Impact of the small molecule Met inhibitor BMS-777607 on the metastatic process in a rodent tumor model with constitutive c-Met activation
Yao Dai • Kyungmi Bae • Christine Pampo •
Dietmar W. Siemann
Received: 22 July 2011 / Accepted: 22 December 2011 / Published online: 28 January 2012
Springer Science+Business Media B.V. 2012
Abstract c-Met tyrosine kinase hyperactivation is strongly associated with tumor metastasis. In a prior study we showed that BMS-777607, a novel selective small molecule Met kinase inhibitor, potently suppressed ligand-mediated functions in prostate cancer cells. Herein we evaluated the impact of this agent on the potential of the highly metastatic murine KHT sarcoma that carries consti-tutive activated c-Met. MET gene knockdown was found to reduce spontaneous cell scatter and motility, suggesting a c-Met-dependent disseminating ability in KHT cells. Fur-thermore, BMS-777607 treatment potently inhibited KHT cell scatter, motility and invasion at doses in the nanomolar range. In contrast, cell proliferation and clonogenicity were modestly affected by BMS-777607. At the molecular level, BMS-777607 potently blocked phosphorylation of c-Met and downstream pathways over the same dose range that impacted metastasis-associated cell functions. In vivo, daily treatment with BMS-777607 (25 mg/kg/day) over the course of the study significantly decreased the number of KHT lung tumor nodules (28.3 ± 14.9%, P \ 0.001) without apparent systemic toxicity. While treatment for short intervals (day 1 or 4) clearly reduced the foci number, delaying the initiation of BMS-777607 treatment until 8 days after tumor cell
Electronic supplementary material The online version of this article (doi:10.1007/s10585-011-9447-z) contains supplementary material, which is available to authorized users.
injection failed to show any reduction, implying that impairment of the initiation phases of the secondary growth via c-Met targeting is required to constrain the formation of macroscopic metastases. Together, the present findings demonstrate that the disruption of c-Met signaling by BMS-777607 significantly impairs the metastatic phenotype, suggesting that this agent may have therapeutic utility in targeting cancer metastasis.
Keywords BMS-777607 c-Met Metastasis KHT Sarcoma
Abbreviations
HGF Hepatocyte growth factor
HGFR Hepatocyte growth factor receptor
PI3K Phosphoinositide 3-kinases
mTOR Mammalian target of rapamycin
MAPK Mitogen-activated protein kinase
ERK Extracellular-signal-regulated kinase
FAK Focal adhesion kinase
STAT3 Signal transducer and activator of transcription 3
S6K S6 kinase
FBS Fetal bovine serum
Introduction
Y. Dai (&) C. Pampo D. W. Siemann
Department of Radiation Oncology, University of Florida, 2033 Mowry Road, Cancer Genetic Research Complex, Room 485E, Gainesville, FL 32610, USA e-mail: [email protected]
K. Bae
Department of Urology, Shands Cancer Center at the University of Florida, Gainesville, FL 32608, USA
The MET oncogene encodes the receptor tyrosine kinase c-Met (also referred to as hepatocyte growth factor receptor, HGFR) that is expressed on epithelial and endothelial cells. While under physiological conditions c-Met is normally activated by its ligand; hepatocyte growth factor (HGF), abnormal c-Met activation is a common feature in human cancers [1]. Alterations including HGF/c-Met autocrine stimulation, crosstalk with other signaling molecules, MET
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gene amplification, activating mutation or overexpression, result in a constitutive activation of c-Met kinase inde-pendent of exogenous HGF ligand [2, 3]. Because dys-regulation of c-Met signaling is strongly associated with metastasis and poor prognosis in a variety of cancers including carcinomas, sarcomas and gliomas, MET acti-vation has been suggested as a possible prognostic indi-cator for cancer progression [4].
c-Met plays a key role in the invasive growth and dis-semination of cancer cells by activating multiple cell functions including proliferation, scattering, migration, invasion and branching morphogenesis [5]. Tumor angio-genesis has also been shown to utilize the HGF/c-Met axis [6]. Mechanistically, c-Met promotes key metastasis-related behaviors by initiating multiple downstream sig-naling pathways such as Ras-Raf-mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathways [7]. In addition, non-receptor tyrosine kinases such as Src, focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3), have also been reported as as downstream effectors of c-Met [8–10].
Given the pivotal role of c-Met signaling, c-Met receptor has been emerged as an attractive target for therapeutic intervention in cancer [11]. Whereas anti-Met neutralizing antibody holds some therapeutic potential, small molecule Met kinase inhibitors, most of which are ATP-competitive, offer the broader application by disrupting both HGF-dependent activation and c-Met-driven functions [12]. A variety of Met kinase inhibitors have entered clinical trials for the treatment of human cancers expressing aberrant c-Met activity [5]. Preclinically, most of these compounds block c-Met autophosphorylation at a nanomolar level, suppress metastasis-associated functions in vitro, and inhi-bit tumor growth in specific xenografted models that harbor activated c-Met [2]. In patients, these agents have shown some benefit in the treatment of sarcoma [13], renal cell carcinoma [13, 14] and non-small cell lung cancer [15]. BMS-777607 is a potent and selective ATP-competitive Met inhibitor that potently blocks phosphorylation of Met family kinases [16]. Similar to other Met inhibitors [17, 18], BMS-777607 has been shown to reduce tumor growth in a MET gene-amplified tumor model [16]. We reported pre-viously that BMS-777607 significantly suppressed HGF-induced cell functions and c-Met signaling pathways in prostate cancer cells, suggesting a therapeutic potential for this agent in ligand-dependent metastasis [19].
KHT is a highly metastatic murine sarcoma tumor model that has been studied in our laboratory [20–22]. Interestingly, these cells exhibit spontaneously scattered phenotype even without addition of the exogenous HGF, and at the molecular level, a certain basal level of
autophosphorylated c-Met can be detected even after serum starvation (data not shown). Since cell scattering is a typ-ical feature associated with HGF/c-Met signaling (HGF is also known as ‘‘scatter factor’’) [23], this observation suggests that KHT cells may possess an inherent capacity to disseminate which perhaps relates with c-Met activity and does not necessarily require HGF. In the current study, the role of c-Met and the impact of BMS-777607 treatment on its signaling and tumor cell associated metastatic characteristics was evaluated in the highly metastatic murine KHT sarcoma tumor model that harbors constitu-tively activated c-Met.
Materials and methods
Reagents
BMS-777607 was kindly provided by Dr. Joseph Fargnoli (Bristol-Myer Squibb, Piscataway, NJ). The powder was dissolved in dimethyl sulfoxide and stored as aliquots (10 mM) at -20LC. Additional chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
Cell culture
Rodent fibrosarcoma KHT cells [21] were maintained in alpha MEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin. Cells were cultured in a 5% CO2 humidified incubator at 37LC.
Cell scattering
Cells were seeded in a six-well plate at a density of 200 cells/well and cultured for 4 days until colonies were formed. Cell colonies were incubated with BMS-777607 for 24 h and then stained with crystal violet (0.1%) and photographed.
Cell migration
Cell migration was evaluated using a ‘‘wound-healing’’ assay. KHT cells were seeded in a six-well plate and allowed to grow for 48 h. Wounds were generated on the confluent cell monolayer by making a 2 mm scratch using a sterilized 1 ml pipette tip. Cells were then treated with BMS-777607 and 24 h later the number of cells that had migrated into the denuded area was counted on 4 random fields (original magnification, 9100). In some circum-stances, migration (%) is calculated as the ratio of migrated cells of treated/untreated control samples.
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Cell invasion
Invasion was examined using a commercial transwell insert (8 lm pore membrane) pre-loaded with Matrigel (BD Bio-sciences, San Diego, CA). Inserts were incubated with serum-free medium in the presence or absence of BMS-777607 at 37LC for 2 h to allow rehydration of Matrigel. Cells suspended in serum-free medium were loaded onto the top chamber (5 9 103/insert). Complete medium (contain-ing 10% FBS) was used in the lower chamber as a chemo-attractant. After incubation for 24 h, the Matrigel was removed and the inserts were stained with crystal violet. Invaded cells on the underside of the filter were photo-graphed and counted. Invasion (%) was calculated as the ratio of invaded cells of treated to untreated control samples.
Cell proliferation
Cells were seeded in a 96-well plate at a density of 5,000 cells/well. After seeding, cells were exposed to serial dilutions of BMS-777607 for 96 h. Cells were then incu-bated with the WST-8 (MTT) reagent in a Cell Counting Kit (Dojindo, Rockville, MD) following the manufacturer’s instruction. Absorbance was determined at 450 nm color-imetrically. Cell proliferation (%) was calculated as the ratio of the absorbance from treated samples compared to that of the untreated control sample. Cell death was examined by trypan blue exclusion.
Colony formation
Cells were seeded into six-well plates (200 and 100/well in triplicate, respectively) and treated with desired doses of BMS-777607. Cells were incubated for 14 days and plates were stained with crystal violet. Cell colonies (C50 cells) were counted macroscopically. Plating efficiency was cal-culated as the percentage of seeded tumor cells forming macroscopic colonies.
siRNA transfection
The siRNA specific for mouse c-Met or control siRNA were purchased from IDT (Coralville, IA). siRNA fragments (5 nmol) were transfected into KHT cells using Oligofec-tAMINE (Invitrogen, Carlsbad, CA), following the manu-facturer’s instructions. Forty-eight hours after transfection, cells were utilized in the ‘‘wound-healing’’ scratch assay. The siRNA knockdown effect was confirmed by western blot.
Western blot
Cells were harvested and disrupted in a radioimmunoprecip-itation assay lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 0.1% SDS, 1% NP-40, 0.25% Sodium deoxycholate and 1 mM EDTA) with protease inhibitor cocktail, 1 mM NaF and 1 mM Na3VO4. Equal amounts (40 lg) of whole cell lysates were resolved on a 10% SDS-PAGE gel (Bio-Rad). After electrophoresis, samples were electrotransferred to a nitrocellulose membrane (Bio-Rad), probed with relevant primary antibodies at 4LC over night, incubated with horse-radish peroxidase conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA), and detected with an enhanced chemiluminescence substrate (Amersham, Piscat-away, NJ). The following primary antibodies were used: phospho-c-Met (Y1234/1235), total c-Met, phospho-Akt (S473), phospho-S6 kinase (S6K) (T389), phospho-S6 (S234/235), phospho-extracellular-signal-regulated kinase (ERK) (T202/Y204), phospho-Src (Y416), and phospho-STAT3 (Y705) (Cell Signaling, Danvers, MA); phospho-FAK (Y397) (Chemicon, Billerica, MA); b-actin (Sigma). The density of the bands was quantified with the aid of the Image J software program (National Institutes of Health, Bethesda, MD).
Experimental metastases
KHT cells (5 9 103) were injected into 6–8 week-old female C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME) via the tail vein (10 mice/group). After tumor cell inoculation, mice were treated with either a vehicle control (PEG 400:water = 3:1) or BMS-777607 by oral gavage once daily with indicated doses and schedules. Mice body weights were monitored twice a week. All mice were euthanized 15 days after cell inoculation. Lungs were removed and fixed in Bouin’s solution overnight and then transferred to isopropyl alcohol (70%). Tumor nodules formed in lungs were counted under a light microscope. Animal studies were governed by the principles of the Guide for the Care and Use of Laboratory Animals, and approved by the University of Florida Institu-tional Animal Care and Use Committee.
Statistical analysis
Two-tailed Student’s t test and non-parametric Wilcoxon rank sum test were employed for analyzing in vitro and in vivo data, respectively, using GraphPad Prism 5.0 software (San Diego, CA). A threshold of P \ 0.05 was defined as statistically significant.
Results
MET gene knockdown attenuates KHT cell scattering and motility
To investigate the role of c-Met in KHT cell functions, the MET gene was silenced by siRNA. Western blot analysis
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confirmed that the c-Met protein as well as its downstream signaling pathways (phospho-Akt and phospho-ERK) was efficiently knocked down (Fig. 1a). Moreover, cells trans-fected by MET-siRNA decreased the scattered and motile ability leading to a significant reduction of migrated cells into the denuded area compared to control cells (treated with scrambled siRNA; Fig. 1b). These data suggest that c-Met is required for the intrinsic mitogenic capacity of KHT cells.
BMS-777607 blocks the c-Met signaling pathway
To explore the effect of BMS-777607 on c-Met signaling, KHT cells were treated with various doses of this agent for a period of 2 h and evaluated by western blot analysis. BMS-777607 potently eliminated basal levels of auto-phosphorylated c-Met (Y1234/1235), with 50% inhibition (IC50) occurring at doses *10 nM and complete suppres-sion being achieved with doses greater than 0.1 lM (Fig. 2; Table 1). Total c-Met was not affected. BMS-777607 treatment also led to dose-dependent inhibition of phosphorylation of c-Met downstream signaling molecules including ERK (T202/Y204), Akt (S473), p70S6K (T389) and S6 (S234/235), whereas autophosphorylation of c-Src (Y416) and FAK (Y397) and phosphorylation of STAT3
(Y705), three other key signaling components potentially associated with c-Met signaling, remained unaffected (Fig. 2).
BMS-777607 prevents spontaneous KHT cell scattering, migration and invasion
To determine the effect of the Met inhibitor BMS-777607 on the scattering behavior of KHT cells, KHT colonies
were treated with the agent for 24 h. Compared to the scattered and ‘‘loose’’ cell clusters normally observed, KHT cell colonies exposed to BMS-777607 clearly were less dissociated as the dose of BMS-777607 increased (Fig. 3a). At a dose of 0.5 lM, cell scattering was largely inhibited by the compound, exhibiting the similar efficacy
BMS-777607
0 0.01 0.1 1 10 (M)
p-c-Met
(Y1234/1235)
1.0 0.5 0.1 0.0 0.0
c-Met
p-Akt (S473)
1.0 0.9 0.9 0.2 0.1
p-ERK(T202/Y204)
1.0 1.1 1.0 0.3 0.3
p-S6K (T389)
p-S6 (S234/235)
p-c-Src (Y416)
p-FAK (Y397)
p-STAT3 (Y705)
Actin
Fig. 2 Effect of BMS-777607 on c-Met signaling pathway. Cells were treated with BMS-777607 for 2 h. Whole cell lysates were processed for western blot analysis with respective primary antibod-ies. Actin is used as a loading control. Data represent one of two independent experiments. The band density of p-c-Met, p-Akt and p-ERK was quantified by Image J and normalized to Actin. The relative expression of the untreated control was defined as ‘‘1.0’’
Fig. 1 Effect of c-Met a b siControl siMET
knockdown on cell scattering
and motility. KHT cells were
transfected by control
(siControl) or c-Met (siMET) Scatter
western blot, with Actin as a
siRNA for 48 h. a Desired
proteins were analyzed by p-c-Met
loading control. b Transfected (Y1234/1235)
cell monolayer was scratched. c-Met
After 24 h, pictures of the
(c-Met/Actin)
‘‘wound’’ (original 1.0 0.3
magnification, 950) and p-Akt (S473)
migrated cells on the frontier
edge (original Motility
magnification, 9100) were p-ERK
shown for cell motility and (T202/Y204)
scattering, respectively
Actin
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Table 1 Fifty percent of inhibition concentration (IC50) range of BMS-777607 in KHT cells
Events IC50 (lM)
Cellular level
Scatter 0.1–0.5
Motility 0.1–0.5
Invasion \0.1
Proliferation 3.3–10
Clonogenicity 3.3–10
Molecular level
p-c-Met (Y1234/5) *0.01
p-Akt (S473) 0.1–1
p-ERK (T202/Y204) 0.1–1
p-c-Src (Y416) [10
p-FAK (Y397) [10
p-STAT3 (Y705) [10
as MET gene ablation; with the highest dose (1 lM), BMS-777607 completely prevented KHT cell spreading (Fig. 3a). To examine the impact of Met inhibition on cell migration, a ‘‘wound-healing’’ assay was performed. As shown in Fig. 3b, 0.1 and 0.5 lM of BMS-777607 signif-icantly decreased KHT cell motility (P \ 0.001), resulting in 41.8 ± 12.9 and 72.0 ± 6.3% of inhibition compared to the control, respectively. Even the lowest dose tested (0.02 lM) affected the scattering of cells that had migrated into the denuded area although at this dose the number of migrated cells was not significantly reduced (Fig. 3b). Consistent with the effect on cell migration, BMS-777607 treatment inhibited cell invasion at nanomolar concentra-tions. Compared to untreated cells, exposure to 0.01, 0.1 and 1 lM BMS-777607 led to 34.0 ± 13.0% (P \ 0.05), 55.7 ± 12.5% (P \ 0.001), and 72.0 ± 7.6% (P \ 0.001) inhibition of invasion, respectively (Fig. 3c).
BMS-777607 modestly inhibits KHT cell proliferation and colony formation
To test whether c-Met inhibition affected cell proliferation, KHT cells were incubated with a range of BMS-777607 for a period of 4 days. The results showed that only at doses exceeding 1 lM did BMS-777607 decrease cell growth (Fig. 4a). No cell death was observed with all tested doses (data not shown). To determine the effect of BMS-777607 on colony forming ability, KHT cells were treated with the agent for 7 days. Only the highest tested dose of BMS-777607 (10 lM) resulted in a significant decrease (P \ 0.001) in tumor cell plating efficiency and drug doses less than 3.3 lM had no effect (Fig. 4b).
BMS-777607 inhibits the ability of KHT sarcoma cells to form pulmonary nodules
To determine whether BMS-777607 could affect the ability of KHT cells to undergo key steps associated with the metastatic process in vivo, tumor cells were injected intra-venously (iv) followed by oral drug administration to the mice. Daily doses of 25 mg/kg of BMS-777607 resulted in a significant reduction in the number of lung nodules formed compared to the vehicle control treatment (P = 0.009) or treatment with a lower dose of BMS-777607 (10 mg/kg, P = 0.027) (Fig. 5a, left panel). The latter treatment (10 mg/kg) also offered a mild but not significant inhibition of lung nodule formation compared to the vehicle control (P = 0.25, Fig. 5a, left panel). Morphologically, multiple nodules accompanied by significant hemorrhage were observed in the lungs of mice that had not received treat-ment; a result which was clearly improved by BMS-777607 exposure (25 mg/kg, Fig. 5a, right panel). Neither 10 nor 25 mg/kg daily treatments of BMS-777607 resulted in a significant body weight loss throughout the course of the experiments (Fig. 5b). To evaluate the affect of dosing period on the drug efficacy, mice were orally given BMS-777607 (25 mg/kg) with various schedules. Daily treat-ments with BMS-777607 commencing immediately after cell inoculation and given for 1 day (D1) or 4 days (D1–4) resulted in a modest reduction in the number of tumor lung nodules formed (P = 0.096 and 0.160 vs. vehicle, respec-tively) (Fig. 5c). Delaying the initiation of treatment for a week proved ineffective at controlling lung colony growth (D8–15, P = 0.283 vs. vehicle, Fig. 5c). However, daily treatment with BMS-777607 throughout the course of the experiment (D1–15) significantly reduced the number of lung nodules formed (P = 0.013 vs. vehicle) (Fig. 5c). Consistently, when normalized to the vehicle control, a daily dose of BMS-777607 (25 mg/kg) throughout the experi-ment reduced the number of pulmonary nodules formed by 28.3 ± 14.9% (P \ 0.001) (Fig. 5d).
Discussion
Tumor invasion and metastasis are two phenotypes evoked by HGF-dependent c-Met signaling [1, 24]. Abnormal MET activation independent of HGF is known to initiate tumorigenesis [25, 26] and cause therapeutic resistance [27, 28]. In this study, we examined the therapeutic potential of a small molecule Met signaling inhibitor (BMS-777607) in a metastatic tumor model expressing constitutive c-Met that is required for the metastatic behaviors (Fig. 1). BMS-777607 was found to potently inhibit tumor cell scatter (Fig. 3a), migration (Fig. 3b) and
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Fig. 3 Effect of Met inhibitor BMS-777607 on cell scattering, migration and invasion. a KHT cells were treated with BMS-777607 or transfected with Control or MET-siRNA for
4 days. Typical picture of each sample was shown (original magnification, 950). Data represent one of three independent experiments.
b Confluent KHT monolayer was scratched and treated with BMS-777607. After 24 h, cells migrated into denuded area were scored. Typical picture of cell migration was shown (original magnification, 950). Data are from three independent experiments. C control,
BMS BMS-777607. Columns mean, bars SD (n = 12).
c Effect of BMS-777607 on cell invasion. KHT cells were seeded into Matrigel-coated transwell inserts (5 9 103/ insert) with or without BMS-777607 in the serum-free medium. Complete medium was loaded on the bottom chamber as a chemo-attractant. The underside of the filter was stained 24 h after cell loading. Typical picture of cell invasion was shown (original magnification, 950). Invading cells were scored from eight random fields (original magnification, 9100). Data are from three independent experiments. Columns mean, bars SD (n = 6). ***P \ 0.001 versus untreated control
a
C
b C
BMS (0.1M)
c C
BMS (0.1 M)
BMS-777607 siRNA
0.1 0.5 1 (µM)Control MET
BMS (0.02M)
120
(%) 100
80 ***
Motility ***
40
60
20
0
(M)
0 0.02 0.1 0.5
BMS (0.5M) BMS-777607
BMS (0.01 M)
120
(%) 100 *
80
Invasion
60 *** ***
40
20
0
(M)
0 0.01 0.1 1
BMS (1M) BMS-777607
invasion (Fig. 3c), at IC50 doses in the nanomolar range (Table 1), and to suppress cell proliferation (Fig. 4a) and clonogenic growth (Fig. 4b) with IC50 doses higher than 3.3 lM (Table 1). These data suggest that functioning as a cytostatic agent, BMS-777607 efficiently interferes with multiple functions that are associated with the metastatic spread of cancer cells.
At the molecular level, BMS-777607 potently impairs the phosphorylation of c-Met, Akt and ERK (Fig. 2), with low IC50 values that are aligned with those of spread-associated behaviors (Table 1). Furthermore, blockade of PI3K (upstream of Akt) or MEK (upstream of ERK) with inhibitors reverses spontaneous cell scattering
(Supplementary Fig. S1), suggesting that both PI3K and MEK signaling are required for c-Met-mediated functions. In contrast, consistent with some studies [19, 29–31] but not with others [17, 32–34], activities of key non-receptor tyrosine kinases such as c-Src, FAK and STAT3 were found not to be perturbed by c-Met inhibition (Fig. 2). Interestingly, while mTOR downstream signaling (p-S6K/ p-S6) is inhibited by BMS-777607 with doses less than
1 lM, no anti-proliferative effect is observed at these doses (Fig. 4), suggesting the redundant role of other signaling pathway(s) that may compensate for the S6K/S6 pathway to maintain survival and growth in response to an upstream c-Met signaling blockade.
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a
120
(%) 100
proliferation 40 ***
80
Cell 60 ***
20
0
10 (M)
0 0.1 0.33 1 3.3
BMS-777607
b 100
(%) 80
efficiency 60
Plating 40
20 ***
0
10 (M)
0 0.1 0.33 1 3.3
BMS-777607
Fig. 4 Effect of BMS-777607 on cell proliferation and clonogenicity. a KHT cells were seeded into a 96-well plate and treated with various doses of BMS-777607. Cell viability was determined by MTT assay after 96 h incubation. Columns mean, bars SD (n = 9). ***P \ 0.001 versus untreated control. b KHT cells were seeded into six-well plates and treated with BMS-777607. After 14 days incubation, cell colonies ([50 cells) were counted macroscopically. Columns mean, bars SD (n = 12). Data (both a and b) are from four independent experiments
Although Met inhibitors have been shown to inhibit growth in primary tumor xenografts [17, 18, 34], limited studies have been reported their possible anti-metastatic potential. In the present investigation we examined the impact of c-Met inhibition on the later stages of the tumor cell disseminating process (extravasation, initiation at the secondary site). BMS-777607 administered only at later times after iv cell inoculation (exposure schedule D8–15) had almost no effect on the formation of lung tumor nod-ules (Fig. 5c, d). In contrast, drug treatments commencing immediately after cell inoculation did affect the ability of KHT cells to form lung tumor nodules (Fig. 5c, d). This effect increased as the number of daily doses of BMS-777607 increased. Nevertheless, although short treatment protocols (D1, D1–4) lowered the average number of lung nodules formed, they did not gain significance (P = 0.096 and 0.160, respectively). These data suggest that early drug
treatment is not sufficient to disrupt the whole metastatic process, however, the anti-metastatic potential of BMS-777607 was still primarily due to an impairment of the relatively early steps (migration, invasion) in the metastatic process rather than subsequent later events (proliferation, clonogenicity). Indeed, the administration of BMS-777607 over the entire treatment period (D1–15) that covered both early and late events resulted in a significant decrease in lung tumor foci (Fig. 5d), suggesting that the late stage intervention is indispensible. However why c-Met activity is necessary for the subsequent other than initial steps of the secondary growth is yet known. One possible inter-pretation could be that the preexistence of exogenous HGF in the lung environment could aid to further enhance c-Met activation albeit its constitutive activity, and further pro-mote c-Met-mediated behaviors, which may attenuate the effect of BMS-777607. Supporting this assumption is the finding by us that mouse HGF does enhance KHT cell proliferation and migration in vitro, and phosphorylate c-Met receptor regardless of the high basal level of c-Met phosphorylation, which renders BMS-777607 to be less effective (data not shown). In addition, the reason why the KHT cells are resistant to the inhibitor in vivo remains to be elucidated. Based on the previous report [16], it is unlikely that the drug has poor pharmacologic profile; also, since the tumor cells are introduced directly into the blood stream, the possibility that the compound is difficult to reach the cells is minimal. Perhaps the most adequate explanation would be that other signaling pathways might be activated when tumor cells are exposed to the external signals (such as soluble growth factors and cytokines) in the lung parenchyma, which would compensate for the therapeutic effect of Met inhibition.
One unanswered question raised by these studies con-cerns how c-Met is activated in KHT cells in the absence of exogenous HGF stimulation. KHT is a fibrosarcoma cell line derived from fibrous connective tissue and fibrous tissue is characterized by immature proliferating fibroblasts that are thought to produce HGF [35]. However, the basal expression level of the HGF gene is barely detectable in KHT cells (data not shown), thus the HGF/c-Met autocrine loop is unlikely to exist. Other possibilities for the regu-lation of c-Met activity due to ligand-independent mecha-nisms such as gene amplification, activating mutation or intracellular crosstalk [3] may also exist, which remains to be examined in the KHT sarcoma model.
In summary, the current study suggested the anti-meta-static potential of the small molecule Met inhibitor BMS-777607 in a highly metastatic rodent tumor model with constitutively activated c-Met. Importantly, this agent impeded the ability of tumor cells to form pulmonary nodules after entering the blood stream in vivo. Although the compound will not advanced for further development,
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a
(number) 50 **
metastases 40
30
20
Lung 10
0
Vehicle 10 mg/kg 25 mg/kg
BMS-777607
b 1.2
weight
1.1
body 1.0
0.9
Relative
0.8 Vehicle
0.7 BMS-777607 (10 mg/kg)
BMS-777607 (25 mg/kg)
0.6
0 5 10 15
Days
(25 mg/kg) BMS-777607 Vehicle
c
Lung metastases (number)
*
100
80
60
40
20
0 Vehicle D1 D1-4 D1-15 D8-15
BMS-777607 (25 mg/kg)
d ***
(%) 120
metastases 100
80
60
Lung 40
20
0
e 1 4 5 5
l D - 1 1
c 1
- -
i D 1 8
h
e D D
V
BMS-777607 (25 mg/kg)
Fig. 5 Efficacy of BMS-777607 on metastatic lung nodules. a Right after intravenous injection of KHT cells, mice were orally given vehicle control or BMS-777607 once daily for 15 days. On day 16, tumor foci on the fixed lung tissue were counted. Three typical lungs from vehicle and BMS-777607 (25 mg/kg) group were shown. Lines median (n = 9–10). b Mouse body weight in A was monitored throughout the experiment. Relative body weight was calculated by normalizing average mice body weight in each group with weight on day of initial treatment (day 1). Lines mean, bars SD (n = 10). c After cell inoculation (day 1, D1), mice were treated with BMS-777607 (25 mg/kg) daily once with four different schedules: one dose
the presented data do support the notion that c-Met tar-geting strategies may offer potential therapeutic options to impair c-Met-dependent metastasis.
on day 1 (D1); four doses from day 1 to day 4 (D1–4); everyday treatment till the end of the experiment (D1–15); first dose started on day 8 till the end of the experiment (D8–15). For control group, mice were fed with vehicles once daily throughout the experiment (D1–15). On day 16, tumor foci on the fixed lung tissues were counted. Lines median (n = 10). d Lung foci after BMS-777607 (25 mg/kg) treatment in a and c were normalized to the vehicle control. Data are depicted in a box plot, with whiskers above and below the box indicating the 90th and 10th percentiles, respectively. P value was obtained by Wilcoxon rank sum test in a, c, d
Acknowledgments The authors acknowledge Sharon Lepler for technical assistance and Dr. Joseph Fargnoli (Bristol-Myer Squibb R&D) for scientific discussion.
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