Clinical Cancer Research The Science of Cancer Health Disparities Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 3051-3057, May 15, 2007. doi: 10.1158/1078-0432.CCR-06-2743
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takeda, M.
Right arrow Articles by Nishio, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takeda, M.
Right arrow Articles by Nishio, K.

Cancer Therapy: Preclinical

AZD2171 Shows Potent Antitumor Activity Against Gastric Cancer Over-Expressing Fibroblast Growth Factor Receptor 2/Keratinocyte Growth Factor Receptor

Masayuki Takeda1,3, Tokuzo Arao1,4, Hideyuki Yokote1,4, Teruo Komatsu5, Kazuyoshi Yanagihara5, Hiroki Sasaki6, Yasuhide Yamada2, Tomohide Tamura2, Kazuya Fukuoka7, Hiroshi Kimura3, Nagahiro Saijo2 and Kazuto Nishio1,4

Authors' Affiliations: 1 Shien Lab and 2 Medical Oncology, National Cancer Center Hospital, Tsukiji, Chuo-ku, Tokyo, Japan; 3 Second Department of Internal Medicine, Nara Medical University; 4 Department of Genome Biology, Kinki University School of Medicine, Ohno-higashi, Osaka-Sayama, Osaka, Japan; and 5 Central Animal Lab and 6 Genetic Division, National Cancer Center Research Institute; and 7 Division of Respiratory Medicine, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

Requests for reprints: Kazuto Nishio, Department of Genome Biology, Kinki University School of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511, Japan. Fax: 81-72-366-0206; E-mail: knishio{at}med.kindai.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer.

Experimental Design: We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo.

Results: AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC50 of 0.15 and 0.37 µmol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus–truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 µmol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar Ki values (~0.05 µmol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts.

Conclusions: AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.

Various anticancer therapies for gastric cancer have been investigated over the past two decades. Despite intensive studies, the prognosis for patients with unresectable advanced or recurrent gastric cancer remains poor (1, 2), and new therapeutic modalities are needed.

Fibroblast growth factors (FGF) and their signaling receptors have been found to be associated with multiple biological activities, including proliferation, differentiation, motility, and transforming activities (3–5). The K-sam gene was first identified as an amplified gene in human gastric cancer cell line KATO-III (6, 7), and its product was later found to be identical to the bacteria-expressed kinase, or keratinocyte growth factor receptor (KGFR), and FGF receptor 2 (FGFR2). FGFR2/KGFR/K-sam is preferentially amplified in poorly differentiated types of gastric cancers with a malignant phenotype, and its protein expression was detected by immunohistochemical staining from 20 of 38 cases of the undifferentiated type of advanced stomach cancer (8, 9). Thus, FGFR2 signaling may be as a promising molecular target for gastric cancer.

AZD2171 is a potent, ATP-competitive small molecule that inhibits all vascular endothelial growth factor receptors [VEGFR-1, VEGFR-2 (also known as KDR), and VEGFR-3]. In vitro studies have shown that recombinant VEGFR-2 tyrosine kinase activity was potently inhibited by AZD2171 (IC50 <1 nmol/L; ref. 10). AZD2171 also showed potent activity versus VEGFR-1 and VEGFR-3 (IC50, 5 and ≤3 nmol/L, respectively). VEGF-stimulated proliferation and VEGFR-2 phosphorylation of human umbilical vascular endothelial cells was inhibited by AZD2171 (IC50, 0.4 and 0.5 nmol/L, respectively). In in vivo studies, inhibition of VEGFR-2 signaling by AZD2171 reduced microvessel density and dose-dependently inhibited the growth of various human tumor xenografts (colon, lung, prostate, breast, and ovary; ref. 10). These data are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. On the other hand, because it was known that AZD2171 also possesses additional activity against FGFR1 (IC50, 26 nmol/L; ref. 10), we hypothesized that AZD2171 may exhibit the additional anticancer activity against FGFR-overexpressing gastric cancer cells.

Our previous studies showed significant activities of the dual VEGFR-2 and epidermal growth factor receptor inhibitor ZD6474 against poorly differentiated gastric cancer (11) and non–small-cell lung cancer with epidermal growth factor receptor mutations (12, 13), both in vitro and in vivo. Based on these findings, we proceeded to investigate the anticancer activity of AZD2171 in preclinical models (gastric cell lines and xenografts).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Anticancer agents. AZD2171 and gefitinib (Iressa) were provided by AstraZeneca. AZD2171 and gefitinib were dissolved in DMSO for the in vitro experiments, and AZD2171 was suspended in 1% (w/v) aqueous polysorbate 80 and administered in a dose of 0.1 mL/10 g per body weight in the in vivo experiments.

Cell culture. Human gastric cancer cell lines 44As3, 58As1, OKAJIMA, OCUM2M, KATO-III, MKN-1, MKN-28, and MKN-74 were maintained in RPMI 1640 (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) and penicillin-streptomycin.

Established highly tumorigenic cell line. Signet ring cell gastric carcinoma cell line KATO-III was gift from Dr. M. Sekiguchi (University of Tokyo, Tokyo, Japan). All of the presented in vitro experiments were done using the KATOIII cell line. We conducted a preliminary experiment to compare the cellular characteristics of TU-KATO-III cells and KATOIII cells, and the results revealed that a high expression level of FGFR2 and high sensitivity to AZD2171 were still maintained in the TU-KATO-III cells (data not shown). KATO-III did not show tumorigenicity following repeated implantation of the cultured cells into BALB/c nude mice. Following s.c. inoculation into nonobese diabetic/severe combined immunodeficient mice, 80% to 100% of the KATO-III cells caused the formation of tumor. Following this result, we cultured the cancer cells isolated from the tumor of mice that developed 2 to 3 months following the implantation of KATO-III cells and attempted s.c. injection into nude mice, in turn, of the incubated cells. This sequence of manipulations was repeated for seven cycles in an attempt to reliably isolate cell lines that would have higher potential to undergo tumor formation over short periods of time. In this way, we obtained a cell line (TU-kato-III) from KATO-III cells that possessed a high tumorigenic potential.

In vitro growth inhibition assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate the growth-inhibitory effect of AZD2171. Cell suspensions (180 µL) were seeded into each well of 96-well microculture plate and incubated in 10% fetal bovine serum medium for 24 h. The cells were exposed to AZD2171 or gefitinib at concentrations ranging from 4 nmol/L to 80 µmol/L and cultured at 37°C in a humidified atmosphere for 72 h. After the culture period, 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added, and the plates were incubated for 4 h. After centrifugation, the culture medium was discarded, and wells were filled with DMSO. The absorbance of the cultures at 562 nmol/L was measured using Delta-soft on a Macintosh computer (Apple) interfaced to a Bio-Tek Microplate Reader EL-340 (BioMatellics). This experiment was done in triplicate.

Reverse-transcription PCR. Using a GeneAmp RNA-PCR kit (Applied Biosystems), 5 µg of total RNA from each cultured cell line was converted to cDNA. The PCR amplification procedure consisted of 28 to 35 cycles (95°C for 45 s, 62°C for 45 s, and 72°C for 60 s) followed by incubation at 72°C for 7 min, and the bands were visualized by ethidium bromide staining. The following primers were used for the PCR: human-specific ß-actin, forward 5-GGAAATCGTGCGTGACATT-3 and reverse 5-CATCTGCTGGAAGGTGGACAG-3; VEGFR-1, forward 5-TAGCGTCACCAGCAGCGAAAGC-3 and reverse 5-CCTTTCTTTTGGGTCTCTGTGC-3; VEGFR-2, forward 5-CAGACGGACAGTGGTATGGTTC-3 and reverse 5-ACCTGCTGGTGGAAAGAACAAC-3; VEGFR-3, forward 5-AGCCATTCATCAACAAGCCT-3 and reverse 5-GGCAACAGCTGGATGTCATA-3; c-KIT, forward 5-GCCCACAATAGATTGGTATTT-3 and reverse 5-AGCATCTTTACAGCGACAGTC-3; FGFR1, forward 5-GGAGGATCGAGCTCACTCGTGG-3 and reverse 5-CGGAGAAGTAGGTGGTGTCAC-3; FGFR2, forward 5-CAGTAGGACTGTAGACAGTGAA-3 and reverse 5-CCGGTGAGGCGATCGCTCCACA-3; FGFR3, forward 5-GGTCAAGGATGGCACAGGGCTG-3 and reverse 5-AGCAGCTTCTTGTCCATCCGCT-3; and FGFR4, forward 5-CCGCCTAGAGATTGCCAGCTTC-3 and reverse 5-AGGCCTGTCCATCCTTAAGCCA-3.

Real-time reverse transcription-PCR. Real-time reverse transcription-PCR amplification was done by using a Premix Ex Taq and Smart Cycler system (Takara Bio, Inc.) according to the manufacturer's instructions. The following primers were used: FGFR2 (IIIb), forward 5-GATAAATAGTTCCAATGCAGAAGTGCT-3 and reverse 5-TGCCCTATATAATTGGAGACCTTACA-3 (7); FGFR2 (COOH-terminal), forward 5-GAATACTTGGACCTCAGCCAA-3 and reverse 5-AACACTGCCGTTTATGTGTGG-3; and human-specific ß-actin, forward 5-GGAAATCGTGCGTGACATT-3 and reverse 5-CATCTGCTGGAAGGTGGACAG-3. The experiment was independently done in triplicate using ß-actin as a reference to normalize the data.

Western blotting. Cells were cultured overnight in 10% serum-containing medium or serum-starved medium and exposed to 0.1 to 10 µmol/L of AZD2171 for 3 h before addition of KGF (100 ng/mL) for 15 min. Immunoblotting was done as described previously (14). In brief, after lysing the cells in radioimmunoprecipitation buffer, the lysate was electrophoresed through 10% (w/v) polyacrylamide gels. The proteins were transferred to polyvinylidene difluoride membranes and reacted with the following antibodies: anti-FGFR2 (H-80) and anti-FGFR2 (C-17) antibody (Santa Cruz Biotechnology, Inc.); anti-phosphotyrosine antibody PY20 (BD Transduction Laboratories); anti–phosphorylated FGFR (Tyr653/654), anti–mitogen-activated protein kinase, anti–phosphorylated mitogen-activated protein kinase antibody, anti-AKT, anti–phosphorylated AKT, and anti-rabbit horseradish peroxidase–conjugated antibody (Cell Signaling Technology); and anti-ß-actin antibody (Sigma). Visualization was achieved with an enhanced chemiluminescent detection reagent (Amersham Bioscience).

FGFR2 kinase assay. FGFR2/KGFR kinase activity was quantified by using a Universal Tyrosine Kinase Assay kit (Takara) according to manufacturer's instructions. FGFR2/KGFR proteins were collected from the KATO-III, OCUM2M, and OKAJIMA cell lysates by overnight immunoprecipitation with an anti-FGFR2 antibody. The FGFR2/KGFR immune complexes were washed thrice with radioimmunoprecipitation assay buffer and diluted kinase reaction buffer. Immobilized tyrosine kinase substrate (poly[Glu-Tyr]) was incubated for 30 min at 37°C with each sample in the presence of kinase-reacting solution and ATP. Samples were washed four times, blocked with blocking solution, and incubated with anti-phosphotyrosine antibody (PY20) conjugated to horseradish peroxidase. The absorbance of the phosphorylated substrate was measured at 450 nm.

Chemical cross-link analysis. The chemical cross-link analysis was carried out as described previously (15). In brief, KATO-III cells and OKAJIMA cells were cultured under serum-starved conditions for 24 h, and after stimulation with KGF (100 ng/mL) for 15 min, they were collected and washed with PBS and incubated for 30 min in PBS containing 1.5 mmol/L of the non-permeable cross-linker bis-(sulfosuccinimidyl) substrate (Pierce). The reaction was terminated by adding 250 mmol/L glycine for 5 min, and the cells were analyzed by immunoblotting with FGFR2 antibody (Sigma).

FGFR2/KGFR gene silencing with small interfering RNA. Pre-designed small interfering RNA (siRNA) targeting FGFR2 was purchased from Ambion. KATO-III cells were plated on a 96-well plate and incubated in serum-containing medium for 24 h. The cells were then transfected with the FGFR2 targeting siRNA or non-silencing siRNA using RNAiFect Transfection Reagent (Qiagen) according to the manufacturer's protocol and incubated another 72 h. Cell growth was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For immunoblotting, 2 x 105 cells per well were plated on a six-well plate for 24 h and transfected with siRNA under the same conditions.

In vivo experiments. Tumorigenic TU-kato-III cells were derived from the gastric cancer cell line KATO-III. Four-week-old female BALB/c nude mice were purchased from CLEA Japan, Inc. and maintained under specific-pathogen-free conditions; 5 x 106 TU-kato-III cells or OCUM2M cells were s.c. injected into both flanks of each mouse. When the tumors had reached a volume of 0.1-0.3 cm3, the mice were randomized into three groups (three per group) and given AZD2171, 1.5 or 6.0 mg/kg/d, or vehicle once daily by oral gavage for 3 weeks. Tumor volume was calculated using the formula: (length x width) x {surd}(length x width) x ({pi}/6), where length is the longest diameter across the tumor, and width is the corresponding perpendicular. All mice were sacrificed on day 21, and the tumors were collected. The protocol of the experiment was approved by the Committee for Ethics in Animal Experimentation and conducted in accordance with the Guidelines for Animal Experiments of National Cancer Center.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
AZD2171 showed growth-inhibitory activity in vitro. To evaluate the growth-inhibitory activity of AZD2171 in vitro, we did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on eight gastric cancer cell lines. The epidermal growth factor receptor–specific tyrosine kinase inhibitor gefitinib was used as a reference. The IC50 of gefitinib for all cell lines was between 7 and 20 µmol/L. AZD2171 inhibited the growth of KATO-III cells and OCUM2M cells (IC50, 0.15 and 0.37 µmol/L, respectively) more potently than the other cell lines (Fig. 1A ).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. A, in vitro growth-inhibitory effect of AZD2171 and gefitinib on eight gastric cancer cell lines. AZD2171 had a growth-inhibitory effect on KATO-III cells and OCUM2M cells (IC50, 0.15 and 0.37 µmol/L, respectively). Columns, mean IC50 of each compound from three independent experiments; bars, SD. {square}, IC50 of gefitinib; {blacksquare}, IC50 of AZD2171. B, the mRNA expression levels of VEGFRs, FGFRs, and c-KIT in gastric cancer cell lines were determined by reverse transcription-PCR. Human umbilical vascular endothelial cells were used as the positive control for the VEGFRs. No mRNA expression of VEGFRs or c-KIT was detected by reverse transcription-PCR in both sensitive cell lines, but FGFR2 was strongly detected; however, little faint or none was detected in the other cell lines.

 
Expression levels of tyrosine kinase receptors. To elucidate the mechanism of action of AZD2171 in the two sensitive cell lines, we measured mRNA expression levels of VEGFRs, FGFRs, and c-KIT, whose kinase activity have been reported to be inhibited by AZD2171 (10). No mRNA expression of VEGFRs or c-KIT was detected by reverse transcription-PCR in either sensitive cell lines. FGFR2 transcripts, however, were strongly expressed in both sensitive cell lines but not strongly in the other cell lines (Fig. 1B). Since we previously found that FGFR2/KGFR/K-sam with a deletion of COOH-terminal exons was amplified in both sensitive cell lines (9), we speculated that amplified FGFR2/KGFR might be associated with sensitivity to AZD2171.

Sensitive cells expressed constitutively active and spontaneously dimerized FGFR2/KGFR. We quantified mRNA expression levels of FGFR2 by real-time reverse transcription-PCR with primers that detect the extracellular domain (IIIb region, see Fig. 2A ) and COOH-terminal region. The results show that KATO-III cells and OCUM2M cells expressed FGFR2 100-fold higher than the other cells tested. The COOH-terminal region of FGFR2 was deleted in the KATO-III cells and OCUM2M cells (Fig. 2B). Overexpression and markedly increased phosphorylation of FGFR2 was observed in the AZD2171-sensitive cell lines (Fig. 2C).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. A, schematic representation of FGFR2 and regions amplified by PCR. B, mRNA expression levels of FGFR2 were quantified by detecting the extracellular domain or COOH-terminal region by real-time reverse transcription-PCR. Expression in the cells is shown as a ratio to expression in OKAJIMA cells. FGFR2 was overexpressed in KATO-III cells and OCUM2M cells by about 100-fold compared with the other cell lines. The majority of the FGFR2 in the sensitive cell lines KATO-III and OCUM2M had no COOH-terminal region. C, protein expression levels of FGFR2 were determined by Western blotting with antibodies to the NH2 or COOH termini. Both AZD2171-sensitive cell lines overexpressed FGFR2, and the phosphorylation levels were markedly higher. D, chemical cross-linking analysis. Cells were cultured under serum-starved conditions for 24 h and then stimulated with KGF (100 ng/mL) for 15 min. After collecting and washing them with PBS, they were incubated for 30 min in PBS containing cross-linker substrate. The reaction was terminated by adding 250 mmol/L glycine for 5 min. In spite of the serum-starved conditions, high levels of expression of the dimerized form were observed in KATO-III cells in the absence of ligand stimulation. This phenomenon was not observed in the control undifferentiated OKAJIMA cell line. Ligand stimulation resulted in a mild increase in the dimerized form in KATO-III cells. Arrows indicate monomer or dimer formation.

 
Immunoblotting with antibodies for the COOH and NH2 termini revealed that almost all the FGFR2 expressed by OCUM2M cells, and about half of FGFR2 expressed by KATO-III cells, were truncated (Fig. 2C). Although the KATO-III cells expressed wild-type receptor to some extent, the COOH-terminal truncated type was dominantly expressed in AZD2171-sensitive cell lines.

A chemical cross-linking analysis was done to evaluate the dimerization of FGFR2. High dimerization of FGFR2 was observed in the KATO-III cells even in the absence of ligand stimulation (Fig. 2D), but no such phenomenon was observed in the control undifferentiated OKAJIMA cell line. Ligand stimulation increased the level of the dimerized-form in KATO-III cells. Taken together, these findings show that the sensitive cell lines expressed high levels of FGFR2 that was highly phosphorylated and spontaneously dimerized without ligand stimulation, suggesting that FGFR2 signaling is constitutively activated in these cells. This evidence is consistent with the widely recognized findings that cancer cells sensitive to other tyrosine kinase inhibitors, such as gefitinib and imatinib, overexpress the highly phosphorylated target receptor with an increased level of dimerization in a ligand-independent manner (12, 16, 17).

FGFR2 targeting siRNA showed a potent growth-inhibitory effect on KATO-III cells. To investigate the dependency of cell growth through activated FGFR2 signaling in the AZD2171-sensitive KATO-III cell line, we evaluated the growth-inhibitory effect of siRNA targeted to FGFR2 in KATO-III cells. Targeted siRNA (5-100 nmol/L) decreased FGFR2 and inhibited cell growth (>80%) in a dose-dependent manner (Fig. 3A ). The results show that most of the growth of KATO-III cells is dependent on activated FGFR2 signaling, suggesting that the FGFR signaling dependency may be responsible for the higher growth-inhibitory effect of AZD2171 on KATO-III cells.


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 3. A, FGFR2 targeting siRNA and cellular growth-inhibitory effect. KATO-III cells were plated on a 96-well plate and incubated in serum-containing medium for 24 h. After incubation, the cells were transfected with FGFR2-targeting or non-silencing siRNA and incubated for another 72 h. Cell growth was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For immunoblotting, 2 x 105 cells per well were plated on a six-well plate and treated similarly. Marked inhibition of cell growth (~80%) was observed by FGFR2 targeting siRNA compared with control siRNA (top). Reduction of FGFR2 protein expression in KATO-III cells was confirmed by immunoblotting (bottom). Columns, % control absorbance in three independent experiments; bars, SD. B, Western blotting for downstream molecules of FGFR2 signaling. Cells were cultured overnight under serum-starved conditions and exposed to 0.1 to 10 µmol/L AZD2171 for 3 h before adding 100 ng/mL KGF for 15 min. AZD2171 completely inhibited KGF-induced phosphorylation of FGFR2 at 1 µmol/L in the sensitive cell lines, compared with 10 µmol/L in the control cell line OKAJIMA. Similar results were observed for FRS-2, AKT, and mitogen-activated protein kinase (MAPK).

 
AZD2171 inhibited FGFR2 signaling. Next, we examined the effect of AZD2171 on FGFR2 downstream phosphorylation signals (i.e., FRS-2, AKT, and mitogen-activated protein kinase). AZD2171 completely inhibited KGF-induced phosphorylation of FGFR2, FRS-2, AKT, and mitogen-activated protein kinase at 1 µmol/L in KATO-III cells, compared with 10 µmol/L in OKAJIMA cells. These results clearly show that AZD2171 possesses inhibitory activity against FGFR2 in cell-based studies and significantly inhibits the phosphorylation of FGFR2 at 1 µmol/L in sensitive cells.

FGFR2 kinase inhibition of AZD2171. To quantify the inhibitory activity of AZD2171 on FGFR2 kinase under cell-free conditions, we calculated the Ki values for immunoprecipitated FGFR2 derived from KATO-III, OCUM2M, and OKAJIMA cells. The Ki values of AZD2171 for FGFR2 in each of these cell lines were 0.067 ± 0.017, 0.072 ± 0.022, and 0.049 ± 0.041 µmol/L, respectively (Table 1 ). In contrast, the Ki value of AZD2171 for recombinant VEGFR-2 was 0.0009 µmol/L (data not shown) and was consistent with previous reports (10). At the cellular level, phosphorylation of FGFR2 was inhibited at 10-fold lower concentrations of AZD2171 in the sensitive cell lines (Fig. 3B), but there were no marked differences between the kinase-inhibitory effects among the proteins derived from the cell lines in this cell-free assay. This discrepancy is discussed in the Discussion.


View this table:
[in this window]
[in a new window]

  		Table 1. In vitro kinase assay of AZD2171 against FGFR2

 
In vivo antitumor activity of AZD2171 against FGFR2-overexpressing gastric cancer. To elucidate the in vivo antitumor activity of AZD2171 in mice bearing gastric cancer tumor xenografts, we used the newly established tumorigenic subline TU-kato-III (derived from KATO-III) and OCCUM2M. We attempted to perform control experiments using OKAJIMA cells in vivo as suggested by the reviewer. Unfortunately, however, the cell lines grew slowly in the mice, and we could not precisely evaluate the antitumor activity of AZD2171 in the model. However, the results of preliminarily experiments showed that AZD2171 seemed to be less effective against OKAJIMA cells than against KatoIII and OCUM2M cell in vivo. Mice implanted the TU-kato-III and OCUM2M tumors were given a low or high dose of AZD2171 (i.e., 1.5 or 6.0 mg/kg/d), or vehicle, orally for 3 weeks. AZD2171 (1.5 mg/kg/d) significantly inhibited tumor growth in the mice bearing TU-kato-III and OCUM2M tumors, and the higher dose (6.0 mg/kg/d) completely inhibited the growth of both tumor models (Fig. 4A ). H&E staining showed broad dose-dependent necrosis of core tumor tissue in mice treated with AZD2171 (Fig. 4B). Thus, AZD2171 showed marked antitumor activity in vivo against both human gastric tumor xenografts.


Figure 4
View larger version (63K):
[in this window]
[in a new window]

  		Fig. 4. A, in vivo growth-inhibitory effect of AZD2171 in a tumor xenograft model. After s.c. injecting 5 x 106 TU-kato-III or OCUM2M cells into both flanks of the mice, AZD2171 (1.5 or 6.0 mg/kg/d), or vehicle, was administered orally once daily for 3 wks. A marked tumor growth-inhibitory effect was observed at the low dose (1.5 mg/kg/d) of AZD2171 in both the TU-kato-III tumors and OCUM2M tumors, and the high dose (6.0 mg/kg/d) of AZD2171 completely inhibited the growth of both cell lines. B, representative H&E staining of tumor tissue from mice treated with AZD2171. Broad dose-dependent necrosis was observed. Original magnification, x 40.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Recent studies have shown that FGFRs and their ligands are promising therapeutic target molecules for various malignant diseases, such as prostate cancer (18), breast cancer (5, 19), endometrial carcinoma (20), synovial sarcomas (21), thyroid carcinoma (22, 23), and hematopoietic malignancies (24–27). These findings are based on the biological properties of malignant cells expressing activated FGFR, like FGFR fusion tyrosine kinase, involved in chromosomal translocations, gene amplification of FGFRs, or overexpression of FGFs (5, 18–27). In the case of gastric cancer, the results of immunohistochemical analysis of clinical samples revealed that 20 of 38 cases of advanced undifferentiated type of gastric cancer were FGFR2/K-sam positive, whereas none of the 11 cases with the differentiated or intestinal type of cancer showed positive staining for K-sam (8). The results suggest that FGFR2/K-sam overexpression is associated with the undifferentiated type of stomach cancers. The results of fluorescence in situ hybridization analysis of the gastric cancer specimens showed gene amplification of FGFR2/K-sam in 2.9% (28). The clinical implication of FGFR2 overexpression/amplification in gastric cancers remains to be fully clarified, and further investigation is needed.

AZD2171 has the most potent kinase-inhibitory activity against VEGFR-2 (IC50 < 1 nmol/L); it also possesses additional activity against VEGFR-1, VEGFR-3, and c-Kit (IC50, 5, ≤3, and 2 nmol/L, respectively; ref. 10). AZD2171 showed antiangiogenic activity and broad antitumor activity consistent with potent inhibition of VEGF-induced angiogenesis. We showed kinase-inhibitory activity of AZD2171 against FGFR2 in the present study. When cancer cells are dependent on FGFR2 signaling, AZD2171 can be expected to give additional therapeutic benefit in addition to its antiangiogenic effects.

A cell-based Western blotting analysis showed that phosphorylation of FGFR2 in KATO-III cells and OCUM2M cells was inhibited by AZD2171 at 10-fold lower dose than in OKAJIMA cells (Fig. 3B). However, there was no significant difference in the Ki values of AZD2171 between the FGFR2 derived from KATO-III, OCUM2M, and OKAJIMA in an in vitro kinase assay. This may be attributable to the different conditions between the cell-based and cell-free assays. For example, undefined intrinsic intracellular factors may influence kinase activity: (a) differences in baseline intracellular FGFR2 phosphatase activity in each cell line, (b) differences in intracellular concentration of ([transporters, such as ATP-binding cassette transporters, may be involved in this phenomenon refs. 29, 30), and (c) undefined intrinsic inhibitory factors that bind the compounds directly may also be involved (e.g., Brehmer D, et al. have identified various gefitinib binding proteins by affinity chromatography; ref. 31).

In conclusion, AZD2171, a potent inhibitor of all VEGFRs (VEGFR-1, VEGFR-2, and VEGFR-3), was found to have antitumor effect against gastric cancer xenografts in line with previous findings in colon, lung, prostate, breast, and ovarian tumor xenografts (10). The results of this study suggest that activation of the FGFR2 pathway may be a promising target for gastric cancer therapy. AZD2171 may provide a clinical benefit to gastric cancer patients.


   Acknowledgments
 
We thank Dr. T. Komatsu and M. Takigahira for the animal study and Dr. K. Hirakawa (Osaka City University Graduate School of Medicine, Osaka, Japan) for providing the OCUM2M cell line.


   Footnotes
 
Grant support: Third-Term Comprehensive 10-Year Strategy for Cancer Control and program for promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and Japan Health Sciences Foundation.

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

Note: M. Takeda and T. Arao are the recipient of a Research Resident Fellowship from the Foundation of Promotion of Cancer Research in Japan.

Received 11/16/06; revised 1/29/07; accepted 2/27/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vanhoefer U, Rougier P, Wilke H, et al. Final results of a randomized phase III trial of sequential high-dose methotrexate, fluorouracil, and doxorubicin versus etoposide, leucovorin, and fluorouracil versus infusional fluorouracil and cisplatin in advanced gastric cancer: a trial of the European Organization for Research and Treatment of Cancer Gastrointestinal Tract Cancer Cooperative Group. J Clin Oncol 2000;18:2/648–57.[Abstract/Free Full Text]
  2. Ohtsu A, Shimada Y, Shirao K, et al. Randomized phase III trial of fluorouracil alone versus fluorouracil plus cisplatin versus uracil and tegafur plus mitomycin in patients with unresectable, advanced gastric cancer: The Japan Clinical Oncology Group Study (JCOG9205). J Clin Oncol 2003;21:54–9.[Abstract/Free Full Text]
  3. Grose R, Dickson C. Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev 2005;16:179–86.[CrossRef][Medline]
  4. Itoh H, Hattori Y, Sakamoto H, et al. Preferential alternative splicing in cancer generates a K-sam messenger RNA with higher transforming activity. Cancer Res 1994;54:3237–41.[Abstract/Free Full Text]
  5. Moffa AB, Tannheimer SL, Ethier SP. Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary epithelial cells. Mol Cancer Res 2004;2:643–52.[Abstract/Free Full Text]
  6. Nakatani H, Sakamoto H, Yoshida T, et al. Isolation of an amplified DNA sequence in stomach cancer. Jpn J Cancer Res 1990;81:707–10.[CrossRef]
  7. Hattori Y, Odagiri H, Nakatani H, et al. K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes. Proc Natl Acad Sci U S A 1990;87:5983–7.[Abstract/Free Full Text]
  8. Hattori Y, Itoh H, Uchino S, et al. Immunohistochemical detection of K-sam protein in stomach cancer. Clin Cancer Res 1996;2:1373–81.[Abstract]
  9. Ueda T, Sasaki H, Kuwahara Y, et al. Deletion of the carboxyl-terminal exons of K-sam/FGFR2 by short homology-mediated recombination, generating preferential expression of specific messenger RNAs. Cancer Res 1999;59:6080–6.[Abstract/Free Full Text]
  10. Wedge SR, Kendrew J, Hennequin LF, et al. AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 2005;65:4389–400.[Abstract/Free Full Text]
  11. Arao T, Yanagihara K, Takigahira M, et al. ZD6474 inhibits tumor growth and intraperitoneal dissemination in a highly metastatic orthotopic gastric cancer model. Int J Cancer 2006;118:483–9.[CrossRef][Medline]
  12. Arao T, Fukumoto H, Takeda M, et al. Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474. Cancer Res 2004;64:9101–4.[Abstract/Free Full Text]
  13. Taguchi F, Koh Y, Koizumi F, et al. Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib ("Iressa")-sensitive and resistant xenograft models. Cancer Sci 2004;95:984–9.[CrossRef]
  14. Koizumi F, Kanzawa F, Ueda Y, et al. Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells. Int J Cancer 2004;108:464–72.[CrossRef][Medline]
  15. Koizumi F, Shimoyama T, Taguchi F, Saijo N, Nishio K. Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int J Cancer 2005;116:36–44.[CrossRef][Medline]
  16. Sakai K, Arao T, Shimoyama T, et al. Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor. FASEB J 2006;20:311–3.[Abstract/Free Full Text]
  17. Duensing A, Heinrich MC, Fletcher CD, Fletcher JA. Biology of gastrointestinal stromal tumors: KIT mutations and beyond. Cancer Invest 2004;22:106–16.[CrossRef][Medline]
  18. Gowardhan B, Douglas DA, Mathers ME, et al. Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer. Br J Cancer 2005;92:320–7.[Medline]
  19. Zang XP, Nguyen TN, Pento JT. Specific and non-specific KGF inhibition of KGF-induced breast cancer cell motility. Anticancer Res 2002;22:2539–45.[Medline]
  20. Taniguchi F, Harada T, Sakamoto Y, et al. Activation of mitogen-activated protein kinase pathway by keratinocyte growth factor or fibroblast growth factor-10 promotes cell proliferation in human endometrial carcinoma cells. J Clin Endocrinol Metab 2003;88:773–80.[Abstract/Free Full Text]
  21. Ishibe T, Nakayama T, Okamoto T, et al. Disruption of fibroblast growth factor signal pathway inhibits the growth of synovial sarcomas: potential application of signal inhibitors to molecular target therapy. Clin Cancer Res 2005;11:2702–12.[Abstract/Free Full Text]
  22. St Bernard R, Zheng L, Liu W, et al. Fibroblast growth factor receptors as molecular targets in thyroid carcinoma. Endocrinology 2005;146:1145–53.[Abstract/Free Full Text]
  23. Ezzat S, Huang P, Dackiw A, Asa SL. Clin Dual inhibition of RET and FGFR4 restrains medullary thyroid cancer cell growth. Cancer Res 2005;11:1336–41.
  24. Chen J, Lee BH, Williams IR, et al. FGFR3 as a therapeutic target of the small molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene 2005;24:8259–67.[CrossRef][Medline]
  25. Trudel S, Li ZH, Wei E, et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma. Blood 2005;105:2941–8.[Abstract/Free Full Text]
  26. Delaval B, Letard S, Lelievre H, et al. Oncogenic tyrosine kinase of malignant hemopathy targets the centrosome. Cancer Res 2005;65:7231–40.[Abstract/Free Full Text]
  27. Chen J, Deangelo DJ, Kutok JL, et al. PKC412 inhibits the zinc finger 198-fibroblast growth factor receptor 1 fusion tyrosine kinase and is active in treatment of stem cell myeloproliferative disorder. Proc Natl Acad Sci U S A 2004;101:14479–84.[Abstract/Free Full Text]
  28. Hara T, Ooi A, Kobayashi M, Mai M, Yanagihara K, Nakanishi I. Amplification of c-myc, K-sam, and c-met in gastric cancers: detection by fluorescence in situ hybridization. Lab Invest 1998;9:43–53.
  29. Yanase K, Tsukahara S, Asada S, Ishikawa E, Imai Y, Sugimoto Y. Gefitinib reverses breast cancer resistance protein-mediated drug resistance. Mol Cancer Ther 2004;3:1119–25.[Abstract/Free Full Text]
  30. Elkind NB, Szentpetery Z, Apati A, et al. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib). Cancer Res 2005;65:1770–7.[Abstract/Free Full Text]
  31. Brehmer D, Greff Z, Godl K, et al. Cellular targets of gefitinib. Cancer Res 2005;65:379–82.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
K. Kunii, L. Davis, J. Gorenstein, H. Hatch, M. Yashiro, A. Di Bacco, C. Elbi, and B. Lutterbach
FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival
Cancer Res., April 1, 2008; 68(7): 2340 - 2348.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
N. Korsisaari, J. Ross, X. Wu, M. Kowanetz, N. Pal, L. Hall, J. Eastham-Anderson, W. F. Forrest, N. Van Bruggen, F. V. Peale, et al.
Blocking Vascular Endothelial Growth Factor-A Inhibits the Growth of Pituitary Adenomas and Lowers Serum Prolactin Level in a Mouse Model of Multiple Endocrine Neoplasia Type 1
Clin. Cancer Res., January 1, 2008; 14(1): 249 - 258.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takeda, M.
Right arrow Articles by Nishio, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takeda, M.
Right arrow Articles by Nishio, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online