L2G7 HGF mAb or 5G8 control treatment. mAb treatment (100 μg per injection) continued until week 15 when the animals were sacrificed. We chose this time point to begin L2G7 treatment to mimic the human setting whereby there is exposure to tobacco prior to chemopreventative measures. In addition, the effect of HGF is more likely to inhibit the promotion phase of tumor cell growth and progression than to inhibit the initiation phase of mutation and establishment of preneoplastic lesions.

The results from two separate experiments were combined for this analysis. The 5G8 control group consisted of 24 mice in total, while the L2G7 treatment group included 22 mice. Seven of the 22 mice (31.8%) in the L2G7 treated arm did not develop any visible lung tumors, while only 4 of the 24 (16.7%) in the 5G8 arm did not develop visible tumors. The mean number of tumors per mouse in the group treated with L2G7 was significantly lower than in the 5G8 control group (P<0.001). The estimated geometric mean, adjusted for sex, of the number of tumors per mouse in the group treated with L2G7 was 1.58 (median 1, range 0-6), while in the control group it was 3.19 (median 3.5, range 0-7) (Fig. 3A).

Figure 3.

Tumor formation following NNK exposure in HGF transgenic mice treated with L2G7 neutralizing antibody to human HGF or control 5G8 isotype-matched antibody. A) Graph of the number of tumors per mouse by treatment group for the combined data set. Each point in the figure represents one mouse (circle= L2G7 treated mouse; triangle= 5G8-treated mouse). The horizontal line within each side of the graph shows the median for the treatment group. *P<0.001. B) Histogram of tumor sizes (mm²) by treatment group for combined data set. Intergroup differences: p-value=0.752.

There was not a significant difference in tumor size (P=0.974) between the L2G7 and 5G8 treated groups. There was one very large outlier (in the 5G8 group) and there was substantial overlap in the data. The median tumor size in the L2G7 treated group was 0.33 mm² (interquartile range: 0.22-0.45 mm², range: 0.12-2.44 mm²); and median size in the 5G8 group also was 0.33 mm² (interquartile range: 0.21-0.55 mm², range: 0.10-5.50 mm²). Figure 3B shows a histogram of the frequency of tumor sizes by treatment group. Although there was no difference in median tumor size among treatment groups, there were fewer larger tumors found in the L2G7 treatment group compared to the 5G8 treatment group based on the distribution frequency. For example, 5 of 36 (13.8%) tumors from the L2G7 treated group were in the 0.7 mm² or greater size categories whereas 16 of 78 (21%) tumors fell in these categories from the 5G8 treated group.

In the multistep carcinogenesis model for lung cancer (20), peripheral adenocarcinoma of the lung develops from noninvasive precursor lesions known as atypical adenomatous hyperplasia. No significant difference was observed in the number of adenomatous lesions or bronchiolar hyperplasia present after 13 weeks of therapy between mice treated with L2G7 or with 5G8 (data not shown), suggesting that L2G7 does not block early carcinogenic events, but more likely affects tumor cell turnover, doubling time, or progression. In addition, in an alternate protocol in which the antibody treatment was initiated 1 week prior to NNK treatment, then continued for an additional 15 weeks, we observed no additional benefit of L2G7 compared to initiating L2G7 treatment during NNK treatment (data not shown). In both protocols, L2G7 treatment reduced tumorigenicity to about half the level observed with control antibody. In previous experiments comparing wild-type littermates to HGF transgenic mice, we also observed about half as many lung tumors in wild-type animals as in carriers of the transgene (14), suggesting L2G7 treatment almost completely inhibited the ability of the human HGF transgene to increase susceptibility to lung cancer.

Signaling in tumors and preneoplastic lesions is affected by L2G7 treatment

We next examined lung tumors removed from mice treated with L2G7 or 5G8 and analyzed the number of proliferating cells by Ki67 immunohistochemistry. The number of Ki67 positive cells in tumors from L2G7 treated mice was 48% lower than that found in tumors from 5G8 treated mice (P<0.05) (Fig. 4A). Additionally, since MAPK is a signaling pathway known to be induced by HGF and we have shown in Fig. 1A that L2G7 can inhibit this signaling pathway in vitro, we examined whether L2G7 reduced the number of phospho-MAPK positive cells found in HGF overexpressing tumors (Fig. 4B). Tumors from L2G7 treated animals demonstrated 84% fewer phospho-MAPK positive cells compared to tumors examined from 5G8 treated mice (P<0.0005). In contrast, the number of apoptotic cells as examined using a Tunel Assay was significantly increased by 5-fold in tumors from L2G7 treated animals compared to tumors from 5G8 treated animals (P<0.005) (Fig. 4C).

Similar effects on expression of signaling molecules were observed in adenomatous hyperplasia lesions of the lung. In this regard, a 34% and 41% decrease was observed in adenomatous hyperplasia lesions of similar size from L2G7 treated animals versus 5G8 treated animals for Ki67 and phospho-MAPK expression, respectively (P<0.05 and P= 0.06, Fig. 5A and B). In addition, the number of apoptotic cells was increased 3.2-fold (P<0.005) in adenomatous hyperplasia lesions of similar size from L2G7 treated animals compared to 5G8 treated animals (Fig. 5C). These findings suggest that even though the number of preneoplastic lesions was not reduced by L2G7 treatment, the ratio of proliferative to apoptotic signals within these lesions was lowered by the HGF neutralizing antibody.

Tumors with K-ras mutation are resistant to L2G7 treatment

Activation of the K-ras protoncogene through mutations is a common event in lung cancer, especially in murine adenocarcinomas induced to NNK, and we examined whether or not L2G7 treatment could inhibit tumors harboring a K-ras activating mutation. Laser capture microdissection was used to isolate tumors from tissue sections for subsequent genomic DNA extraction and K-ras mutation analysis in codons 12 and 13. Greater than 95% of ras mutations are found in these codons (21). All activating mutations switch Ras to its GTP-bound, constitutively active state. Thirty-four tumors were isolated from the L2G7 treated group and 27 tumors from the 5G8 treated group. Some tumors had already been depleted from the previous assays thus the entire dataset could not be analyzed. Table I shows that in the presence of L2G7, which produces about a 50% inhibition of tumorigenesis, tumors that did form were more likely to be K-ras mutant. K-ras mutations were found in 73.5 % (25 of 34) of tumors from L2G7 treated animals and 44.4 % (12 of 27) of tumors from 5G8 treated animals (P=0.034, Fishers exact test). All K-ras mutations found were located in codon 12 and corresponded to a GGT to GAT transition of glycine to aspartate (G12D). K-ras mutation status in FVB/N wild-type littermates exposed to NNK showed a 40% incidence rate of K-ras mutations (data not shown), similar to that observed in HGF transgenic animals treated with 5G8. This suggests that the HGF transgene in general promotes both K-ras mutant and wild-type tumors, but that K-ras mutant tumors are less responsive to withdrawal of HGF signaling. These results suggest that targeting the HGF/c-Met pathway in patients may not be as effective if tumors contain activating K-ras mutations. We did however note that Ki67 and apoptotic cell labeling showed equal alterations from L2G7 treatment in some tumors that formed which had K-ras activating mutations or K-ras wild-type status (not shown).

To further examine the effects of HGF signaling withdrawal in lung tumor cells that express c-Met and contain a K-ras mutation, MAPK phosphorylation was measured at baseline and following HGF stimulation in three NSCLC cell lines containing K-ras point mutations (16, 17). In 343T cells containing the K-ras mutation G12C, basal phospho-MAPK was highly expressed, no increase in phospho-MAPK was observed with HGF, and there was no appreciable decrease in phospho-MAPK with L2G7 (Supplemental Fig. 1A). In 91T cells, containing the G12V K-ras mutation, basal phospho-MAPK was low, there was a strong 8-fold increase with HGF, but the neutralizing antibody L2G7 was much less effective (maximum inhibition 38%) (Supplemental Fig. 1B). A similar result was observed with A549 cells, K-ras mutation G12S (data not shown). Dependence on HGF signaling or an ability to inhibit HGF signaling may be reduced in NSCLC cell lines with K-ras mutation compared to wild-type K-ras cells, such as 201T (Fig. 1).

Discussion

Because of the overwhelming evidence for the role of the HGF/c-Met pathway in the pathogenesis of human cancers, therapeutic inhibitors that target this pathway are being developed for clinical use. MAbs and small molecule tyrosine kinase inhibitors (TKI) represent the most feasible approaches and are the most clinically relevant agents at this time. Currently, there are phase I clinical trials for cancer therapy in progress for both of these classes of drugs. This pathway may be desirable for cancer therapeutic inhibition because side effects associated with inhibition of this pathway in the absence of wound healing would potentially be low.

We have demonstrated here that a human HGF mAb, L2G7, can profoundly inhibit HGF-induced activation of MAPK as well as biological responses in NSCLC in vitro (wound healing and cell invasion). L2G7 was inactive against either EGF or recombinant murine HGF, thus showing specificity for human HGF. The anti-HGF neutralizing antibody also substantially inhibited tumor formation in transgenic mice overexpressing the human HGF gene in the airways; there was about a 2-fold difference between tumor formation with the control antibody treatment compared to neutralizing antibody. This is the same order of magnitude as the observed 2-fold difference in lung tumor formation between HGF transgenic and wild-type mice (14), suggesting almost all the biological effect of the human HGF transgene was abolished by L2G7. L2G7 treatment also significantly altered critical signaling pathways within the tumors. In this regard, proliferation index as measured by Ki67 immunostaining was significantly decreased as well as phospho-MAPK expression, a well established signaling pathway involved in lung tumor cell growth. The HGF signaling pathway also demonstrates anti-apoptotic effects (22). Treatment with L2G7 blocked this function as well. These effects were observed in tumors and to a lesser extent in hyperplastic lesions of the lung. The inhibition of signaling occurred in both K-ras wild-type tumors as well as K-ras mutant tumors. Therefore, even though L2G7 appears to have less overall effect on reducing formation of K-ras mutant tumors detected by this protocol, downstream signaling is still diminished by L2G7 even in the presence of mutant K-ras in this model.

The effect of L2G7 did not appear to be related to tumor initiation, but rather to tumor progression since the number of preneoplastic areas in the lungs from L2G7 versus 5G8 treated animals was not significantly altered, but the number of adenomas was significantly decreased. Additional evidence for an effect on the promotional phase was seen in that starting the L2G7 treatments prior to NNK exposure did not exhibit a greater inhibitory effect compared to starting L2G7 treatment during the course of NNK exposure. Median tumor size was not affected by L2G7 treatment, however, the largest tumors were observed in the 5G8 control antibody treated group. We also compared K-ras status versus tumor size in a subset of tumors where both parameters were known. No difference in mean or median tumor size between treatment groups was observed when analyzing K-ras mutants only or K-ras wild-type tumors only, suggesting both groups have equal ability to proliferate.

Mutations in ras genes are found in around 30% of human cancers (23). K-ras mutations are the most linked to NSCLC and occurs in 20-30% of lung adenocarcinoma (17, 23). Overall, K-ras appears to be a weak negative prognostic marker in adenocarcinoma of the lung (24, 25). The major type of K-ras mutation that we report in lung tumors is similar to other reports (26, 27). In the studies presented here, the K-ras wild-type tumors are selectively targeted with the L2G7 neutralizing antibody to human HGF, suggesting that downstream constitutive activation of the ras pathway in K-ras mutant tumors make them more resistant to inhibition that blocks an upstream tyrosine kinase receptor. We also observed that NSCLC cell lines with K-ras mutations either showed high basal phospho-MAPK with little induction by HGF or HGF signaling could not be effectively inhibited by the neutralizing antibody. When signaling to phospho-MAPK was HGF dependent in K-ras mutants, it is not clear what would cause the resistance to HGF neutralizing antibody. The K-ras mutant cells showed similar HGF concentration dependence as K-ras wild-type cells. It is possible that the physical association between activated K-ras and c-Met compared to wild-type K-ras and c-Met has different dynamics in the presence of the HGF neutralizing antibody complex. The exact mechanism warrants further study. Both the in vivo and in vitro observations suggest that effective targeting of c-Met signaling could be accomplished more efficiently when there is not a K-ras mutation present. This is similar to inhibition of other RTK pathways; there is an apparent negative association between K-ras mutations and mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain that determine patient response to EGFR TKIs (16, 28). This may prove to be the case with HGF/c-Met inhibition as well.

c-Met activation is involved in different steps of tumorigenesis including tumor formation, growth, and spreading in many different solid tumor types. In addition, this pathway interacts with several other tumorigenic pathways including the EGFR signaling pathway making this pathway an attractive target for therapy. For example, when EGFR is overexpressed in tumor cells, it can directly associate with and phosphorylate c-Met (29). Additionally, amplification of c-Met is a mechanism of resistance to the EGFR TKI gefitinib (30). With the anticipation that combinations of pathway selective therapies will most likely be necessary for many cancer types, this therapy may also be beneficial for combined therapy with other drugs, in particular with agents that target the EGFR, COX-2 or vascular endothelial growth factor (VEGF) pathways. We have recently shown that HGF can induce COX-2 expression and release PGE₂ which can then act to directly activate c-Met in lung cancer cells in an HGF-independent manner (19) suggesting that dual targeting of HGF/c-Met with COX-2 may be beneficial. There is also evidence that HGF can induce angiogenesis independently of the VEGF pathway (31) indicating that inhibition of both the HGF and VEGF pathways may be advantageous as well. The HGF transgenic mouse model used in these studies would be an extremely useful model system to study these types of combination therapies.

In summary, this work demonstrates that therapeutic targeting of the HGF pathway with L2G7 or other agents targeting this pathway has potential to move to human clinical trials for NSCLC. Additionally, K-ras mutation status will most likely be a critical part of a panel of markers that will have predictive value for HGF/c-Met targeted therapy. Future challenges will be to accurately identify patients who are most likely to respond to HGF/c-Met targeted therapies, understand the effect of long-term blockade of this pathway, and identify other pathways that would be beneficial to target in combination treatment regimens for enhanced anti-tumorigenic effects. Understanding these combined issues might enable us to better understand, control, and treat NSCLC.

Supplementary Material

Supplemental Figure Legend

Figure 1. L2G7 effects in K-ras mutant cell lines. A) 343T cells or B) 91T cells were serum deprived for 48 h followed by treatment with 0-300ng/ml L2G7 and the addition of 10ng/ml rhHGF. Cell lysates were prepared and analyzed for phospho-MAPK (P-MAPK) expression. Separate gels were run for total MAPK (T-MAPK) expression. Relative quantitation by densitometry and ImageQuant analysis is shown beneath each blot.

List of Abbreviations

HGF

hepatocyte growth factor

NNK

nitrosoamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone

i.p

intraperitoneal

RTK

receptor tyrosine kinase

mAb

monoclonal antibody

EGFR

epidermal growth factor receptor

VEGF

vascular endothelial growth factor

NSCLC

non-small cell lung cancer

rhHGF

recombinant human HGF

rmHGF

recombinant mouse HGF

rhEGF

recombinant human epidermal growth factor

AS LMD

application solution laser microdissection

DGGE

denaturing gradient gel electrophoresis

TKI

tyrosine kinase inhibitor

DGGE

denaturing gradient gel electrophoresis