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Author Manuscript Published OnlineFirst on April 2, 2013; DOI: 10.1158/1535-7163.MCT-12-1011 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Novel Inhibitor of c-Met and VEGF Receptor Tyrosine Kinases with a Broad

Spectrum of In Vivo Antitumor Activities

Yoshiko Awazu1, Kazuhide Nakamura1, Akio Mizutani1, Yuichi Kakoi1, Hidehisa

Iwata2, Seiji Yamasaki2, Naoki Miyamoto3, Shinichi Imamura1, Hiroshi Miki2, and

Akira Hori1

1Oncology Drug Discovery Unit, 2Discovery Research Laboratories, and 3Medicinal

Chemistry Laboratories, Takeda Pharmaceutical Company Co. Ltd., Fujisawa,

Kanagawa, Japan

Note: Supplementary data for this article are available at Molecular Cancer

Therapeutics.

Online (http://mct.aacrjournals.org/).

Y. Awazu, K. Nakamura, and A Mizutani contributed equally to this work.

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 2, 2013; DOI: 10.1158/1535-7163.MCT-12-1011 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Corresponding Author: Kazuhide Nakamura, Oncology Drug Discovery Unit, Takeda

Pharmaceutical Company, Ltd., 2-26-1, Muraokahigashi, Fujisawa, Kanagawa,

251-8555, Japan. Phone: 81-466-32-2609; Fax: 81-466-29-4410; E-mail:

[email protected]

Running Title: Wide Antitumor Spectrum of a Dual c-Met and VEGFR2 Inhibitor

Key Words: c-Met inhibitor; VEGF receptor inhibitor; T-1840383; hypoxia; invasive

growth

Word count, excluding references but including main figure legends: 5,512

Total number of figures and tables: 6 figures, 1 table

Supplemental figures: 5

Supplemental table: 2

Disclosure of potential conflict of interest: All of the authors are employees of Takeda

Pharmaceuticals Company, Ltd.

Abstract

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF),

are dysregulated in a wide variety of human cancers and are linked with tumorigenesis

and metastatic progression. Vascular endothelial growth factor (VEGF) also plays a key

role in tumor angiogenesis and progression by stimulating the proangiogenic signaling

of endothelial cells via activation of VEGF receptor tyrosine kinases (VEGFRs).

Therefore, inhibiting both HGF/c-Met and VEGF/VEGFR signaling may provide a

novel therapeutic approach for treating patients with a broad spectrum of tumors.

Toward this goal, we generated and characterized T-1840383, a small-molecule kinase

inhibitor that targets both c-Met and VEGFRs. T-1840383 inhibited HGF-induced c-Met

phosphorylation and VEGF-induced VEGFR-2 phosphorylation in cancer epithelial

cells and vascular endothelial cells, respectively. It also inhibited constitutively

activated c-Met phosphorylation in c-met-amplified cancer cells, leading to suppression

of cell proliferation. In addition, T-1840383 potently blocked VEGF-dependent

proliferation and capillary tube formation of endothelial cells. Following oral

administration, T-1840383 showed potent antitumor efficacy in a wide variety of human

tumor xenograft mouse models, along with reduction of c-Met phosphorylation levels

and microvessel density within tumor xenografts. These results suggest that the efficacy

of T-1840383 is produced by direct effects on tumor cell growth and by an

antiangiogenic mechanism. Furthermore, T-1840383 showed profound antitumor

activity in a gastric tumor peritoneal dissemination model. Collectively, our findings

indicate the therapeutic potential of targeting both c-Met and VEGFRs simultaneously

with a single small-molecule inhibitor for the treatment of human cancers.

Introduction

The c-Met receptor tyrosine kinase (RTK) is the receptor for hepatocyte growth

factor/scatter factor (HGF; reviewed in ref. 1). Binding of HGF to c-Met induces c-Met

dimerization and phosphorylation of multiple tyrosine residues in the intracellular

region, leading to activation of c-Met signaling pathways. c-Met signaling plays

important roles in cell growth, survival, motility, and morphogenesis during embryonic

development as well as in cancer cell proliferation, invasion, metastasis, tumor

angiogenesis, and drug resistance (reviewed in refs. 1 and 2). Aberrant c-Met signaling,

resulting from met genomic amplification, c-Met or HGF overexpression, or c-Met

mutations, has been observed in a variety of human cancers (1, 2). Clinically,

dysregulation of c-Met is correlated with a poor prognostic outcome (1, 2). Recently,

constitutive activation of c-Met due to met amplification was also found to be a driver

of cell proliferation and survival in several gastric cancer cell lines (3, 4) and has

additionally been linked to acquired resistance to epidermal growth factor inhibitors in

lung cancers (5, 6). Thus, c-Met has been considered an appealing molecular target for

cancer therapy (2, 7–9).

The receptors for vascular endothelial growth factor (VEGF), VEGF receptor 1

(VEGFR-1) and VEGF receptor 2 (VEGFR-2), are members of the RTK family; they

are mostly located in endothelial cells (reviewed in ref. 10). VEGFR activation occurs

through VEGF binding, which triggers receptor dimerization, tyrosine kinase activation,

and phosphorylation of tyrosine residues. Of the two VEGFRs, VEGFR-2 is believed to

be a major driver of tumor angiogenesis, which plays important roles in tumor

malignancy (such as sustaining tumor growth) and in blood-borne metastasis (11).

VEGF expression is upregulated by changes associated with cancer, including hypoxia,

proto-oncogene activation, loss of tumor suppressor gene expression, and growth factor

stimuli in tumors (12, 13). Its overexpression has been reported to correlate with the

degree of vascularity, aggressive disease, and poor prognosis in the majority of human

solid tumors (14–17), making the VEGF/VEGFR axis an attractive molecular target for

cancer therapy. Indeed, the monoclonal anti-VEGF antibody bevacizumab (18) and

several RTK inhibitors targeting VEGFRs, such as sunitinib (19), sorafenib (20), and

pazopanib (21), have proven to be efficacious in clinical settings.

The above-mentioned findings highlight the possibility that simultaneous inhibition

of c-Met and VEGFRs by a small-molecule inhibitor may result in broader and more

potent antitumor efficacy. Here, we report the characterization of T-1840383, a novel

ATP-competitive multitargeted kinase inhibitor that preferentially inhibits c-Met and

VEGFR-2, in various functional cellular assays and the evaluation of its in vivo

antitumor activities in various tumor xenograft models.

Materials and Methods

T-1840383

N-[4-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-a]pyridin-6-yl}oxy)-3-fluoroph

enyl]-6-methyl-2-oxo-1-phenyl-1,2-dihydropyridine-3-carboxamide hydrochloride

(T-1840383, Fig. 1A) was synthesized at Takeda Pharmaceutical Company, Ltd. For in

vitro studies, T-1840383 was dissolved in dimethyl sulfoxide (DMSO) and diluted in

culture medium immediately before use. For in vivo studies, T-1840383 was suspended

in vehicle (0.5% methyl cellulose in distilled water), and the suspension was

administered to animals within a week of preparation.

Kinase inhibition assays

Kinase inhibition of c-Met, VEGFRs, Tie-2 and Flt-3 was investigated using

AlphaScreen tyrosine kinase technology (PerkinElmer). To evaluate kinase selectivity, a

single concentration (1 µmol/L) of T-1840383 was tested using 256 kinases by

TM KinaseProfiler (Millipore), and then IC50 values for the selected 66 kinases were

determined using KinaseProfiler IC50Express™ (Millipore).

Cell lines

Unless otherwise mentioned, cells were obtained from the American Type Culture

Collection. Five gastric cancer cell lines (GSU, Kato III, KE-97, MKN45, and

NUGC-4) were obtained from the RIKEN BioResource Center, and four gastric cancer

cell lines (SNU-484, -620, -638, and -668) were obtained from the Korean Cell Line

Bank. EBC-1 squamous cell lung cancer cells and SUIT-2 pancreatic cancer cells were

obtained from the Health Science Research Resources Bank. OE33 esophageal

adenocarcinoma cells were obtained from the European Collection of Animal Cell

Cultures. Human umbilical vein endothelial cells (HUVECs) and normal human dermal

fibroblasts (NHDFs) were purchased from Cambrex. All cells were cultured in

recommended media and serum concentration, unless otherwise indicated. No

authentication was done by the authors.

Tube formation assay

HUVECs (1,000 cells/well) and NHDFs (10,000 cells/well) were co-cultured in

96-well plates with 60 ng/mL VEGF (R&D Systems) and designated concentrations of

T-1840383 for 7 days. After fixation with 70% ethanol, the cells were incubated with

anti-human CD31 monoclonal antibody (R&D Systems) followed by Alexa Fluor

488-conjugated anti-mouse IgG polyclonal antibody (Invitrogen). Tube formation by

endothelial cells was visualized using fluorescence imaging (Discovery-1 system,

Molecular Devices) according to the manufacturer’s protocol, “Angiogenesis Tube

Formation.” The filament length and number of branch points were quantified by using

Metamorph® software (Molecular Devices).

Cell scattering assay and E-cadherin expression analysis

A549 cells were seeded onto poly-lysine-coated glass-bottom dishes (2,000

cells/dish) and were allowed to form colonies of appropriate size for 5 days. Controls or

T-1840383 were added to each dish. After a 1-h incubation, 50 ng/mL HGF (R&D

Systems) was added to the dishes. Cell scattering was stimulated for 48 h under a mild

hypoxic condition (3% O2). Cells were stained using a Diff-Quik stain kit (Sysmex). For

E-cadherin expression analysis, scattering-stimulated cells were fixed with 4%

paraformaldehyde and were subjected to immunofluorescence staining using

anti-E-cadherin primary antibody and Alexa Fluor 488-conjugated anti-rabbit IgG

antibody. Nucleic acid was stained with propidium iodide.

Matrigel invasion assay

Cell invasion was assessed using Matrigel Invasion Chambers (BD Biosciences).

A549 cells or SUIT-2 cells were placed in the upper chamber, and T-1840383 was added

to both the upper and lower chambers. Cell invasion was then initiated by adding HGF

to the bottom chamber (50 ng/mL). After 72 h, noninvading cells were removed with

cotton swabs. Cells that invaded through the Matrigel were stained using a Diff-Quik

stain kit, and the number of invading cells was quantified using WinROOF software

(Mitani Corp.).

Cell proliferation assay

Cancer cells and HUVECs were seeded in 96-well plates in appropriate media with

10% fetal bovine serum (FBS) and human endothelial serum-free medium (Invitrogen),

respectively. The following day, serial dilutions of T-1840383 or 0.1% DMSO were

added to each well. Cancer cells were then incubated for a further 72 h. Proliferation of

HUVECs was stimulated with 60 ng/mL recombinant human VEGF for 120 h. After

incubation, cell proliferation was determined using a Cell Counting Kit-8 (DOJINDO

Laboratories). IC50 values were calculated by nonlinear regression analysis using

GraphPad Prism (GraphPad Software, Inc.).

Western blotting

Antibodies were purchased from Cell Signaling Technology. For analysis of A549

cells and HUVECs, c-Met and VEGFR2 phosphorylation were stimulated with 50

ng/mL HGF for 10 min and 100 ng/mL VEGF for 5 min, respectively. All cells were

lysed in RIPA buffer (Pierce) with protease and phosphatase inhibitors. In the case of

the tumor samples, tissues were homogenized in RIPA buffer with protease and

phosphatase inhibitors. Samples were then subjected to western blot analysis. Detection

was performed using enhanced chemiluminescence reagent (GE Healthcare).

Tumor xenograft models

All animal experiments were conducted according to the guidelines of the Takeda

Experimental Animal Care and Use Committee. Athymic nude mice (BALB/cA

Jcl-nu/nu) were purchased from Japan CLEA. For tumor models, cancer cells in Hanks’

balanced salt solution (HBSS; Gibco, Invitrogen Corp.) were subcutaneously (s.c.)

inoculated into the hind flank of 6- to 7-week-old mice (day 0). For the study with

MKN45, cells were suspended in a 1:1 mixture of Matrigel and HBSS and then

inoculated. After the tumors were established, mice bearing tumors of appropriate size

were randomized. Once-daily oral administration of T-1840383 or vehicle was initiated

on the next day after randomization. Tumor volume was measured twice weekly with

Vernier calipers and calculated as (length × width2) × 0.5. Percentage treated/control

ratio (T/C%) was calculated by dividing the change in volume of the tumors in the

treated mice by the change in volume in the mice treated by vehicle, and statistical

analysis was performed as described previously (22).

For a peritoneal dissemination model, NUGC-4 cells expressing luciferase

(NUGC-4-luc cells) were generated by stable transfection and inoculated via

intraperitoneal (i.p.) injection into nude mice (1 × 106 cells). Daily oral administration

(5 days per week) of T-1840383 at 5 mg/kg or vehicle was initiated 2 weeks after

inoculation. Tumor growth was monitored based on determination of emitted

bioluminescence (photons/s) 10 min after i.p. administration of D-luciferin (1.5 mg/kg).

Mice were euthanized when they became moribund. Statistical analysis was performed

by a Log-rank test using GraphPad Prism.

Immunohistochemistry

Mice (BALB/cA Jcl-nu/nu) bearing A549 tumors were treated with T-1840383 at the

indicated doses for 3 days. The dissected tumors were fixed in paraformaldehyde and

then paraffin-embedded. For visualization of the vessels, immunohistochemical staining

for CD31 was performed using rabbit anti-CD31 polyclonal antibody (Spring

Bioscience) and the immunoperoxidase technique with diaminobenzidine

tetrahydrochloride as the chromagen. Ki67 staining for proliferating cells and TUNEL

for apoptotic cells were performed using anti-Ki67 antibody (Dako) and ApopTag®

Peroxidase In Situ Apoptosis Detection Kit (Millipore), respectively. Stained sections

were scanned with a NanoZoomer Digital Pathology system (Hamamatsu Photonics).

Image processing was performed by analyzing four or five randomly selected fields of

each tumor using WinROOF software (Mitani Corporation).

Results

T-1840383 is a potent kinase inhibitor that targets both c-Met and VEGF

receptors.

The selectivity of T-1840383 (Fig. 1A) was profiled against a panel of protein

kinases and lipid kinases (Table 1). T-1840383 inhibited c-Met tyrosine kinase with an

IC50 value of 1.9 nmol/L. It also inhibited VEGF receptor tyrosine kinases with IC50

values of 7.7, 2.2, and 5.5 nmol/L for VEGFR-1, -2, and -3, respectively. The structure

of T-1840383 has little in common with other VEGFR or c-Met selective inhibitors,

except for TAK-593 (Supplementary Fig. S1). T-1840383 was found to inhibit several

kinases including c-Mer, Ret, Ron, Res, Tie-2, and TrkA as well as mutants of Abl,

c-Kit, FGFR2, and PDGFRα with similar IC50 values ( 10 nmol/L) to those for c-Met

and VEGFRs.

T-1840383 inhibits HGF-induced c-Met phosphorylation and HGF-dependent

cellular phenotypes

In cell-based assays using A549 cells, T-1840383 inhibited HGF-stimulated c-Met

phosphorylation with an IC50 value of 8.8 nmol/L (Fig. 1B). It also inhibited A549 cell

scattering stimulated with HGF (Fig. 1C). Since HGF signaling has been reported to

trigger destabilization of adherens junctions by downregulation of E-cadherin (23),

expression level of E-cadherin was examined in a similar cell-scattering assay. As

expected, HGF-stimulated cell scattering was accompanied by diminished expression of

E-cadherin on cell membrane, which was inhibited by T-1840383 in a

concentration-dependent manner (Fig. 1D). T-1840383 also displayed similar potency

against HGF-induced A549 cell invasion in a Matrigel invasion assay (Fig. 1E and

Supplementary Fig. S2).

T-1840383 inhibits c-Met-dependent cell proliferation in vitro

The effects of T-1840383 on cell proliferation were evaluated using a panel of cancer

cell lines and HUVECs. The antiproliferative potency varied widely, with the IC50

values ranging from 1.8 nmol/L to >10,000 nmol/L (Fig. 1F). With the exception of

HUVECs, all T-1840383-sensitive cell lines (IC50 ≤25 nmol/L; SNU-5, SNU-638,

MKN45, NUGC-4, and Hs746T gastric cancer cells as well as EBC-1 non-small cell

lung carcinoma cells) were shown to overexpress constitutively activated c-Met protein

(Supplementary Fig. S3A). These cell lines are also known to harbor met genomic

amplification (3, 4, 24, 25), suggesting that constitutive activation of c-Met is dependent

on genomic amplification. In MKN45 cells, T-1840383 inhibited activation of c-Met

and the downstream effectors ERK1/2 and AKT in a concentration-dependent manner

(Supplementary Fig. S3B). In contrast, OE33 esophageal adenocarcinoma cells and

KATOIII and SNU-620 gastric cancer cells were much less sensitive to T-1840383

despite the fact that c-Met was constitutively active in those cell lines. Other insensitive

cell lines (IC50 >10,000 nmol/L) did not show constitutively activated c-Met. These

results suggest that constitutively active c-Met is a necessary but not sufficient predictor

of in vitro cancer cell response to T-1840383.

T-1840383 inhibits VEGF receptor-2 phosphorylation and VEGF signaling in

HUVECs

Because T-1840383 also potently inhibits VEGF receptor tyrosine kinases, the effects

on VEGF signaling were tested in endothelial cells. As shown in Fig. 2A, T-1840383

inhibited VEGF-induced phosphorylation of VEGF receptor-2 (IC50 = 0.86 nmol/L). It

also blocked VEGF-induced activation of the downstream effectors ERK1/2 and AKT

in a concentration-dependent manner. Consistent with this, T-1840383 inhibited

VEGF-driven HUVEC proliferation with an IC50 value of 1.8 nmol/L (Fig. 1E). When

co-cultured with fibroblasts, HUVECs formed capillary-like structures in response to

VEGF, which was, however, abolished by the presence of T-1840383 (Fig. 2B).

T-1840383 inhibited the VEGF-induced increase in total length of tubes and number of

blanch points with an IC50 value of 12 and 8.3 nmol/L, respectively (Fig. 2C).

T-1840383 inhibits in vivo c-Met signaling and tumor growth in mouse xenograft

models

To assess the in vivo potency of T-1840383 for inhibiting c-Met activation, the level

of c-Met phosphorylation in MKN45 tumors was measured at several time points

following a single oral administration of T-1840383 (Fig. 3A). T-1840383

administration at a dose of 2 mg/kg resulted in substantial inhibition of c-Met

phosphorylation from 2 to 8 h, with full recovery by 24 h. In addition, dose-dependent

inhibition of c-Met, AKT, and ERK1/2 phosphorylation was observed 4 h after

T-1840383 administration (Fig. 3B). Pharmacokinetic analysis showed that the plasma

concentration of T-1840383 increased in a dose-dependent manner (over the dose range,

1–20 mg/kg; Supplementary Table S1). The tumor concentration of T-1840383 was

lower than that in plasma, but relatively sustained (Supplementary Fig. S4). Tmax in

tumors was achieved between 2 and 8 h after dosing. Tmax and the inhibition of c-Met

phosphorylation in tumors were well correlated (Fig. 3A). In the MKN45 tumor

xenograft mouse model, once-daily 2-week administration resulted in a dose-dependent

inhibition of tumor growth with the T/C values of 57%, 30%, and 4% at doses of 0.5,

1.5, and 5 mg/kg, respectively (Fig. 3C). T-1840383 caused tumor regression at 15

mg/kg. Additional tumor models were employed to assess the potency of T-1840383

against c-Met-dependent or -independent tumors: EBC-1 cells expressed elevated levels

of constitutively active c-Met and were highly sensitive to T-1840383 in vitro (Fig. 1F

and Supplementary Fig. S3A), while COLO 205 cells showed no sign of constitutively

active c-Met and were insensitive to T-1840383 in vitro (Fig. 1F and Supplementary Fig.

S3A). U-87 MG cells are reported to express both HGF and c-Met, and that their

proliferation and/or tumor growth are at least partially driven by the HGF/c-Met

autocrine loop (26); in the present study, T-1840383 showed marked antitumor activities

in a dose-dependent manner in c-Met-dependent EBC-1 and U-87 MG tumor models

(Fig. 3D and 3E). The T/C values for EBC-1 tumors were 85% and 26% at 0.5 and 1.5

mg/kg, respectively, and the tumors regressed by 22% and 52% at 5 mg/kg and 15

mg/kg, respectively. The T/C values for U-87 MG tumors were 72%, 32%, and 10% at

0.5, 1.5, and 5 mg/kg, respectively, and the tumors were almost static at 15 mg/kg. In

the c-Met-independent COLO 205 tumor model, T-1840383 showed slightly less potent

but still substantial antitumor activity than it did in the c-Met-dependent tumor models,

where the T/C values were 30% and 13% at 5 mg/kg and 15 mg/kg, respectively (Fig.

3F). A slight but statistically significant difference was observed in the body weight

between vehicle- and T-1840383-treated EBC-1 tumor xenograft model mice at 15

mg/kg (2.3% loss vs. 7.9% gain compared to their initial body weight). However,

T-1840383 was generally well tolerated at the efficacious doses without obvious signs

of animal discomfort in all subcutaneous tumor xenograft models.

T-1840383 inhibits tumor angiogenesis

To further understand the mechanism of tumor growth inhibition by T-1840383,

especially in c-Met-independent tumor models, tumor angiogenesis was analyzed. A

single oral administration of T-1840383 at 2 mg/kg resulted in a significant decrease in

phosphorylated VEGFR-2 levels in the tumors of ectopically VEGFR-2-expressing 293

cells (Supplementary Fig. S5), suggesting that T-1840383 inhibits in vivo VEGFR

activation at the efficacious doses. We next tested the antiangiogenic activities in tumor

xenografts of the in vitro T-1840383-insensitive cell line A549. Following a 3-day

treatment, T-1840383 exhibited significant antitumor activities with T/C values of 64%,

51%, and 35% at 0.5, 1.5, and 5 mg/kg, respectively (data not shown). T-1840383

treatment resulted in a dose-dependent reduction in the microvessel density in viable

regions of the tumors by 29%, 50%, and 65% at 0.5, 1.5, and 5 mg/kg, respectively (Fig.

4A and 4B). In similar dose-dependent manners, T-1840383 treatment also led to a

decreased tendency of cell mitosis (Ki67) and a significant increase in apoptosis

(TUNEL) in the A549 tumor xenografts (Fig. 4C and 4D). Together, these results

suggest that targeting tumor angiogenesis is one of the mechanisms by which

T-1840383 inhibits growth of c-Met-independent tumors in vivo.

Antitumor activities of T-1840383 in a gastric tumor peritoneal dissemination

model

To extend the finding that T-1840383 potently inhibits the growth c-Met-dependent

cancer cells, T-1840383 was further evaluated in a mouse model of peritoneal

dissemination using c-Met-dependent gastric cancer NUGC-4-luc cells. In this model,

tumor growth was monitored noninvasively with bioluminescence emitted from

NUGC-4-luc cells in mice. As shown in Fig. 5A and 5B, vehicle-treated mice showed a

significant increase in tumor burden over the monitoring time from day 14 to day 41

(1.26 × 107/s vs. 3.63 × 107/s, p <0.01). In contrast, the T-1840383-treated mice did not

show a statistically significant increase in tumor burdens (1.30 × 107/s vs. 2.02 × 107/s).

In addition, T-1840383 treatment resulted in a prolonged survival of mice compared to

vehicle treatment (Fig. 5C, median survival: 46.5 days vs. 108.5 days, p <0.01). In

vehicle-treated mice, on the basis of in vivo imaging analysis, one mouse apparently

became tumor-free and showed an exceptionally longer survival than the others,

probably due to rejection of the tumor challenge.

c-Met expression and activation under hypoxia

Hypoxia, which is known to be a major stimulator of VEGF and consequent

angiogenesis, has been shown to promote invasive tumor growth by transcriptional

activation of c-met and by sensitizing cells to HGF stimulation (27). To explore this

finding in our systems, we investigated c-Met expression and HGF-induced cell

invasion in response to hypoxia. Hypoxia upregulated c-Met expression compared to

normoxia (Fig. 6A). Moreover, we found that hypoxia-induced c-Met phosphorylation

in A549 cancer cells without HGF stimulation while serum starvation had no such

effects on c-Met expression and activation. Similar effects were seen in Caki-1 renal

cell carcinoma, SUIT-2 pancreatic cancer, and U-87 MG glioma cells (Fig. 6B). In

addition, hypoxia augmented SUIT-2 cell invasion induced by HGF compared to

normoxia (Fig. 6C), suggesting functional relevance for the c-Met pathway in cancer

cell invasive growth under hypoxia.

Discussion

Here, we have characterized T-1840383, a novel multitargeted kinase inhibitor, in

various functional assays. T-1840383 potently blocked c-Met activation and

downstream signaling in cancer cells while cell proliferation assay revealed that

T-1840383 selectively inhibited cancer cells that harbored c-met gene amplification.

Recent studies have demonstrated that some gastric and lung cancer types harboring

c-met amplification are “addicted” to the constitutively activated c-Met signals and that

targeting such activated c-Met with small-molecule inhibitors results in marked

inhibition of cell growth and survival (3, 28). Thus, the response profile of T-1840383

may well reflect the pharmacologic consequence of inhibition of c-Met rather than other

targets. However, T-1840383 failed to display antiproliferative activities against some

c-met-amplified cancer cells despite the fact that they expressed constitutively activated

c-Met, suggesting that c-Met amplification/activation alone may not necessarily

determine the response. Such resistance may be attributed to collateral activation of

HER family members (29, 30), amplification of wild-type KRAS (31), or c-Met

mutations that affect the inhibitor binding (32, 33), although these speculations need to

be further investigated. The response profile we observed here may provide a tool to

help identify reliable predictive markers, sensitive patient populations for T-1840383 or

other c-Met inhibitors currently under clinical evaluation (reviewed in refs. 2, 7–9), and

effective combination therapy for specific patients in the clinical setting.

We also demonstrated the in vivo antitumor efficacies of T-1840383. In the tumor

xenograft models using in vitro T-1840383-sensitive cancer cells, T-1840383 treatment

resulted in potent tumor growth inhibition, in agreement with attenuation of c-Met

phosphorylation levels and downstream signaling in the tumors, suggesting that such

potent efficacies primarily depend on direct inhibition of c-Met-driven tumor cell

growth. In clinical settings, patients with gastric cancers having scirrhous-type stroma

particularly showed poor prognosis even after curative resection; they also showed

highly progressed peritoneal dissemination (34). This type of cancer has been observed

to often possess c-met gene amplification (35). T-1840383 showed a potent antitumor

efficacy in a peritoneal tumor dissemination model for a c-met-amplified gastric cancer,

leading to a survival benefit in those mice. These results provide a rationale for further

investigation of T-1840383 as an antitumor agent for patients with such types of gastric

cancer.

In addition, our findings indicate that in vivo efficacy may also depend on the

antiangiogenic properties. Indeed, T-1840383 potently inhibited endothelial cell

responses to VEGF stimuli in vitro and decreased tumor microvessel density in vivo at

the efficacious doses, even in the c-Met-independent tumor models. It is therefore clear

that T-1840383 affects tumor angiogenesis as a VEGFR inhibitor. In addition, the c-Met

inhibitory activity may contribute to angiogenesis inhibition because HGF/c-Met

signaling induces tumor angiogenesis by directly inducing proliferation and migration

of endothelial cells; by inducing expression of VEGF; as well as by downregulating

thrombospondin 1, a negative regulator of angiogenesis (36). T-1840383 has a strong

inhibitory effect on other angiokinases such as Tie-2 and PDGFR and thus such

inhibitory activities may also be involved in the antiangiogenic property of T-1840383.

We believe that inhibition of angiogenesis represents a mechanism by which T-1840383

manifests a wide spectrum of its antitumor activities. Indeed, compared to the selective

inhibitors of VEGFR or c-Met, T-1840383 showed a broad in vivo antitumor spectrum

(Supplementary Table S2).

Several small-molecule inhibitors, including cabozantinib/XL184 (37) and

foretinib/XL880 (38), that simultaneously target c-Met and VEGFRs have been

previously reported. Cabozantinib appears to have a stronger inhibitory activity against

VEGFR2 than against c-Met (e.g., IC50 values: 0.035 vs. 1.3 nmol/L, respectively),

whereas T-1840383 has very similar activities against them (2.2 vs. 1.9 nmol/L). Similar

to T-1840383, foretinib has similar inhibitory activities against VEGFR2 and c-Met (0.4

vs. 0.86 nmol/L). While foretinib is required to be administered at a dose of

approximately 100 mg/kg to effectively suppress c-Met and VEGFR activity in vivo,

T-1840383 suppresses their activities in similar in vivo models at much lower doses

(around 2 mg/kg). This finding may be attributed to more potent cellular activities

and/or favorable PK profile of T-1840383.

HGF is a potent inducer of epithelial-mesenchymal transition (EMT) (39) that

facilitates most of the invasive growth functions of cancer, such as loss of adhesive

junctions, motility, angiogenesis, and survival (reviewed in ref. 40). Thus, the ability of

T-1840383 to block HGF-induced cell scattering and invasion may have important

implications for treatment of the invasive growth and metastasis of cancer, although this

remains to be confirmed experimentally. In the tumor xenograft models we used here,

the HGF/c-Met paracrine loop was nearly absent due to the low affinity of mouse HGF

derived from host stromal cells to the human c-Met on implanted cancer cells (41). It is

therefore necessary to employ relevant preclinical models, such as tumor allograft

models or xenograft models in HGF-transgenic or knock-in mice (42), where the

HGF/c-Met paracrine loop is functional between stroma and cancer cells.

We further investigated c-Met signaling in response to hypoxia because hypoxia has

been reported to promote invasive growth by transcriptional activation of c-Met (27). In

agreement with previous studies on this subject, hypoxia indeed upregulated c-Met

expression and promoted HGF-induced cell invasion. Moreover, we found that hypoxia

elevated c-Met phosphorylation levels even in the absence of HGF stimuli, although this

may be partly explained through the recent findings that hypoxia promotes interaction

between c-Met and neuropilin-1, a coreceptor for VEGF-A, which in turn facilitates

VEGF-induced c-Met activation (43). These results indicate a key role of c-Met

activation in response to the hypoxic stress of cancer cells. Importantly, it has been

suggested that hypoxia generated by inhibition of angiogenesis triggers pathways that

make tumors more aggressive and metastatic (44, 45). Thus, it is possible that c-Met

activation under hypoxia is a mechanism of refractoriness to antiangiogenic treatment

by inducing a tumor-invasive switch (46). In addition, a recent study suggests that an

alternative angiogenic pathway, via c-Met activation in endothelial cells, is involved in

resistance/low-responsiveness to antiangiogenic treatment (47). Collectively,

pharmacological intervention of c-Met signaling is a potential strategy to prevent

tumors from progressing under hypoxia, an adverse tumor microenvironment condition,

and to circumvent resistance to angiogenesis inhibitors. This strategy clearly warrants

further exploration.

In summary, T-1840383 is a novel, highly potent inhibitor of c-Met and VEGFR

tyrosine kinases. Through this unique spectrum of dual kinase inhibition, T-1840383

showed in vivo broad and potent antitumor activities by targeting tumor epithelial cells

and vasculature. Our findings indicate the therapeutic potential of targeting both c-Met

and VEGFRs simultaneously with a single small-molecule inhibitor for treatment of

various human cancers.

Acknowledgements

We would like to thank Yuka Yarita for excellent technical assistance; Shigemitsu

Matsumoto, Hideyuki Oki, and Takeshi Watanabe for helpful discussion; Ryujiro Hara

for critical reading of the manuscript; and Yuichi Hikichi, Osamu Nakanishi, Syuichi

Furuya, and Hideaki Nagaya for their guidance and support during the course of this

work.

References

1. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility

and more. Nat Rev Mol Cell Biol 2003;4:915–25.

2. Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and

characterization of inhibitors for therapeutic intervention. Cancer Lett 2005;225:1–26.

3. Smolen GA, Sordella R, Muir B, Mohapatra G, Barmettler A, Archibald H, et al.

Amplification of MET may identify a subset of cancers with extreme sensitivity to the

selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci U S A

2006;103:2316–21.

4. Lutterbach B, Zeng Q, Davis LJ, Hatch H, Hang G, Kohl NE, et al. Lung cancer cell

lines harboring MET gene amplification are dependent on Met for growth and survival.

Cancer Res 2007;67:2081–8.

5. Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET

amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.

Science 2007;316:1039–43.

6. Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L, et al. MET amplification occurs

with or without T790M mutations in EGFR mutant lung tumors with acquired resistance

to gefitinib or erlotinib. Proc Natl Acad Sci U S A 2007;104:20932–7.

7. Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors:

targeting oncogene addiction and expedience. Nat Rev Drug Discov 2008;7:504–16.

8. Eder JP, Vande Woude GF, Boerner SA, LoRusso PM. Novel therapeutic inhibitors of

the c-Met signaling pathway in cancer. Clin Cancer Res 2009;15:2207–14.

9. Cecchi F, Rabe DC, Bottaro DP. Targeting the HGF/Met signalling pathway in cancer.

Eur J Cancer 2010;46:1260–70.

10. Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation of angiogenesis

via vascular endothelial growth factor receptors. Cancer Res 2000;60:203–12.

11. Shibuya M, Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of

angiogenesis and lymphangiogenesis. Exp Cell Res 2006;312:549–60.

12. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med

2003;9:669–76.

13. Harris AL. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer

2002;2:38–47.

14. Weidner N. Intratumor microvessel density as a prognostic factor in cancer. Am J

Pathol 1995;147:9–19.

15. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical

cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin

Oncol 2002;20:4368–80.

16. Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic,

predictive, and therapeutic implications. Lancet Oncol 2001;2:667–73.

17. Hasan J, Byers R, Jayson GC. Intra-tumoural microvessel density in human solid

tumours. Br J Cancer 2002;86:1566–77.

18. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al.

Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal

cancer. N Engl J Med 2004;350:2335–42.

19. Motzer RJ, Michaelson MD, Redman BG, Hudes GR, Wilding G, Figlin RA, et al.

Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor

receptor and platelet-derived growth factor receptor, in patients with metastatic renal

cell carcinoma. J Clin Oncol 2006;24:16–24.

20. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in

advanced clear-cell renal-cell carcinoma. N Engl J Med 2007;356:125–34.

21. Sternberg CN, Davis ID, Mardiak J, Szczylik C, Lee E, Wagstaff J, et al. Pazopanib in

locally advanced or metastatic renal cell carcinoma: results of a randomized phase III

trial. J Clin Oncol, 2010;28:1061–8.

22. Awazu Y, Mizutani A, Nagase Y, Iwata H, Oguro Y, Miki H, et al. A novel pyrrolo[3,

2-d]pyrimidine derivative, as a vascular endothelial growth factor receptor and

platelet-derived growth factor receptor tyrosine kinase inhibitor, shows potent antitumor

activity by suppression of tumor angiogenesis. Cancer Sci. 2012;103:939–44.

23. Miura H, Nishimura K, Tsujimura A, Matsumiya K, Matsumoto K, Nakamura T, et al.

Effects of hepatocyte growth factor on E-cadherin-mediated cell-cell adhesion in

DU145 prostate cancer cells. Urology 2001;58:1064–9.

24. Park M, Park H, Kim WH, Cho H, Lee JH. Presence of autocrine hepatocyte growth

factor-Met signaling and its role in proliferation and migration of SNU-484 gastric

cancer cell line. Exp Mol Med 2005;37:213–9.

25. Asaoka Y, Tada M, Ikenoue T, Seto M, Imai M, Miyabayashi K, et al. Gastric cancer

cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain

deletion. Biochem Biophys Res Commun 2010;394:1042–6.

26. Burgess T, Coxon A, Meyer S, Sun J, Rex K, Tsuruda T, et al. Fully human monoclonal

antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte

growth factor/c-Met-dependent human tumors. Cancer Res 2006;66:1721–9.

27. Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, Comoglio PM.

Hypoxia promotes invasive growth by transcriptional activation of the met

protooncogene. Cancer Cell 2003;3:347–61.

28. McDermott U, Sharma SV, Dowell L, Greninger P, Montagut C, Lamb J, et al.

Identification of genotype-correlated sensitivity to selective kinase inhibitors by using

high-throughput tumor cell line profiling. Proc Natl Acad Sci U S A

2007;104:19936–41.

29. Bachleitner-Hofmann T, Sun MY, Chen CT, Tang L, Song L, Zeng Z, et al. HER kinase

activation confers resistance to MET tyrosine kinase inhibition in MET

oncogene-addicted gastric cancer cells. Mol Cancer Ther 2008;7:3499–508.

30. McDermott U, Pusapati RV, Christensen JG, Gray NS, Settleman J. Acquired resistance

of non-small cell lung cancer cells to MET kinase inhibition is mediated by a switch to

epidermal growth factor receptor dependency. Cancer Res 2010;70:1625–34.

31. Cepero V, Sierra JR, Corso S, Ghiso E, Casorzo L, Perera T, et al. MET and KRAS gene

amplification mediates acquired resistance to MET tyrosine kinase inhibitors. Cancer

Res 2010;70:7580–90.

32. Berthou S, Aebersold DM, Schmidt LS, Stroka D, Heigl C, Streit B, et al. The Met

kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different

receptor mutated variants. Oncogene 2004;23:5387–93.

33. Zimmer Y, Vaseva AV, Medová M, Streit B, Blank-Liss W, Greiner RH, et al.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor

SU11274. Cancer Lett 2010;289:228–36.

34. Maehara Y, Moriguchi S, Orita H, Kakeji Y, Haraguchi M, Korenaga D, et al. Lower

survival rate for patients with carcinoma of the stomach of Borrmann type IV after

gastric resection. Surg Gynecol Obstet 1992;175:13–6.

35. Kuniyasu H, Yasui W, Kitadai Y, Yokozaki H, Ito H, Tahara E. Frequent amplification of

the c-met gene in scirrhous type stomach cancer. Biochem Biophys Res Commun

1992;189:227–32.

36. Zhang YW, Su Y, Volpert OV, Vande Woude GF. Hepatocyte growth factor/scatter factor

mediates angiogenesis through positive VEGF and negative thrombospondin 1

regulation. Proc Natl Acad Sci U S A 2003;100:12718–23.

37. Yakes FM, Chen J, Tan J, Yamaguchi K, Shi Y, Yu P, et al. Inhibition of tumor cell

growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel

inhibitor of HGF and VEGF receptor tyrosine kinases. Mol Cancer Ther

2011;10:2298–308.

38. Qian F, Engst S, Yamaguchi K, Yu P, Won KA, Mock L, et al. Inhibition of tumor cell

growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel

inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res 2009;69:8009–16.

39. Birchmeier C, Birchmeier W, Brand-Saberi B. Epithelial-mesenchymal transitions in

cancer progression. Acta Anat (Basel) 1996;156:217–26.

40. Desiderio MA. Hepatocyte growth factor in invasive growth of carcinomas. Cell Mol

Life Sci 2007;64:1341–54.

41. Rong S, Bodescot M, Blair D, Dunn J, Nakamura T, Mizuno K, et al. Tumorigenicity of

the met proto-oncogene and the gene for hepatocyte growth factor. Mol Cell Biol

1992;12:5152–8.

42. Zhang YW, Staal B, Essenburg C, Su Y, Kang L, West R, et al. MET kinase inhibitor

SGX523 synergizes with epidermal growth factor receptor inhibitor erlotinib in a

hepatocyte growth factor-dependent fashion to suppress carcinoma growth. Cancer Res

2010;70:6880–90.

43. Zhang S, Zhau HE, Osunkoya AO, Iqbal S, Yang X, Fan S, et al. Vascular endothelial

growth factor regulates myeloid cell leukemia-1 expression through

neuropilin-1-dependent activation of c-MET signaling in human prostate cancer cells.

Mol Cancer 2010;9:9.

44. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS.

Accelerated metastasis after short-term treatment with a potent inhibitor of tumor

angiogenesis. Cancer Cell 2009;15:232–9.

45. Pàez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals F, et al.

Antiangiogenic therapy elicits malignant progression of tumors to increased local

invasion and distant metastasis. Cancer Cell 2009;15:220–31.

46. Boccaccio C, Comoglio PM. Invasive growth: a MET-driven genetic programme for

cancer and stem cells. Nat Rev Cancer 2006;6:637–45.

47. Shojaei F, Lee JH, Simmons BH, Wong A, Esparza CO, Plumlee PA, et al. HGF/c-Met

acts as an alternative angiogenic pathway in sunitinib-resistant tumors. Cancer Res

2010;70:10090–100.

Table 1: Inhibitory activities of T-1840383 against 72 kinases

IC50 IC50 IC50 Kinase Kinase Kinase (nmol/L) (nmol/L) (nmol/L) Abl (H396P) 2.8 EphB1 13 Mnk2 100 Abl (M351T) 1 EphB2 19 MuSK 51 Abl (Q252H) 4.9 EphB4 79 PDGFRα(D842V) 3 Abl 13 Fer 110 PDGFRα 270 Abl(T315I) 1.6 FGFR1 370 PDGFRα(V561D) 2 Arg 26 FGFR2 270 PDGFRβ 290 Aurora-B 210 FGFR2(N549H) 5.6 PTK5 93 Axl 19 Fgr 81 Pyk2 590 Bmx 110 Flt3* 19 Ret (V804L) 58 BRK 100 Fms 11 Ret 2 c-Kit(D816H) 200 Fms(Y969C) 36 Ret(V804M) 160 c-Kit 2300 Fyn 180 Ron 3 c-Kit(V560G) 2.7 Hck 34 Rse 2 c-Kit(V654A) 1100 IKKβ 110 SIK 290 c-Mer <1 IR 1400 Tie2* 1.2 c-RAF 100 IRAK1 870 Tie2(R849W) 10 DDR2 120 Itk 93 Tie2(Y897S) 14 EphA1 40 Lck 11 TrkA 4 EphA2 12 LIMK1 160 TrkB 10 EphA3 96 LOK 11 Txk 37 EphA4 76 Lyn 65 VEGFR1* 7.7 EphA5 17 MELK 540 VEGFR2* 2.2 EphA7 120 Met* 1.9 VEGFR3* 5.5 EphA8 26 MLK1 43 Yes 73

* Note: For these 6 kinases, the IC50 values were determined by the AlphaScreen assay.

For the others, the IC50 values were determined using KinaseProfiler IC50Express™

(Millipore).

Figure 1. T-1840383 inhibits HGF-induced c-Met phosphorylation, cell scattering,

and invasion. A, Chemical structure of T-1840383. B, A549 cells were treated with

T-1840383 for 1 h prior to HGF stimulation. c-Met phosphorylation (p-Met) levels were

determined by western blotting. Total c-Met levels are shown as a loading control.

Western blot was done in quadruplicate with very similar results. C, T-1840383 was

added to the cell culture prior to HGF stimulation. Scattering-stimulated cells were then

stained with a Diff-Quik staining kit. Magnification, ×100. D, Scattering-stimulated

cells were subjected to immunofluorescence stain for E-cadherin expression (green) and

nuclear stain with propidium iodide (red). Magnification, ×400. E, Cell invasion was

assessed using a Matrigel invasion chamber for 72 h under mild hypoxia (3% O2). The

invading cells were stained with a Diff-Quik staining kit and quantified. Columns, mean

(n = 4); bars, SD. F, Cell viabilities were determined in appropriate media containing

various concentration of T-1840383. Shown are the mean of the IC50 values (n = 2-6).

Rows labeled as “>10,000” indicate that the IC50 values are more than 10,000 nmol/L.

Figure 2. T-1840383 inhibits VEGF receptor-2 phosphorylation and VEGF

signaling in HUVECs. A, HUVECs were treated with T-1840383 at the indicated

concentration for 1 h. Phosphorylation of VEGFR2 (Tyr1175), Akt (Ser473), and

ERK1/2 (Thr202/Tyr204) following VEGF stimulation were analyzed by western

blotting. Western blotting was done in quadruplicate with very similar results. B,

HUVECs were co-cultured with fibroblasts. T-1840383 was added to the medium

followed by VEGF stimulation. Shown are representative images after endothelial

cell-specific staining (n = 4). C, Total filament length and number of branch points of

the stained tube-like structures were measured using image analysis software in 4–10

different fields for each condition. Columns, means; bars, SD.

Figure 3. In vivo inhibition by T-1840383 of c-Met downstream signaling pathways

and antitumor efficacy of T-1840383 in tumor xenograft models. A, Mice bearing

MKN45 tumors were administered a single oral dose of T-1840383 (2 mg/kg). At the

indicated time points after administration, the tumors were collected and homogenized.

c-Met phosphorylation were determined by western blotting. B, c-Met signaling in

MKN45 tumors from mice that had been received a single oral dose of T-1840383 or

vehicle were analyzed by western blotting. Mice bearing MKN45 (C), EBC-1 (D),

U87MG (E), or COLO 205 (F) tumors were orally administered T-1840383 once daily

at the indicated dose levels or vehicle alone for 2 weeks. Data represent the means ±

SEM for each group over the treatment period (n = 5 or 6).

Figure 4. Effects of T-1840383 on tumor microvessel density, tumor cell

proliferation, and tumor cell apoptosis in A549 tumors. A, Mice bearing A549

tumors were treated with T-1840383 at 0.5, 1.5, and 5 mg/kg/day or vehicle for 3 days.

Tumors were fixed, paraffin-embedded, sectioned, and immunostained for CD31. Scale

bars, 400 µm. B, Quantitative analysis of CD31-positive endothelial cells were

performed. C, Tumor specimens were immunostained with anti-Ki67 antibody, and

Ki67-positive, proliferating cells were quantified. D, Apoptotic tumor cells were

visualized and quantified by TUNEL assay. Column, mean (n = 4 or 5); bars, SEM. * p

≤ 0.025 versus the vehicle control group by the one-tailed Williams’ test.

Figure 5. Antitumor efficacy of T-1840383 in a peritoneal dissemination model. A,

Photon emission from individual mice in which NUGC-4-luc cells had been inoculated

intraperitoneally on day 0 was visualized before administration of the first dose of

T-1840383 or vehicle (day 14) and over the course of treatment (days 27, 34, and 41). B,

Emitted bioluminescence from vehicle- or 5 mg/kg T-1840383-treated mice at days 14

and 41. C, Survival curves of T-1840383-treated NUGC-4-bearing mice. Daily

administration (5 days a week) of T-1840383 or vehicle was initiated at day 14 and

lasted until the mice were euthanized (up to day 110).

Figure 6. Hypoxia-induced c-Met expression and activation in cancer cells. A, A549

cells were cultured in media containing 0.1%, 1%, or 10% FBS in collagen-type

I-coated plates under normoxia or hypoxia (2% O2) for 24 h. B, Caki-1, SUIT-2, and

U87-MG cells were cultured in appropriate media containing 10% FBS under normoxia

or hypoxia (3% O2) for 48 h. c-Met phosphorylation were determined by western

blotting. C, SUIT-2 cell invasion was stimulated with HGF under normoxia or hypoxia

(2% O2) for 72 h. The invading cells were stained with a Diff-Quik staining kit and

quantified. Columns, mean (n = 4); bars, SD.

A Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. E Author ManuscriptPublishedOnlineFirstonApril2,2013;DOI:10.1158/1535-7163.MCT-12-1011 800 700 mct.aacrjournals.org 600 500 sion per field) aa B 400 300 Cell inv 100 300 T-1840383 (nmol/L) 0 0 0.03 0.1 0.3 1 3 10 30 1000 200 on October 3,2021. ©2013 American Association forCancer Research. (cell number HGF-+++++++++++ 100 p-Met (Tyr1234/1235) 0 0 1 10 100 T-1840383 concentration (nmol/L) Total Met F

C HUVEC (endothelial) 1.8 SNU-5 (gastric) 343.4 T-1840383 (nmol/L) 0 0 1 10 100 SNU-638 (gastric) 4.2 MKN45 (gastric) 5 HGF -++ + + EBC-1 (NSCLC) 8.4 NUGC-4 (gastric) 10 Hs746T (gastric) 25 OE33 (oesophagus) 570 KATOⅢ (gastric) 2400 SNU-620 (gastric) >10000 SNU-668 (gastric) >10000 D U-87 MG (GBM) >10000 T-1840383 (nmol/L) 0 0 1 10 100 SNU-484 (gastric) >10000 AGS (gastric) >10000 HGF -+ + + + A549 (NSCLC) >10000 Colo 205 (CRC) >10000 MRC-5 (fibrob rast) >10000 1 10 100 1000 10000 IC50 (nmol/L) Figure 2 Nakamura et al., Downloaded from Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. A T-1840383 (nmol/L) 0 0 0.03 0.1 0.3 1 3 10 30 100 300 1000 Author ManuscriptPublishedOnlineFirstonApril2,2013;DOI:10.1158/1535-7163.MCT-12-1011 VEGF -++++++ ++++ + p-VEGFR2 (Tyr1175) mct.aacrjournals.org Total VEGFR2 p-Akt (Ser473) p-ERK1/2 (Thr202/Tyr204)

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CDEF 2000 900 1400 1200 Vehicle Vehicle Vehicle Vehicle 800 05mg/kg0.5 mg/kg 05mg/kg0.5 mg/kg 1200 050.5 mg /kg 050.5 mg /kg 1.5 mg/kg 1.5 mg/kg 1000 700 1.5 mg/kg 1.5 mg/kg 5 mg/kg 1500 5 mg/kg 1000 5 mg/kg 5 mg/kg 15 mg/kg 15 mg/kg * 600 15 mg/kg 800 15 mg/kg 500 800 1000 600 400 600 300 * 400 * * 400 * 500 200 Tumor volume (mm3) volume Tumor Tumor volume (mm3) volume Tumor Tumor volume (mm3) volume Tumor Tumor volume (mm3) volume Tumor * * * 200 * 200 * 100 * * 0 0 0 0 8 1216202428 12 16 20 24 28 32 21 25 29 33 37 41 7 1115192327

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A Serum (0.1%) Serum (1%) Serum (10%) Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonApril2,2013;DOI:10.1158/1535-7163.MCT-12-1011 HHNNHHNNHHNN c-Met mct.aacrjournals.org p-Met (Tyr1234/1235) p-Met (Tyr1003) Actin

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C 450 400 Normoxia 350 Hypoxia 300 250 200 150 ll number / field) Cell invasion 100 (Ce 50 0 0 12.5 25 50 100 HGF concentration (ng/mL) Author Manuscript Published OnlineFirst on April 2, 2013; DOI: 10.1158/1535-7163.MCT-12-1011 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Novel Inhibitor of c-Met and VEGF Receptor Tyrosine Kinases with a Broad Spectrum of In Vivo Antitumor Activities

Yoshiko Awazu, Kazuhide Nakamura, Akio Mizutani, et al.

Mol Cancer Ther Published OnlineFirst April 2, 2013.

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