Amplification of human epidermal growth factor receptor 2 (HER2) has been detected in 20% to 30% of gastric cancers and is associated with a poor outcome. Combination therapies with HER2-targeting agents and cytotoxic agents are considered a potential therapeutic option for gastric cancer with HER2 amplification. We have now investigated the effects of combination treatment with the oral fluoropyrimidine S-1 and the HER2-targeting agents lapatinib or trastuzumab in gastric cancer cells with or without HER2 amplification. We used 5-fluorouracil (5FU) instead of S-1 for in vitro experiments, given that tegafur, a component of S-1, is metabolized to 5FU in the liver. The combination of 5FU and HER2-targeting agents synergistically inhibited cell proliferation and exhibited an enhanced proapoptotic effect in gastric cancer cells with HER2 amplification, but not in those without it. Lapatinib or trastuzumab also induced downregulation of thymidylate synthase (TS) expression and activity only in cells with HER2 amplification. The combination of 5FU and TS depletion by RNA interference also exhibited an enhanced proapoptotic effect in cells with HER2 amplification. These observations thus suggest that lapatinib-induced or trastuzumab-induced downregulation of TS is responsible, at least in part, for the synergistic antitumor effect of combined treatment with 5FU and HER2-targeting agents. The antitumor effect of the combination of S-1 and HER2-targeting agents in vivo was also greater than that of either drug alone. Our preclinical findings thus indicate that the combination of S-1 and HER2-targeting agents is a promising treatment option for gastric cancer with HER2 amplification. Mol Cancer Ther; 9(5); 1198–207. ©2010 AACR.

Gastric cancer is the second leading cause of cancer mortality worldwide, with 700,000 confirmed deaths annually (1, 2). Advanced gastric cancer is treated predominantly by combination chemotherapy that includes fluoropyrimidine derivatives, but overall survival time remains <1 year (3, 4). Further improvement in such therapy is therefore warranted. S-1 is a novel oral anticancer drug that combines tegafur, a prodrug of 5-fluorouracil (5FU), with 5-chloro-2,4-dihydropyrimidine and potassium oxonate. 5-Chloro-2,4-dihydropyrimidine increases the plasma concentration of 5FU through competitive inhibition of dihydropyrimidine dehydrogenase, which catalyzes 5FU catabolism (5), whereas potassium oxonate reduces the gastrointestinal toxicity of 5FU (6). Clinical trials have revealed response rates of ∼30% to 50% for S-1 in advanced gastric cancer (69), and S-1 is now recognized as one of the standard chemotherapeutic drugs for this condition, especially in East Asia (911).

Recent years have seen substantial advances in the development of molecularly targeted therapy for various types of cancer. Amplification of human epidermal growth factor receptor 2 (HER2) has been detected in 20% to 30% of gastric cancers and is associated with a poor outcome and aggressiveness of the disease (12, 13). Targeting of HER2 is therefore thought to be beneficial for those gastric cancer patients with HER2 amplification. Clinical trials to evaluate the efficacy of HER2-targeting agents—including lapatinib, a dual tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) and HER2, and trastuzumab, a humanized monoclonal antibody to HER2—in individuals with gastric cancer positive for HER2 amplification are under way. However, the development of HER2-targeted therapy for gastric cancer lags behind that for breast cancer, for which trastuzumab is now recognized as a standard therapy for HER2-positive patients. Preclinical studies of HER2-targeting agents with gastric cancer cells positive for HER2 amplification are still limited (1417), with further investigations to clarify the efficacy and mechanism of action of HER2-targeting agents alone or in combination with cytotoxic drugs being required. We have now investigated the effects of combination treatment with S-1 (or 5FU) and the HER2-targeting agents lapatinib or trastuzumab in gastric cancer cells with or without HER2 amplification, and we have further examined the mechanism of such effects.

Cell culture and reagents

Human gastric cancer cell lines were obtained from the following sources: NCI-N87 from American Type Culture Collection; MKN-1, MKN-7, and AZ-521 from Health Science Research Resources Bank; MKN-28 from Immuno-Biological Laboratories; and SNU-216 from Korean Cell Line Bank. All cells were cultured under a humidified atmosphere of 5% CO2 at 37°C in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum. The human gastric cancer line 4-1ST was obtained from Central Institute for Experimental Animals and was maintained in BALB/c-nu/nu mice by s.c. injection of tumor pieces. Lapatinib was obtained from Sequoia Research Products, trastuzumab was from Hoffmann-La Roche, and 5FU and S-1 were from Wako. Tegafur, gimeracil, and oteracil, all of which are components of S-1, were synthesized by Taiho Pharmaceutical.

Fluorescence in situ hybridization analysis

The gene copy number per cell for HER2 was determined by fluorescence in situ hybridization with the use of HER2/neu (17q11.2-q12) Spectrum Orange and CEP17 (chromosome 17 centromere) Spectrum Green probes (Vysis; Abbott). Cells were centrifuged onto glass slides with a Shandon cytocentrifuge (Thermo Electron) and were fixed by consecutive incubations with ice-cold 70% ethanol for 10 minutes, 85% ethanol for 5 minutes, and 100% ethanol for 5 minutes. The slides were stored at −20°C until analysis. Cells were subsequently subjected to digestion with pepsin for 10 minutes at 37°C, washed with water, dehydrated with a graded series of ethanol solutions, denatured with 70% formamide in 2× SSC for 5 minutes at 72°C, and dehydrated again with a graded series of ethanol solutions before incubation with a hybridization mixture consisting of 50% formamide, Cot-1 DNA, and labeled DNA in 2× SSC. The slides were washed for 5 minutes at 73°C with 3× SSC, for 5 minutes at 37°C with 4× SSC containing 0.1% Triton X-100, and for 5 minutes at room temperature with 2× SSC before counter-staining with antifade solution containing 4′,6-diamidino-2-phenylindole. Hybridization signals were scored in 40 nuclei with the use of a 100× immersion objective lens. Nuclei with a disrupted boundary were excluded from the analysis. Gene amplification was defined as a mean HER2/chromosome 17 copy number ratio of >2.0 (18).

Growth inhibition assay in vitro

Cells were plated in 96-well flat-bottomed plates and cultured for 24 hours before exposure to various concentrations of drugs for 72 hours. TetraColor One (5 mmol/L tetrazolium monosodium salt and 0.2 mmol/L 1-methoxy-5-methyl phenazinium methylsulfate; Seikagaku) was then added to each well, and the cells were incubated for 3 hours at 37°C before measurement of absorbance at 490 nm with a Multiskan Spectrum instrument (Thermo Labsystems). Absorbance values were expressed as a percentage of that for untreated cells, and the concentration of tested drugs resulting in 50% growth inhibition (IC50) was calculated. Data were analyzed by the median-effect method (CalcuSyn software; Biosoft) to determine the combination index (CI), a well-established index of the interaction between two drugs (19). CI values of <1, 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively.

Annexin V binding assay

Binding of Annexin V to cells was measured with the use of an Annexin V-FLUOS Staining kit (Roche). Cells were harvested by exposure to trypsin-EDTA, washed with PBS, and centrifuged at 200 × g for 5 minutes. The cell pellets were resuspended in 100 μL of Annexin V-FLUOS labeling solution, incubated for 10 to 15 minutes at 15°C to 25°C, and then analyzed for fluorescence with a flow cytometer (FACSCalibur) and Cell Quest software (Becton Dickinson).

Assay of caspase-3 activity

The activity of caspase-3 in cell lysates was measured with the use of a CCP32/Caspase-3 Fluometric Protease Assay kit (MBL). Fluorescence attributable to cleavage of the Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) substrate was measured at excitation and emission wavelengths of 390 and 460 nm, respectively.

Immunoblot analysis

Cells were washed twice with ice-cold PBS and then lysed in a solution containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, and leupeptin (1 μg/mL). The protein concentration of cell lysates was determined with the Bradford reagent (Bio-Rad), and equal amounts of protein were subjected to SDS-PAGE on a 7.5% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then incubated with Blocking One solution (Nacalai Tesque) for 20 minutes at room temperature before incubation overnight at 4°C with primary antibodies including those to phosphorylated AKT (1:1,000 dilution; Cell Signaling Technology), to AKT (1:1,000 dilution, Cell Signaling Technology), to phosphorylated extracellular signal-regulated kinase (ERK; 1:1,000 dilution; Santa Cruz Biotechnology), to ERK (1:1,000 dilution, Santa Cruz Biotechnology), to E2F1 (1:1,000 dilution, Santa Cruz Biotechnology), to thymidylate synthase (TS; 1:1,000 dilution, Santa Cruz Biotechnology), or to β-actin (1:500 dilution, Sigma). The membrane was then washed with PBS containing 0.05% Tween 20 before incubation for 1 hour at room temperature with horseradish peroxidase–conjugated antibodies to rabbit IgG (Sigma). Immune complexes were finally detected with ECL Western Blotting Detection Reagents (GE Healthcare).

TS activity assay

TS activity was quantified with the use of a tritiated fluoro-dUMP binding assay (20). Cells were harvested and disrupted by ultrasonic treatment in a solution containing 0.2 mol/L Tris-HCl (pH 7.4), 20 mmol/L 2-mercaptoethanol, 15 mmol/L CMP, and 100 mmol/L NaF. The cell lysate was centrifuged at 1,630 × g for 15 minutes at 4°C, and the resulting supernatant was centrifuged at 105,000 × g for 1 hour at 4°C. A portion (50 μL) of the final supernatant was mixed with 50 μL of a solution containing 600 mmol/L NH4HCO3 buffer (pH 8.0), 100 mmol/L 2-mercaptoethanol, 100 mmol/L NaF, and 15 mmol/L CMP. After the addition of 50 μL of [6-3H]fluoro-dUMP (7.8 pmol, 0.12 μCi) plus 25 μL of cofactor solution containing 50 mmol/L potassium phosphate buffer (pH 7.4), 20 mmol/L 2-mercaptoethanol, 100 mmol/L NaF, 15 mmol/L CMP, 2% bovine serum albumin, 2 mmol/L tetrahydrofolic acid, 16 mmol/L sodium ascorbate, and 9 mmol/L formaldehyde, the mixture was incubated for 20 minutes at 30°C. The reaction was terminated by the addition of 100 μL of 2% bovine serum albumin and 275 μL of 1 mol/L HClO4 followed by centrifugation at 1,630 × g for 15 minutes at 4°C. The resulting pellet was resuspended in 2 mL of 0.5 mol/L HClO4, and the suspension was subjected to ultrasonic treatment followed by centrifugation at 1,630× g for 15 minutes at 4°C. The final precipitate was solubilized in 0.5 mL of 98% formic acid, mixed with 10 mL of ACS II scintillation fluid (GE Healthcare), and assayed for radioactivity.

Gene silencing

Cells were plated at 50% to 60% confluence in six-well plates or 25-cm2 flasks and then incubated for 24 hours before transient transfection for 48 hours with small interfering RNAs (siRNA) mixed with the Lipofectamine reagent (Invitrogen). An siRNA specific for TS mRNA (5′-CAAUCCGCAUCCAACUAUUTT-3′) and a nonspecific siRNA (control) were obtained from Nippon EGT.

Animals

Male athymic nude mice were exposed to a 12-h light/12-h dark cycle and provided with food and water ad libitum in a barrier facility. All animal experiments were done in compliance with the regulations of the Animal Experimentation Committee of Taiho Pharmaceutical Co. Ltd.

Growth inhibition assay in vivo

Cubic fragments of tumor tissue (∼2 × 2 × 2 mm) were implanted s.c. into the axilla of 5-week-old to 6-week-old male athymic nude mice. Treatment was initiated when tumors in each group achieved an average volume of 50 to 200 mm3. Treatment groups consisted of control, S-1 alone, lapatinib alone, trastuzumab alone, and the combination of S-1 and either lapatinib or trastuzumab. Each treatment group contained seven mice. S-1 and lapatinib were given by oral gavage daily for 28 days; control animals received a 0.5% (w/v) aqueous solution of hydroxypropylmethylcellulose as vehicle. Trastuzumab was given i.p. weekly. Tumor volume was determined from caliper measurements of tumor length (L) and width (W) according to the formula LW2/2. Both tumor size and body weight were measured twice per week.

Statistical analysis

Unless indicated otherwise, data are presented as means ± SEM from three independent experiments or for seven animals per group. The unpaired two-tailed Student's t test was used to evaluate the significance of differences in the percentage of Annexin V–positive cells, relative caspase-3 activity, or tumor volume. A P value of <0.05 was considered statistically significant.

Synergistic antiproliferative effect of 5FU and either lapatinib or trastuzumab in gastric cancer cells positive for HER2 amplification

We first examined the effect of the combination of 5FU and either lapatinib or trastuzumab on the growth in vitro of gastric cancer cells positive or negative for HER2 amplification. We used 5FU instead of S-1 for in vitro experiments, given that tegafur, a component of S-1, is metabolized to 5FU in the liver. The combined effect of each pair of drugs was evaluated on the basis of the CI. The combination of 5FU and lapatinib exhibited a synergistic inhibitory effect (CI < 1.0) on the growth of cells with HER2 amplification, including NCI-N87, SNU-216, and MKN-7 cells, but not on that of cells without HER2 amplification, including AZ-521, MKN-28, and MKN-1 cells (Fig. 1A and B). A synergistic interaction between 5FU and trastuzumab was also apparent in cells with HER2 amplification but not in those without it (Fig. 1C). The combination of 5FU with either lapatinib or trastuzumab thus exerted a synergistic antiproliferative effect in gastric cancer cells positive for HER2 amplification but not in those negative for HER2 amplification.

Figure 1.

Effect of the combination of 5FU and HER2-targeting agents on the growth in vitro of gastric cancer cells positive or negative for HER2 amplification. A, fluorescence in situ hybridization analysis of gastric cancer cell lines. The indicated cell lines were subjected to hybridization with a HER2/neu probe (orange) and a chromosome 17 centromere probe (green). B and C, gastric cancer cells with or without HER2 amplification were incubated for 72 hours with lapatinib (B) or trastuzumab (C) together with 5FU at a fixed lapatinib/5FU molar ratio of 1:10 or a fixed trastuzumab/5FU molar ratio of 15:1, after which cell viability was measured. The interaction between the two drugs in each combination was evaluated on the basis of the CI. CI values of <1, 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. Data are means of triplicates from a representative experiment.

Figure 1.

Effect of the combination of 5FU and HER2-targeting agents on the growth in vitro of gastric cancer cells positive or negative for HER2 amplification. A, fluorescence in situ hybridization analysis of gastric cancer cell lines. The indicated cell lines were subjected to hybridization with a HER2/neu probe (orange) and a chromosome 17 centromere probe (green). B and C, gastric cancer cells with or without HER2 amplification were incubated for 72 hours with lapatinib (B) or trastuzumab (C) together with 5FU at a fixed lapatinib/5FU molar ratio of 1:10 or a fixed trastuzumab/5FU molar ratio of 15:1, after which cell viability was measured. The interaction between the two drugs in each combination was evaluated on the basis of the CI. CI values of <1, 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. Data are means of triplicates from a representative experiment.

Close modal

Enhanced induction of apoptosis by the combination of 5FU and either lapatinib or trastuzumab in gastric cancer cells positive for HER2 amplification

To investigate the mechanism of the synergistic growth inhibition induced by the combination of 5FU and either lapatinib or trastuzumab, we examined the effects of each agent alone and in combination on apoptosis in gastric cancer cells. An assay based on the binding of Annexin V to the cell surface revealed that the frequency of apoptosis was markedly greater for HER2 amplification–positive cells treated with the combination of 5FU and either lapatinib or trastuzumab than for those treated with either agent alone (Fig. 2A and B). Such an effect was not apparent in cells negative for HER2 amplification. To confirm the results of the Annexin V binding assay, we measured the activity of caspase-3. Again, the combination of 5FU and either lapatinib or trastuzumab induced an increase in caspase-3 activity greater than that apparent with either agent alone in cells with HER2 amplification but not in those without it (Fig. 2C). Together, these data thus indicated that the combination of 5FU and either lapatinib or trastuzumab exhibits an enhanced proapoptotic effect in gastric cancer cells positive for HER2 amplification but not in those negative for this genetic change.

Figure 2.

Effect of the combination of 5FU and HER2-targeting agents on apoptosis in gastric cancer cells positive or negative for HER2 amplification. A, cells were incubated for 72 hours with lapatinib, trastuzumab, or 5FU at their IC50 concentrations unless indicated otherwise: 0.02 μmol/L, 1.5 μg/mL, and 2.5 μmol/L, respectively, for NCI-N87 cells and 2.0 μmol/L, 200 μg/mL (IC50 not determined), and 4.5 μmol/L, respectively, for AZ-521 cells. The proportion of apoptotic cells was then assessed by staining with FITC-conjugated Annexin V and propidium iodide (PI) followed by flow cytometry. B, the proportion of apoptotic cells in experiments similar to that shown in A was determined. Data are means ± SEM from three independent experiments. C, lysates prepared from cells exposed to drugs as in A for 48 hours were assayed for caspase-3 activity. Data are expressed relative to the corresponding value for the control condition and are means ± SEM from three independent experiments. *, P < 0.05, for the indicated comparisons.

Figure 2.

Effect of the combination of 5FU and HER2-targeting agents on apoptosis in gastric cancer cells positive or negative for HER2 amplification. A, cells were incubated for 72 hours with lapatinib, trastuzumab, or 5FU at their IC50 concentrations unless indicated otherwise: 0.02 μmol/L, 1.5 μg/mL, and 2.5 μmol/L, respectively, for NCI-N87 cells and 2.0 μmol/L, 200 μg/mL (IC50 not determined), and 4.5 μmol/L, respectively, for AZ-521 cells. The proportion of apoptotic cells was then assessed by staining with FITC-conjugated Annexin V and propidium iodide (PI) followed by flow cytometry. B, the proportion of apoptotic cells in experiments similar to that shown in A was determined. Data are means ± SEM from three independent experiments. C, lysates prepared from cells exposed to drugs as in A for 48 hours were assayed for caspase-3 activity. Data are expressed relative to the corresponding value for the control condition and are means ± SEM from three independent experiments. *, P < 0.05, for the indicated comparisons.

Close modal

Downregulation by lapatinib or trastuzumab of the expression and activity of TS in gastric cancer cells positive for HER2 amplification

To investigate further the molecular mechanism of the synergistic antiproliferative effect of the combination of 5FU and HER2-targeting agents, we next examined the effects of lapatinib and trastuzumab on TS expression and activity in gastric cancer cells, given that a reduced level of TS expression has been associated with a higher response rate to 5FU-based chemotherapy (21, 22). Exposure of HER2 amplification–positive cells to either lapatinib or trastuzumab resulted in downregulation of TS expression in a concentration-dependent manner, whereas TS expression was not affected by these agents in cells without HER2 amplification (Fig. 3A and B). Consistent with these results, lapatinib or trastuzumab reduced TS activity in cells with HER2 amplification but not in those without it (Fig. 3C). Furthermore, lapatinib or trastuzumab downregulated the expression of E2F1, a transcription factor that promotes expression of the TS gene (23), in cells positive for HER2 amplification but not in those negative for this genetic change (Fig. 3A and B).

Figure 3.

Effect of HER2-targeting agents on E2F1 and TS expression or activity in gastric cancer cells positive or negative for HER2 amplification. A and B, cells were incubated with the indicated concentrations of lapatinib for 24 hours (A) or trastuzumab for 48 hours (B), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of AKT or ERK as well as with those to E2F1, TS, and β-actin (loading control). C, cells were treated with lapatinib (1 μmol/L) for 24 hours or with trastuzumab (200 μg/mL) for 48 hours, after which cell lysates were prepared and assayed for TS activity. Data are expressed as a percentage of the corresponding value for control cells and are means ± SEM from three independent experiments.

Figure 3.

Effect of HER2-targeting agents on E2F1 and TS expression or activity in gastric cancer cells positive or negative for HER2 amplification. A and B, cells were incubated with the indicated concentrations of lapatinib for 24 hours (A) or trastuzumab for 48 hours (B), after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of AKT or ERK as well as with those to E2F1, TS, and β-actin (loading control). C, cells were treated with lapatinib (1 μmol/L) for 24 hours or with trastuzumab (200 μg/mL) for 48 hours, after which cell lysates were prepared and assayed for TS activity. Data are expressed as a percentage of the corresponding value for control cells and are means ± SEM from three independent experiments.

Close modal

To explore the mechanism of TS downregulation by HER2-targeting agents, we examined the effects of these agents on the phosphoinositide 3-kinase (PI3K)–AKT signaling pathway as well as on signaling by the mitogen-activated protein kinase ERK. Immunoblot analysis showed that phosphorylation of AKT in HER2 amplification–positive cells was inhibited by lapatinib or trastuzumab, whereas phosphorylation of ERK in these cells was inhibited only by lapatinib (Fig. 3A and B). Phosphorylation of AKT or ERK was not affected by either HER2-targeting agent in cells without HER2 amplification. These data thus suggested that lapatinib and trastuzumab each induce downregulation of TS expression and activity in HER2 amplification–positive gastric cancer cells and that this effect is attributable to downregulation of E2F1, possibly mediated by inhibition of the PI3K-AKT signaling pathway.

Enhancement of 5FU-induced apoptosis by depletion of TS in gastric cancer cells positive for HER2 amplification

To investigate whether the downregulation of TS by lapatinib or trastuzumab indeed contributes to the synergistic antiproliferative effect of these drugs with 5FU in gastric cancer cells positive for HER2 amplification, we depleted such cells of TS by transfection with an siRNA specific for TS mRNA (Fig. 4A). Similar to the action of lapatinib or trastuzumab, RNA interference–mediated depletion of TS enhanced the effects of 5FU treatment on the number of apoptotic cells and the activity of caspase-3 compared with those apparent in cells transfected with a control siRNA (Fig. 4B–D). These data thus indicated that downregulation of TS by lapatinib or trastuzumab contributes, at least in part, to the observed synergistic antiproliferative and proapoptotic interaction of these drugs with 5FU.

Figure 4.

Effect of RNA interference–mediated depletion of TS on the proapoptotic action of 5FU in gastric cancer cells positive for HER2 amplification. A, cells were transfected with nonspecific (control) or TS siRNAs for 48 hours, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS and to β-actin. B, cells were transfected with nonspecific or TS siRNAs as in A, replated and incubated for 72 hours in complete medium in the absence or presence of 5FU at IC50 concentrations (2.5 and 1.5 μmol/L for NCI-N87 and SNU-216 cells, respectively), and then evaluated for apoptosis by staining with Annexin V. C, the proportion of apoptotic cells in experiments similar to that in B was determined. Data are means ± SEM from three independent experiments. D, cells treated as in B were lysed and assayed for caspase-3 activity after exposure to 5FU for 48 hours. Data are means ± SEM from three independent experiments. *, P < 0.05 for the indicated comparisons.

Figure 4.

Effect of RNA interference–mediated depletion of TS on the proapoptotic action of 5FU in gastric cancer cells positive for HER2 amplification. A, cells were transfected with nonspecific (control) or TS siRNAs for 48 hours, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS and to β-actin. B, cells were transfected with nonspecific or TS siRNAs as in A, replated and incubated for 72 hours in complete medium in the absence or presence of 5FU at IC50 concentrations (2.5 and 1.5 μmol/L for NCI-N87 and SNU-216 cells, respectively), and then evaluated for apoptosis by staining with Annexin V. C, the proportion of apoptotic cells in experiments similar to that in B was determined. Data are means ± SEM from three independent experiments. D, cells treated as in B were lysed and assayed for caspase-3 activity after exposure to 5FU for 48 hours. Data are means ± SEM from three independent experiments. *, P < 0.05 for the indicated comparisons.

Close modal

Enhanced inhibition of the growth of HER2 amplification–positive gastric cancer cells in vivo by combined treatment with S-1 and either lapatinib or trastuzumab

Finally, we investigated the effect of combined treatment with S-1 and either lapatinib or trastuzumab on the growth in vivo of gastric cancer cells positive for HER2 amplification. Mice with palpable tumors formed by NCI-N87 or 4-1ST cells were divided into groups for treatment with vehicle, S-1, lapatinib, trastuzumab, or the combination of S-1 and either lapatinib or trastuzumab for 4 weeks. Combination therapy with S-1 and lapatinib (Fig. 5A) or with S-1 and trastuzumab (Fig. 5B) inhibited the growth of tumors formed by NCI-N87 or 4-1ST cells to a significantly greater extent than did treatment with either drug alone. All treatments were well tolerated by the mice, with no signs of toxicity or weight loss during therapy (data not shown). These findings thus suggested that combination therapy with S-1 and either lapatinib or trastuzumab exhibits an enhanced antitumor effect in gastric cancer xenografts positive for HER2 amplification, consistent with the results obtained in vitro.

Figure 5.

Effect of the combination of S-1 and HER2-targeting agents on the growth in vivo of gastric cancer cells with HER2 amplification. Nude mice with tumor xenografts established by s.c. implantation of NCI-N87 cells were treated for 4 weeks by daily oral gavage with vehicle (control), S-1 (10 mg/kg), or lapatinib (50 × 2 mg/kg, twice a day; A) or by weekly i.p. administration of trastuzumab (20 mg/kg on days 1, 8, 15, and 22; B), as indicated. Nude mice with 4-1ST xenografts were similarly treated with vehicle (control), S-1 (8.3 mg/kg), lapatinib (30 × 2 mg/kg, twice a day; A), or trastuzumab (10 mg/kg on days 1, 8, 15, and 22; B). Tumor volume was determined at the indicated times after the onset of treatment. Data are means ± SEM of values from seven mice per group. *, P < 0.05, for the combination of S-1 plus lapatinib or trastuzumab at 28 days versus the corresponding value for S-1, lapatinib, or trastuzumab alone.

Figure 5.

Effect of the combination of S-1 and HER2-targeting agents on the growth in vivo of gastric cancer cells with HER2 amplification. Nude mice with tumor xenografts established by s.c. implantation of NCI-N87 cells were treated for 4 weeks by daily oral gavage with vehicle (control), S-1 (10 mg/kg), or lapatinib (50 × 2 mg/kg, twice a day; A) or by weekly i.p. administration of trastuzumab (20 mg/kg on days 1, 8, 15, and 22; B), as indicated. Nude mice with 4-1ST xenografts were similarly treated with vehicle (control), S-1 (8.3 mg/kg), lapatinib (30 × 2 mg/kg, twice a day; A), or trastuzumab (10 mg/kg on days 1, 8, 15, and 22; B). Tumor volume was determined at the indicated times after the onset of treatment. Data are means ± SEM of values from seven mice per group. *, P < 0.05, for the combination of S-1 plus lapatinib or trastuzumab at 28 days versus the corresponding value for S-1, lapatinib, or trastuzumab alone.

Close modal

HER2 amplification is a frequent molecular abnormality in gastric cancer as well as in various other cancers. Trastuzumab is widely used as a standard therapy for HER2-positive patients with breast cancer, with the drug showing clinical efficacy both alone and in combination with chemotherapeutic agents (24, 25). HER2 is thus considered to be a potential target for the treatment of gastric cancer positive for HER2 amplification. A recently reported phase III clinical trial showed a significant gain in overall survival for HER2-positive patients with advanced gastric cancer who received combined treatment with trastuzumab and fluoropyrimidine-cisplatin compared with those treated without trastuzumab (26). However, there has been limited examination of HER2-targeting agents in gastric cancer models, and most such studies have been restricted to cells with HER2 amplification. Furthermore, the mechanisms of action of HER2-targeting agents in combination with cytotoxic agents have remained unclear.

In the present study, we have shown that the combination of S-1 (or 5FU) and HER2-targeting agents exerts a synergistic antitumor effect in gastric cancer cells with HER2 amplification but not in those without it. We found that HER2-targeting agents inhibit TS activity as well as TS expression in HER2 amplification–positive gastric cancer cells, but not in cells without HER2 amplification. Lapatinib is a dual inhibitor of EGFR and HER2, and so its downregulation of TS might be attributable to inhibition of either of these tyrosine kinases. However, given that trastuzumab downregulated TS expression and activity to an extent similar to that observed with lapatinib, the effects of both lapatanib and trastuzumab on TS are likely mediated by inhibition of HER2. This conclusion is further supported by the observation that transfection of HER2 amplification–positive gastric cancer cells with an siRNA specific for HER2 mRNA resulted in marked inhibition of TS expression, whereas transfection with an EGFR siRNA had no such effect (data not shown). Downregulation of TS by HER2-targeting agents was accompanied by a reduction in the abundance of E2F1, suggesting that this effect on TS results from attenuation of E2F1-dependent transcription of the TS gene. Although the mechanism responsible for regulation of TS and E2F1 remains unclear, our observations indicate that inhibition of the PI3K-AKT pathway contributes, at least in part, to the downregulation of TS by HER2-targeting agents. Activation of PI3K-AKT signaling has been found to result in E2F1 accumulation (27, 28), supporting the notion that inhibition of such signaling by HER2-targeting agents leads to downregulation of E2F1 and TS. We previously showed that inhibition of EGFR by EGFR–tyrosine kinase inhibitors results in downregulation of TS and E2F1 expression in non–small cell lung cancer cells (29, 30). Given that downregulation of TS was induced by HER2-targeting agents in gastric cancer cells with HER2 amplification and by EGFR–tyrosine kinase inhibitors in non–small cell lung cancer cells, the expression of TS is likely dependent on receptor tyrosine kinase signaling, which is essential for cell survival.

Downregulation of TS expression has been found to enhance the efficacy of 5FU, possibly as a direct result of the decrease in the amount of this protein target of 5FU (31). In the present study, we found that depletion of TS by RNA interference enhanced the induction of apoptosis by 5FU in gastric cancer cells with HER2 amplification, suggesting that the proapoptotic effect of the combination of 5FU and HER2-targeting agents is attributable to TS inhibition. The abundance of TS in neoplastic cells has been found to increase after exposure to 5FU, resulting in maintenance of the amount of the free enzyme in excess of that of enzyme bound to 5FU (3234). Such an increase in TS expression and activity has been viewed as a mechanistic driver of 5FU resistance in cancer cells (22, 3539). Downregulation of TS by HER2-targeting agents might thus contribute to reversal of the 5FU-induced increase in TS expression, resulting in enhancement of 5FU-induced apoptosis. In addition, prolonged inhibition of TS has been shown to trigger apoptosis by inducing an imbalance in the deoxyribonucleoside pool and consequent disruption of DNA synthesis and repair (4042). Given that the TS siRNA itself induced apoptosis in gastric cancer cells positive for HER2 amplification in the present study, the depletion of TS by HER2-targeting agents might also contribute directly to the combined proapoptotic action with 5FU.

The HER2 amplification–positive gastric cancer cell line MKN-7 has been found to be insensitive to trastuzumab. In contrast to their insensitivity to trastuzumab, we found that MKN-7 cells retain sensitivity to lapatinib (IC50 values of >200 μg/mL and 0.99 ± 0.055 μmol/L for trastuzumab and lapatinib, respectively; data not shown). Most HER2-positive breast cancer patients who initially respond to trastuzumab ultimately develop resistance to this drug (25). Preclinical studies have indicated several molecular mechanisms that might contribute to the development of trastuzumab resistance, including signaling by a HER2-HER3-PI3K-PTEN pathway (43, 44). One possible explanation for trastuzumab resistance in MKN-7 cells is activation of the EGFR signaling pathway (45, 46). MKN-7 cells might prove to be a good model for the study of trastuzumab-resistant cells positive for HER2 amplification. We found that lapatinib and trastuzumab each inhibit TS expression and activity in MKN-7 cells, likely accounting for the synergistic antiproliferative effect observed with 5FU. These data suggest that the synergistic antitumor effect of the combination of 5FU and HER2-targeting agents is conserved in trastuzumab-resistant cells with HER2 amplification.

In conclusion, we have shown that the combination of S-1 and HER2-targeting agents exerts a synergistic antitumor effect mediated by TS inhibition in gastric cancer cells with HER2 amplification, but not in those negative for HER2 amplification. Our observations provide a rationale for clinical evaluation of combination chemotherapy with S-1 and HER2-targeting agents according to HER2 amplification status.

No potential conflicts of interest were disclosed.

We thank M. Iki for helpful discussion and E. Hatashita, K. Kuwata, and H. Yamaguchi for technical assistance.

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.

1
Kelley
JR
,
Duggan
JM
. 
Gastric cancer epidemiology and risk factors
.
J Clin Epidemiol
2003
;
56
:
1
9
.
2
Kamangar
F
,
Dores
GM
,
Anderson
WF
. 
Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world
.
J Clin Oncol
2006
;
24
:
2137
50
.
3
Hartgrink
HH
,
Jansen
EP
,
van Grieken
NC
,
van de Velde
CJ
. 
Gastric cancer
.
Lancet
2009
;
374
:
477
90
.
4
Wesolowski
R
,
Lee
C
,
Kim
R
. 
Is there a role for second-line chemotherapy in advanced gastric cancer?
Lancet Oncol
2009
;
10
:
903
12
.
5
Tatsumi
K
,
Fukushima
M
,
Shirasaka
T
,
Fujii
S
. 
Inhibitory effects of pyrimidine, barbituric acid and pyridine derivatives on 5-fluorouracil degradation in rat liver extracts
.
Jpn J Cancer Res
1987
;
78
:
748
55
.
6
Shirasaka
T
,
Shimamato
Y
,
Ohshimo
H
, et al
. 
Development of a novel form of an oral 5-fluorouracil derivative (S-1) directed to the potentiation of the tumor selective cytotoxicity of 5-fluorouracil by two biochemical modulators
.
Anticancer Drugs
1996
;
7
:
548
57
.
7
Sakata
Y
,
Ohtsu
A
,
Horikoshi
N
,
Sugimachi
K
,
Mitachi
Y
,
Taguchi
T
. 
Late phase II study of novel oral fluoropyrimidine anticancer drug S-1 (1 M tegafur-0.4 M gimestat-1 M otastat potassium) in advanced gastric cancer patients
.
Eur J Cancer
1998
;
34
:
1715
20
.
8
Koizumi
W
,
Kurihara
M
,
Nakano
S
,
Hasegawa
K
. 
Phase II study of S-1, a novel oral derivative of 5-fluorouracil, in advanced gastric cancer. For the S-1 Cooperative Gastric Cancer Study Group
.
Oncology
2000
;
58
:
191
7
.
9
Koizumi
W
,
Narahara
H
,
Hara
T
, et al
. 
S-1 plus cisplatin versus S-1 alone for first-line treatment of advanced gastric cancer (SPIRITS trial): a phase III trial
.
Lancet Oncol
2008
;
9
:
215
21
.
10
Sakuramoto
S
,
Sasako
M
,
Yamaguchi
T
, et al
. 
Adjuvant chemotherapy for gastric cancer with S-1, an oral fluoropyrimidine
.
N Engl J Med
2007
;
357
:
1810
20
.
11
Boku
N
. 
Chemotherapy for metastatic disease: review from JCOG trials
.
Int J Clin Oncol
2008
;
13
:
196
200
.
12
Gravalos
C
,
Jimeno
A
. 
HER2 in gastric cancer: a new prognostic factor and a novel therapeutic target
.
Ann Oncol
2008
;
19
:
1523
9
.
13
Tanner
M
,
Hollmen
M
,
Junttila
TT
, et al
. 
Amplification of HER-2 in gastric carcinoma: association with topoisomerase IIα gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab
.
Ann Oncol
2005
;
16
:
273
8
.
14
Kim
JW
,
Kim
HP
,
Im
SA
, et al
. 
The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines
.
Cancer Lett
2008
;
272
:
296
306
.
15
Fujimoto-Ouchi
K
,
Sekiguchi
F
,
Yasuno
H
,
Moriya
Y
,
Mori
K
,
Tanaka
Y
. 
Antitumor activity of trastuzumab in combination with chemotherapy in human gastric cancer xenograft models
.
Cancer Chemother Pharmacol
2007
;
59
:
795
805
.
16
Kim
HP
,
Yoon
YK
,
Kim
JW
, et al
. 
Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, downregulates thymidylate synthase by inhibiting the nuclear translocation of EGFR and HER2
.
PLoS One
2009
;
4
:
e5933
.
17
Kim
SY
,
Kim
HP
,
Kim
YJ
, et al
. 
Trastuzumab inhibits the growth of human gastric cancer cell lines with HER2 amplification synergistically with cisplatin
.
Int J Oncol
2008
;
32
:
89
95
.
18
Wolff
AC
,
Hammond
ME
,
Schwartz
JN
, et al
. 
American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer
.
Arch Pathol Lab Med
2007
;
131
:
18
43
.
19
Chou
TC
,
Talalay
P
. 
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
.
Adv Enzyme Regul
1984
;
22
:
27
55
.
20
Spears
CP
,
Gustavsson
BG
,
Mitchell
MS
, et al
. 
Thymidylate synthetase inhibition in malignant tumors and normal liver of patients given intravenous 5-fluorouracil
.
Cancer Res
1984
;
44
:
4144
50
.
21
Johnston
PG
,
Fisher
ER
,
Rockette
HE
, et al
. 
The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer
.
J Clin Oncol
1994
;
12
:
2640
7
.
22
Johnston
PG
,
Drake
JC
,
Trepel
J
,
Allegra
CJ
. 
Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines
.
Cancer Res
1992
;
52
:
4306
12
.
23
DeGregori
J
,
Kowalik
T
,
Nevins
JR
. 
Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes
.
Mol Cell Biol
1995
;
15
:
4215
24
.
24
Cobleigh
MA
,
Vogel
CL
,
Tripathy
D
, et al
. 
Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease
.
J Clin Oncol
1999
;
17
:
2639
48
.
25
Slamon
DJ
,
Leyland-Jones
B
,
Shak
S
, et al
. 
Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2
.
N Engl J Med
2001
;
344
:
783
92
.
26
Van Cutsem
E
,
Kang
Y
,
Chung
H
. 
Efficacy results from the TOGA trial: a phase III study of trastuzumab added to standard chemotherapy (CT) in first-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC)
.
J Clin Oncol
2009
;
27
:
18s (abstr LBA4509)
.
27
Hallstrom
TC
,
Nevins
JR
. 
Specificity in the activation and control of transcription factor E2F-dependent apoptosis
.
Proc Natl Acad Sci U S A
2003
;
100
:
10848
53
.
28
Liu
K
,
Paik
JC
,
Wang
B
,
Lin
FT
,
Lin
WC
. 
Regulation of TopBP1 oligomerization by Akt/PKB for cell survival
.
EMBO J
2006
;
25
:
4795
807
.
29
Okabe
T
,
Okamoto
I
,
Tsukioka
S
, et al
. 
Addition of S-1 to the epidermal growth factor receptor inhibitor gefitinib overcomes gefitinib resistance in non-small cell lung cancer cell lines with MET amplification
.
Clin Cancer Res
2009
;
15
:
907
13
.
30
Okabe
T
,
Okamoto
I
,
Tsukioka
S
, et al
. 
Synergistic antitumor effect of S-1 and the epidermal growth factor receptor inhibitor gefitinib in non-small cell lung cancer cell lines: role of gefitinib-induced downregulation of thymidylate synthase
.
Mol Cancer Ther
2008
;
7
:
599
606
.
31
Ferguson
PJ
,
Collins
O
,
Dean
NM
, et al
. 
Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells
.
Br J Pharmacol
1999
;
127
:
1777
86
.
32
Washtien
WL
. 
Increased levels of thymidylate synthetase in cells exposed to 5-fluorouracil
.
Mol Pharmacol
1984
;
25
:
171
7
.
33
Spears
CP
,
Gustavsson
BG
,
Berne
M
,
Frosing
R
,
Bernstein
L
,
Hayes
AA
. 
Mechanisms of innate resistance to thymidylate synthase inhibition after 5-fluorouracil
.
Cancer Res
1988
;
48
:
5894
900
.
34
Chu
E
,
Zinn
S
,
Boarman
D
,
Allegra
CJ
. 
Interaction of γ interferon and 5-fluorouracil in the H630 human colon carcinoma cell line
.
Cancer Res
1990
;
50
:
5834
40
.
35
Copur
S
,
Aiba
K
,
Drake
JC
,
Allegra
CJ
,
Chu
E
. 
Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil
.
Biochem Pharmacol
1995
;
49
:
1419
26
.
36
Chu
E
,
Koeller
DM
,
Johnston
PG
,
Zinn
S
,
Allegra
CJ
. 
Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-γ
.
Mol Pharmacol
1993
;
43
:
527
33
.
37
Chu
E
,
Voeller
DM
,
Jones
KL
, et al
. 
Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells
.
Mol Cell Biol
1994
;
14
:
207
13
.
38
Longley
DB
,
Harkin
DP
,
Johnston
PG
. 
5-Fluorouracil: mechanisms of action and clinical strategies
.
Nat Rev Cancer
2003
;
3
:
330
8
.
39
Kawate
H
,
Landis
DM
,
Loeb
LA
. 
Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine
.
J Biol Chem
2002
;
277
:
36304
11
.
40
Yoshioka
A
,
Tanaka
S
,
Hiraoka
O
, et al
. 
Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death
.
J Biol Chem
1987
;
262
:
8235
41
.
41
Ayusawa
D
,
Shimizu
K
,
Koyama
H
,
Takeishi
K
,
Seno
T
. 
Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells
.
J Biol Chem
1983
;
258
:
12448
54
.
42
Wyatt
MD
,
Wilson
DM
 III
. 
Participation of DNA repair in the response to 5-fluorouracil
.
Cell Mol Life Sci
2009
;
66
:
788
99
.
43
Kruser
TJ
,
Wheeler
DL
. 
Mechanisms of resistance to HER family targeting antibodies
.
Exp Cell Res
2010
;
316
:
1083
100
.
44
Nahta
R
,
Yu
D
,
Hung
MC
,
Hortobagyi
GN
,
Esteva
FJ
. 
Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer
.
Nat Clin Pract Oncol
2006
;
3
:
269
80
.
45
Lane
HA
,
Beuvink
I
,
Motoyama
AB
,
Daly
JM
,
Neve
RM
,
Hynes
NE
. 
ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency
.
Mol Cell Biol
2000
;
20
:
3210
23
.
46
Lewis
GD
,
Figari
I
,
Fendly
B
, et al
. 
Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies
.
Cancer Immunol Immunother
1993
;
37
:
255
63
.