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Clinical Cancer Research Vol. 12, 6161-6169, October 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Effect of Simultaneous Inhibition of Epidermal Growth Factor Receptor and Cyclooxygenase-2 in HER-2/Neu-Positive Breast Cancer

Susan Lanza-Jacoby1, Randy Burd2, Francis E. Rosato, Jr.1, Kandace McGuire1, James Little1, Noel Nougbilly1 and Sheldon Miller1

Authors' Affiliations: Departments of 1 Surgery and 2 Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania

Requests for reprints: Susan Lanza-Jacoby, Department of Surgery, Jefferson Medical College, 1025 Walnut Street, Philadelphia, PA 19107. Phone: 215-955-7903; Fax: 215-923-1429; E-mail: Susan.Lanza-Jacoby{at}jefferson.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: HER-2/erbB2/neu is overexpressed in 25% to 30% of all invasive breast cancers and is associated with an aggressive course and reduced survival. HER-2/erbB2/neu breast tumors are frequently associated with up-regulation of cyclooxygenase (COX)-2 and activation of the epidermal growth factor receptor (EGFR) pathway, which promote enhanced cell growth and resistance to apoptosis. This study investigated whether simultaneously blocking both EGFR and COX-2 pathways with ZD1839 and celecoxib, respectively, would be more effective in inhibiting cell growth and inducing apoptosis than either agent alone.

Experimental Design: The effects of ZD1839, celecoxib, and their combination on cell survival, cell cycle progression, and apoptosis were determined in NMF11.2 mouse mammary tumor cells. We also investigated the potential downstream EGFR-activated pathways that are altered by simultaneous inhibition of COX-2 and EGFR.

Results: Celecoxib alone and ZD1839 alone produced a concentration- and time-dependent inhibition of cell survival. Both agents combined produced supra-additive inhibitory effects on cell survival and apoptosis compared with either agent alone. This effect was associated with increased cleaved poly(ADP-ribose)polymerase and reduced protein expression of bcl-2. Phosphorylation of extracellular signal-regulated kinase 1/2 was partially blocked by ZD1839 and celecoxib alone and was completely blocked by the combination of both agents. The enhanced proapoptotic effects of the combined agents were also associated with decreased phosphorylation of Akt and increased phosphorylation of p38.

Conclusions: These findings show that both COX-2 and EGFR are important targets for inhibiting survival and inducing apoptosis in breast cancer. The data suggest a potential cross-talk between COX-2 and EGFR signaling in breast cancer cells overexpressing HER-2/erbB2/neu.

Breast tumors with overexpression of the receptor tyrosine kinase erbB-2/neu/HER-2 occur in ~25% to 30% of human breast cancers and are associated with increased metastasis, increased resistance to chemotherapy, and poor clinical outcome (1). Overexpression of HER-2/neu in transgenic mice leads to spontaneous development of mammary tumors and metastasis to the lung (2). HER-2 is a member of the erbB family of receptors, which includes epidermal growth factor receptor (EGFR)/erbB1/HER-1, erbB-2/neu/HER-2, erbB3/HER-3, and erbB4/HER-4. Specific ligands bind to these receptors, resulting in activation of the erbB receptors, which trigger multiple, diverse downstream signals that regulate survival and apoptosis. Although HER-2 has no known ligand, it is the preferred heterodimer of the other receptors (3). Breast tumors that overexpress HER-2 frequently coexpress other erbB receptors; coexpression of EGFR and HER-2 predicts aggressive tumors with poor prognosis (1, 4). HER-2 overexpression strengthens the signal intensity of its heterdimer, leading to aberrant responses to mitogenic and survival stimuli (5, 6). These observations provide evidence for the role of HER-2 in mammary tumor development and progression.

Recent studies have indicated that overexpression of HER-2 up-regulated cyclooxygenase-2 (COX-2) transcription (7, 8), increased prostaglandin E2 (PGE2) production, and enhanced HER-2/neu protein and mRNA levels. This effect could be blocked by selective inhibition of COX-2 (9). Coexpression of HER-2 and COX-2 was observed in breast cancer cells (8), human breast cancers (10, 11), and in mammary tumors from MMTVneu202 transgenic mice (12, 13). There is considerable preclinical evidence to indicate that COX-2 has an important role in many cancers including breast. In human breast tumors, COX-2 localized to breast cancer cells rather than the surrounding stroma and was associated with metastases and poor survival (14). Increased expression of COX-2 was observed in ductal carcinoma in situ compared with normal breast tissue, a fact suggesting that COX-2 is an early event in the development of breast cancer and is an appropriate target in breast cancer prevention (15). The most direct evidence for the link between COX-2 and tumorigenesis was shown in a study showing that overexpression of COX-2 in the mammary gland induces mammary tumors in transgenic mice (16).

Pharmacologic studies indicate that COX-2 and EGFR may be targets for breast cancer prevention. Dietary administration of celecoxib had a significant chemopreventive effect on the development of 7,12-dimethylbenzanthracene-formed rat mammary tumors. Recent findings (12, 13) show that celecoxib prevented the development of spontaneous mammary tumors in MMTVneu202 transgenic mice. Another study showed that an EGFR tyrosine kinase inhibitor, ZD1839 (gefitinib, Iressa), significantly delayed mammary tumor development and tumor multiplicity in MMTVneu202 transgenic mice (17).

Recent studies have suggested that there is cross-talk between the EGFR and the COX-2 pathways (18, 19). Because there also seems to be an interaction between HER-2 and COX-2, and EGFR preferentially forms heterodimers with HER-2, we investigated whether simultaneous inhibition EGFR and COX-2 will have greater growth inhibitory and proapoptotic effects than blocking each pathway alone. In this study, we showed that the combination of the EGFR tyrosine inhibitor ZD1839 and the COX-2 inhibitor celecoxib led to a supra-additive apoptotic effect in HER-2/neu mouse mammary tumor cells. The effectiveness of the combined treatment was also associated with greater inhibition of EGF-activated EGFR and extracellular signal-regulated kinase (ERK)-1/2 than either agent alone.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures, inhibitors, and antibodies. The MMTV-HER-2/neu cell line NMF11.2 (provided by Sandra J. Gendler, Mayo Clinic, Scottsdale, AZ) was derived from mammary gland tumors from MMTV-HER-2/neu mice. Cells were cultured in Improved MEM Zinc Option Media without phenol red (Life Technologies, Inc., Grand Island, NY). The medium was supplemented with 5% fetal bovine serum, which had been pretreated with charcoal and dextran (Hyclone, Logan, UT) and 1% GlutaMax-1 supplement solution (Life Technologies) as an additional source of glutamine. Cells were maintained at 37°C in an atmosphere of 5% CO2. To remove the adherent cells from the flasks for passaging or counting, cells were washed with HBSS without calcium or magnesium, incubated with a small volume of 0.25% trypsin-EDTA (Sigma Chemical Co., St. Louis, MO) for 5 to 10 minutes, and washed with culture medium by centrifugation.

The human breast cancer cell line BT474 was obtained from the American Type Culture Collection (Manassas, VA). BT474 cells were maintained in DMEM:Ham's F-12 containing 10% fetal bovine serum with insulin (0.01 µg/mL).

Celecoxib was purchased from LKT Laboratories (St. Paul, MN) and ZD1839 was provided by AstraZeneca Pharmaceuticals (Macclesfield, United Kingdom). Purified EGF was obtained from Sigma Chemical.

For immunoblot analysis, the following antibodies were used: anti-COX-2, anti-bcl-2, anti-bax, anti-poly(ADP-ribose)polymerase, anti-EGFR, anti-phospho-EGFR, anti-erbB2/HER-2, anti-phospho-erbB2/HER-2, anti-p38, anti-phospho-p38, biotinylated antimouse or antirabbit secondary antibodies (Santa Cruz Biotechnology Corp., Santa Cruz, CA), anti-Akt, anti-phospho-Akt473 (Cell Signaling Technology, Beverly, MA), and anti-ß-actin (Sigma Chemical).

Cell viability. Cell viability was determined with the CellTiter assay (Promega Corp., Madison, WI). Cells were seeded on a 96-well plate at a concentration of 9.4 x 103 per well in 100 µL. Twenty-four hours later, each well was incubated for 24, 48, or 72 hours with the various concentrations of ZD1839, celecoxib, or the combination of both agents dissolved in DMSO (<0.1%). At the end of the experiment, 20 µL of CellTiter reagent were added to each well and the plates were placed in a 37°C incubator for 4 hours. After incubation, the absorbance of the supernatant was determined at 475 nm. This assay is based on the reduction of a water-soluble tetrazolium salt, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt, by cellular enzymes in viable cells. A graph of the absorbance versus cell number was constructed. Various numbers of cells were added to the 96-well plate; after attachment, 20 µL of Procheck reagent were added and the absorbance was determined. This graph served as a standard curve for all experiments.

Cell cycle analysis. Cells (1 x 106 per flask) were plated onto T175 flasks and allowed to attach overnight. The next day, NMF11.2 cells were placed in Improved MEM Zinc Option Medium without phenol red and supplemented with 1% fetal bovine serum and treated with celecoxib, ZD1839, or the combination of these agents. After 48 hours of incubation, attached and floating cells were harvested and fixed in 70% ethanol, digested with RNase A, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting using a Beckman Coulter XL-MCL flow cytometer with System II Software.

M30 assay. Cytokeratin 18 is a particular cytokeratin that is affected early in apoptosis. Cytodeath monoclonal antibody M30 recognizes a specific caspase cleavage site in cytokeratin 18. After trypsinization, the attached and detached NMF11.2 fractions in the culture supernatant were combined, resuspended in PBS, and fixed in 1% paraformaldehyde while on ice. After 15 minutes, the cells were resuspended in ice-cold 70% ethanol and incubated for 30 to 60 minutes. Cells were washed in PBS, centrifuged, and incubated in 100 µL of M30 CytoDeath antibody, 1:100 (Roche Diagnostic Corp., Indianapolis, IN), at room temperature for 60 minutes. To visualize the M30 CytoDeath antibody, a FITC-conjugated second antibody was used and the FITC signal was evaluated using a Beckman Coulter XL-MCL flow cytometer with System II Software.

Apoptosis. A terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay was done with ApopTag plus Fluorescein In Situ Detection kit (Chemicon International, Temecula, CA). Cells (1 x 106 per flask) were seeded onto T175 flasks and allowed to attach overnight. The next day, cells were placed in Improved MEM Zinc Option Medium without phenol red, supplemented with 1% fetal bovine serum, and treated as described under the specific protocols. After incubation, attached and floating cells were harvested and washed in PBS. The 3'-OH ends of degraded DNA were reacted with terminal deoxynucleotidyl transferase to form a digoxigenin nucleotide and allowed to bind to an anti-digoxigenin antibody that was conjugated to fluorescein. Cells were washed in PBS, stained with propidium iodide, and analyzed using a Beckman Coulter XL-MCL flow cytometer with System II Software. Background staining was determined with a control without the terminal deoxynucleotidyl transferase enzyme.

Western blot analysis. For immunoblotting, cells were lysed by adding 1x Cell Lysis Buffer (Na2EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin; Cell Signaling) containing 1 mmol/L phenylmethylsulfonyl fluoride and protease inhibitors cocktail (Sigma Chemical) according to the instructions of the manufacturer. Proteins were electrophoresed under reducing conditions on a NuPAGE Invitrogen gel (3-8% Tris-acetate, 10% and 12% Bis-Tris; NuPAGE Novex, Invitrogen, Carlsbad, CA) at a constant voltage (150 or 200 V depending on the gel) using NuPAGE MOPS Running Buffers. Then, proteins were transferred to a polyvinylidene difluoride membrane and blocked with specific antibodies in 5% dry milk (Vector Laboratories, Burlingame, CA) for 30 minutes before being washed thrice with washing buffer. Blots were incubated for another 30 minutes with a solution containing the avidin/biotinylated enzyme complex in 5% milk, which binds to the biotinylated antimouse or antirabbit secondary antibodies. The blots were given a final wash (3x) before bands were visualized by chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Biosciences Corp., Piscataway, NJ) and scanned with a densitometer (Sharp JX-330), quantitated by Image Master 1D, and expressed as arbitrary units relative to ß-actin or the specific unphosphorylated protein.

Production of PGE2. NMF11.2 cells were treated with 0.5 µmol/L ZD1839, 20 µmol/L celecoxib, or their combination for 48 hours. The medium was harvested by centrifugation at 5,000 x g at 4°C. The amount of PGE2 released in the medium was measured with enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI).

Statistical analysis. The data shown are mean values of at least three replicate experiments and expressed as means ± SE. Cell survival data were analyzed using simple linear regression equivalent to two-way ANOVA to compare the four groups. ANOVA and Bonferroni correction were used to compare data for cell cycle, apoptosis, and protein expression. Statistical analyses were conducted using SAS 8.2 software package (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Celecoxib plus ZD1839 inhibited cell growth in NMF11.2 cells in a supra-additive manner. NMF11.2 cells were incubated with different concentrations of celecoxib (0-40 µmol/L) for 24, 48, and 72 hours or ZD1839 (0-5 µmol/L) for 24, 48, and 72 hours. Analysis of cell growth using the CellTiter assay showed that celecoxib and ZD1839 produced a concentration-dependent and time-dependent inhibition of cell growth. The dose of 20 µmol/L celecoxib alone and 0.5 µmol/L ZD1839 alone inhibited cell growth by ~50% (Fig. 1A and B ). These concentrations were used as single or combined agents in the following experiments. Figure 1C shows that after 72 hours of incubation, cell growth was inhibited by 50%, 65%, and 89% with 20 µmol/L celecoxib, 0.5 µmol/L ZD1839, and the combination of the two agents, respectively.


Figure 1
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  		Fig. 1. Effects of celecoxib, ZD1839, and celecoxib plus ZD1839 on cell survival. Cells were treated with 0 to 40 µmol/L of celecoxib (A), with 0 to 5 µmol/L ZD1839 (B), or with 0.5 µmol/L ZD1839 + 20 µmol/L celecoxib (C) for 24, 48, and 72 hours. Columns, mean of triplicate determinations of three experiments; bars, SE. *, P < 0.05, versus control; §, P < 0.01, versus control, celecoxib, and ZD1839.

 
The combination of celecoxib plus ZD1839 enhanced apoptosis. To determine whether the growth inhibitory effects of the combined treatment could be attributed to a delay in the cell cycle or increased apoptosis, we evaluated cell cycle progression and apoptosis with flow cytometric assays. NMF11.2 cells were treated with celecoxib, ZD1839, and the combination of these agents. Table 1 shows that celecoxib induced a G2 block; adding ZD1839 negated this effect. Celecoxib in combination with ZD1839 led to an accumulation of cells in the G0-G1 phase of the cell cycle in comparison with each of the single agents. Accordingly, a smaller fraction of NMF11.2 cells entered the DNA/synthesis phase.


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  		Table 1. Effect of ZD1839 and celecoxib on cell cycle distribution of NMF11.2 cells

 
Cytokeratin-18 degradation, identified with the M30 monoclonal antibody, was used to determine whether celecoxib and ZD1839 altered the early phase of apoptosis. Figure 2A shows that ZD1839, celecoxib, and the combination produced a 5-, 2-, and 16-fold increase, respectively, in the level of M30 antigen. We also determined cleavage of poly(ADP-ribose)polymerase as a marker of early apoptosis with Western blot analysis. Poly(ADP-ribose)polymerase cleavage was increased by the combination of ZD1839 and celecoxib (Fig. 2B).


Figure 2
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  		Fig. 2. Combined treatment with celecoxib + ZD1839 supra-additively induce apoptosis in NMF11.2 cells. Attached and floating cells were pooled and apoptosis was evaluated using M30 CytoDeath assay (A), cleavage of poly(ADP-ribose)polymerase (PARP; B), and ApopTag plus Fluorescein In Situ Detection kit (C). Columns, mean of triplicate determinations of three experiments; bars, SE. *, P < 0.01, versus vehicle; §, P < 0.001, versus vehicle, celecoxib, and ZD1839.

 
Apoptosis was confirmed by the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay. Figure 2C illustrates that the apoptotic population of cells increased in a supra-additive manner with the combined treatment as compared with either agent alone (control, 2.4%; celecoxib, 7.6%; ZD1839, 8.0%; and celecoxib + ZD1839, 20.9%). These three methods of evaluating apoptosis provide strong evidence that combining ZD1839 with celecoxib enhances the apoptotic effects of each agent alone.

The combination ZD1839 plus celecoxib decreased bcl-2. Both bcl-2 and bax are involved in the balance between antiapoptotic and proapoptotic signals; bcl-2 prevents apoptosis and bax induces cell death. We determined the effects of ZD1839, celecoxib, and the combination of both agents on the protein expression of bcl-2 and bax. Quantification of the Western blots by densitometry is shown in Fig. 3 . The combined treatments significantly decreased the relative density of bcl-2 more than the individual treatments (P = 0.004). There was no change in the protein levels of bax. The ratio of the relative densities of bax/bcl-2 (vehicle, 1.8; ZD1839, 2.5; celecoxib, 2.4; and the combination, 10.4) was increased by the combination of ZD1839 and celecoxib in favor of apoptosis.


Figure 3
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  		Fig. 3. Combined treatment with celecoxib + ZD1839 down-regulates bcl-2. NMF11.2 cells treated with 0.5 µmol/L ZD1839, 20 µmol/L celecoxib, or their combination for 48 hours. Cell lysates were subject to immunoblotting with anti-bcl-2 and anti-bax antibodies (top). Blots were stripped and reprobed for ß-actin (middle). Bottom, quantitative analysis determined by the ratio of bcl-2 or bax antibody to ß-actin done in three separate experiments (columns, mean; bars, SE). *, P < 0.04, versus vehicle, ZD1839, and ZD1839 + celecoxib; §, P < 0.004, versus vehicle, ZD1839, and celecoxib.

 
ZD1839 decreased COX-2 protein expression. NMF11.2 cells were incubated for 48 hours with ZD1839 and celecoxib. The effect of the treatments on COX-2 protein expression was evaluated by Western blot. Treating the cells with ZD1839 significantly decreased protein expression of COX-2 in comparison with the vehicle-treated and celecoxib-treated samples (P < 0.04), which suggests that EGFR signaling may be involved in the regulation of COX-2 (Fig. 4 ). Celecoxib treatment produced a small increase in the level of COX-2 protein. The observed inhibition of COX-2 protein with the combination treatment seemed to be due to the effect of ZD1839.


Figure 4
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  		Fig. 4. ZD1839 decreases protein expression of COX-2. NMF11.2 cells treated with 0.5 µmol/L ZD1839, 20 µmol/L celecoxib, or their combination for 48 hours. Cell lysates were subject to immunoblotting with anti-COX antibody (top). Blots were stripped and reprobed for ß-actin (middle). Bottom, quantitative analysis determined by the ratio of COX-2 antibody to ß-actin done in three separate experiments (columns, mean; bars, SE). *, P < 0.04, versus vehicle and celecoxib.

 
Celecoxib reduced PGE2 production. To determine the effect of the combined treatments on COX-2 activity, the NMF11.2 cells were treated with ZD1839, celecoxib, and the combined agents for 48 hours; the medium was obtained by centrifugation and analyzed for PGE2 by an ELISA. Celecoxib alone significantly (P < 0.01) decreased the production of PGE2; the decrease in PGE2 production observed with the combination treatment seems to be due to celecoxib as ZD1839 had a minimal effect on PGE2 production (Table 2 ).


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  		Table 2. Effect of celecoxib, ZD1839, and celecoxib + ZD1839 on COX-2 activity

 
ZD1839 and celecoxib decreased phosphorylation of EGFR and HER-2/neu. To investigate the potential interaction of the COX-2 and EGFR signaling pathways in HER-2-overexpressing mammary tumors, we determined whether the combined treatment with ZD1839 and celecoxib decreased EGF-induced phosphorylation of EGFR and HER-2 to a greater extent than either agent alone. NMF11.2 cells were serum starved overnight and pretreated with ZD1839 and celecoxib followed by EGF stimulation. Figure 5A shows that EGF-stimulated EGFR phosphorylation was almost completely blocked by 0.5 µmol/L of ZD1839 (P < 0.0001), partially blocked by the celecoxib treatment (P < 0.001), and completely blocked by the combined treatment (P < 0.0001). The treatments did not alter total EGFR protein expression. Phosphorylation of HER-2/neu was reduced by each treatment alone and completely blocked by the combination of the treatments (Fig. 5B and C). To provide additional evidence that targeting EGFR and COX-2 activities is effective in breast cancer cells overexpressing HER-2, we tested the effect of the ZD1839 + celecoxib combination in BT474 cells, which also express high levels of HER-2. Similar to the effect in NMF11.2 cells, treating BT474 cells with ZD1839 + celecoxib produced a greater inhibition of HER-2/neu phosphorylation than each individual treatment alone (Fig. 5C).


Figure 5
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  		Fig. 5. Combined treatment with celecoxib + ZD1839 reduces phosphorylation of EGFR and HER-2. NMF11.2 cells were treated with 0.5 µmol/L ZD1839, 20 µmol/L celecoxib, or their combination for 48 hours and stimulated with 25 ng/mL EGF for 15 minutes. Cell lysates were subject to immunoblotting with p-EGFR and EGFR (A) or p-HER-, and HER-2 antibodies (B). Top, phosphorylated antibodies were determined by immunoblotting with specific anti–phospho antibody (phospho-EGFR or phospho HER-2). Middle, total antibody was determined by reprobing with respective antibody (EGFR or HER-2). Bottom, quantitative analysis determined by the ratio of phosphorylation of the antibody to the total protein done in three separate experiments (columns, mean; bars, SE). For (A), EGFR phosphorylation: *, P < 0.0001, versus vehicle and celecoxib; §, P < 0.001, versus vehicle, ZD1839, and ZD1839 + celecoxib. For (B), HER-2/neu phosphorylation of NMF11.2 cells: *, P < 0.01, versus vehicle and ZD1839 + celecoxib; §, P < 0.001, versus vehicle, ZD1839, and celecoxib. For (C), HER-2/neu phosphorylation of BT474 cells: *, P < 0.01, versus vehicle and ZD1839 + celecoxib; §, P < 0.001, versus vehicle, ZD1839, and celecoxib.

 
ZD1839 and celecoxib decrease phosphorylation of ERK1/2 and Akt. To evaluate whether the growth inhibitory effects of the combination of ZD1839 and celecoxib were associated with the ERK and phosphatidylinositol 3-kinase pathways, we determined the effects of combining both ZD1839 and celecoxib on EGF-induced activation of ERK1/2 and Akt using phospho-specific antibodies. ZD1839 dramatically decreased ERK phosphorylation; celecoxib also decreased the level of phospho-ERK, but to a lesser extent than ZD1839 (Fig. 6A ). Statistical analysis of the relative density indicated that vehicle, ZD1839, and celecoxib were significantly different from the combined treatments (P < 0.0001), indicating that the combination of ZD1839 + celecoxib produced a greater reduction in phospho-ERK than ZD1839 or celecoxib alone. There was no change in ERK protein levels.


Figure 6
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  		Fig. 6. Combined treatment with celecoxib + ZD1839 reduces phosphorylation of ERK1/2 and activates p38. NMF11.2 cells were treated with 0.5 µmol/L ZD1839, 20 µmol/L celecoxib, or their combination for 48 hours and stimulated with 25 ng/mL EGF for 15 minutes. Cell lysates were subject to immunoblotting with phospho-ERK1/2 and ERK1/2 (A) or phospho-Akt and Akt antibodies (B), or phospho-p38 and p38 antibodies (C). Top, phosphorylated antibodies were determined by immunoblotting with specific anti–phospho antibody (phospho-ERK1/2, phospho-Akt, and phospho-p38). Middle, total antibody was determined by reprobing with respective antibody (ERK1/2, Akt, and p38). Bottom, quantitative analysis determined by the ratio of phosphorylation of the antibody to the total protein done in three separate experiments (columns, mean; bars, SE). For (A), ERK1/2 phosphorylation: *, P < 0.001, versus vehicle and ZD1839 + celecoxib; §, P < 0.0001, versus vehicle, ZD1839, and celecoxib. For (B), Akt phosphorylation: *, P < 0.05, versus vehicle and ZD1839 + celecoxib; §, P < 0.01, versus vehicle, ZD1839, and celecoxib. For (C), p38 phosphorylation: *, P < 0.01, versus vehicle, ZD1839, and ZD1839 + celecoxib; §, P < 0.001, versus vehicle, ZD1839, and celecoxib.

 
Because HER-2 overexpression promotes activation of the phosphatidylinositol 3-kinase/Akt pathway and enhances cell survival, we determined whether celecoxib and ZD1839 blocked EGF-induced activation of Akt. Figure 6B shows that ZD1839 alone and celecoxib alone partially decreased the levels of phospho-Akt compared with the control group (P < 0.05). The combined treatment further inhibited Akt phosphorylation to a greater extent than the individual treatments (P < 0.01).

The stress-activated p38 mitogen-activated protein kinases are associated with both cell survival (20) and apoptosis (21). The response may vary with different cell types, the external stimulus, or the presence of different isoforms (22). For example, it has been reported that the p38{alpha} and p38ß isoforms are involved in apoptosis (23). When NMF11.2 cells were treated with ZD1839, there was no change in p38 phosphorylation (Fig. 6C). In contrast, celecoxib treatment decreased p38 phosphorylation (P < 0.01) compared with vehicle, ZD1839, or ZD1839 + celecoxib treatments. However, the combination of ZD1839 and celecoxib increased p38 phosphorylation 3-fold (P < 0.001) compared with the other treatments.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There is recent evidence to suggest a cross-talk between the EGFR and COX-2 signaling pathways in a number of tumor cells. Studies have found that COX-2-derived PGE2 can transactivate EGFR in colon cancer cells (18, 24) and in cholangiocarcinoma cancer cells (25). In turn, activation of EGFR increased COX-2 expression and PGE2 production in squamous carcinoma cells (26) and colon cancer cells (27). Inhibition of EGFR tyrosine kinase in squamous carcinoma cells has been shown to decrease COX-2 expression (26).

In the present study, we provide the first evidence in breast cancer cells that the combination of the EGFR tyrosine inhibitor ZD1839 and the COX-2 inhibitor celecoxib leads to a supra-additive induction of apoptosis and inhibition of EGFR phosphorylation. Our findings also show that ZD1839 in combination with celecoxib reduces downstream EGF-induced phosphorylation of ERK and Akt and stimulates p38 phosphorylation in breast cancer cells.

Both the EGFR and the PG signaling pathways promote cell survival through activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways. Overexpression of HER-2/neu enhances the EGFR signaling response (5, 6), resulting in activation of the Ras/Raf/mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and phospholipase C pathways, and up-regulates COX-2 expression (9, 10). In colon and cholangiocarcinoma cells, PGE2 transactivates EGFR leading to downstream signaling through four PGE (EP)1-4 receptors (18, 24, 25). We have also observed that PGE2 transactivated EGFR in NMF11.2 cells (data not shown). This evidence provides a strong rational for targeting both EGFR and COX-2 in HER-2-overexpressing breast cancer.

As single agents, COX-2 and EGFR tyrosine kinase inhibitors have had limited success in preventing the development of spontaneous mammary tumors in mice. We observed that diets containing 900 ppm of celecoxib reduced the incidence of mammary tumors by 29% in HER-2/neu transgenic mice at 14 months of age (13). Another study by Howe et al. (12) found that celecoxib fed at 500 ppm reduced mammary tumor incidence by ~15% at 12 months of age. When HER-2/neu transgenic mice were fed a high dose of ZD1839 (100 mg/kg), tumor incidence was reduced by 75% at 10 months of age (17). The improved efficiency of combining these agents was shown in a study where the combination of an EGFR tyrosine inhibitor with a nonspecific nonsteroidal anti-inflammatory drug sulindac protected APCMin/+ mice from intestinal neoplasia (28). More recent studies have shown that the combination of EGFR and COX-2 inhibitors has a greater antitumor effect in colon cancer (29) and in squamous cell carcinoma of the head and neck (30) than each agent alone.

In this study, we observed a greater apoptotic effect when treating NMF11.2 cells for 48 hours with the combination of ZD1839 plus celecoxib (30%) than ZD1839 treatment alone (8%) or celecoxib alone (7.8%). Consistent with our previous observation, inhibition of COX-2 led to a G2-M block in the cell cycle (31). Combining ZD1839 with celecoxib abrogated the G2-M block, forcing the cells into the cell cycle before repair was completed, thus inducing apoptosis. Several pathways leading to apoptosis converge at the mitochondrial membrane; the breakdown of the mitochondrial membrane is controlled, in part, by the balance of bcl-2 and bax in cells (32). The protein expression of bcl-2 decreased in cells treated with celecoxib alone or the combination of ZD1839 + celecoxib, whereas there was no change in bax protein expression. Thus, the ratio of bax to bcl-2 was altered in favor of apoptosis. The role of bcl-2 in celecoxib-mediated apoptosis may be cell type specific. In prostate (33) and colon cancer cells (34), decreased bcl-2 expression was associated with COX-2 inhibition. In contrast, induction of apoptosis by celecoxib in cholangiocarcinoma cells was independent of bcl-2 (35). The enhanced apoptotic effect of celecoxib treatment alone seemed to be associated with a COX-2-independent effect; the concentration of celecoxib (20 µmol/L) that induced apoptosis was higher than the concentration (5 µmol/L) found to inhibit PGE2 production from NMF11.2 cells (31). Celecoxib has been shown to inhibit tumor cell growth and induce apoptosis by targeting non-COX-2 pathways such as Akt (36), 3-phosphoinositide-dependent kinase-1 activity (37), nuclear factor {kappa}B (38), the mitochondrial membrane potential, activation of caspase-9 (39), peroxisome proliferator–activated receptor-{gamma} (40), and ß-catenin/adenomatous polyposis coli signaling (41).

In the NMF11.2 cells, celecoxib treatment increased COX-2 whereas ZD1839 decreased COX-2 expression (Fig. 4). Although the COX-2 protein increased, celecoxib significantly reduced the production of PGE2. The mechanism for the celecoxib-induced increase in COX-2 expression is not known but may be due to the removal of a negative feedback regulator(s) that controls COX-2 protein synthesis and/or breakdown. Other studies also reported that celecoxib increased COX-2 protein expression in squamous cell carcinoma (42) and MDA-MB231 breast cancer cells (43).

Previous studies have found that breast cancer cells with HER-2 overexpression are particularly sensitive to ZD1839 (44, 45). EGF, transforming growth factor-{alpha}, and amphiregulin stimulate phosphorylation of EGFR, which activates downstream mitogen-activated protein kinase/ERK, phosphatidylinositol 3-kinase/Akt, and phospholipase C pathways. We have observed that treating NMF11.2 cells with 0.5 µmol/L ZD1839 before EGF stimulation reduced phosphorylation of both EGFR and HER-2, which was accompanied by a decrease in cell survival. NMF11.2 cells express both EGFR and HER-2 and have very low levels of HER-3 and HER-4, which suggests that ZD1839 disrupts cell survival by targeting EGFR/HER-2 heterodimers. Selective inhibition of COX-2 with celecoxib also reduced EGF-induced phosphorylation of EGFR, although not to the same extent as ZD1839. Recent reports have documented in colon cancer cells (18, 24) and in cholangiocarcinoma cancer cells (19) that COX-2-derived PGE2 transactivated EGFR through the G protein–coupled receptor EP1. PGs mediate their signaling effects by interacting with specific EP1-4 receptors (46). Because EGF has been shown to stimulate COX-2 expression and PGE2 production, the reduction in EGF-induced phosphorylation of EGFR observed with celecoxib treatment suggests that the increase in EGF-induced PGE2 contributes to transactivation of EGFR in these cells.

Studies have reported that ZD1839 decreased HER-2 phosphorylation in BT474 (47) and SkBr3 (44) breast cancer cells with HER-2 overexpression. ZD1839 at 0.5 µmol/L also reduced HER-2 phosphorylation in NMF11.2 cells, whereas our laboratory and others (47) have found that a higher concentration of ZD1839 (10 µmol/L) was required to inhibit HER-2 phosphorylation in BT474 cells. In NMF11.2 and BT474 cells, celecoxib partially reduced HER-2/neu phosphorylation and the combination of ZD1839 with celecoxib completely blocked phosphorylation of HER-2/neu. These observations support the premise that there is interaction of COX-2 and EGFR signaling in breast cancer cells with overexpression of HER-2/neu.

Blocking EGFR alone or COX-2 alone has been shown to reduce EGF-induced activation of the mitogen-activated protein kinase and Akt pathways and induce apoptosis in cancer cells (36, 37, 44, 47). In this study, we observed a supra-additive inhibitory effect on EGF-induced phosphorylation of ERK1/2 and Akt when ZD1839 and celecoxib were combined.

We next examined the effect of inhibiting EGFR and COX-2 on p38 phosphorylation because of the importance of p38 in mediating stress response, which may occur when receptor tyrosine kinase survival pathway is disrupted. Treatment with ZD1839 alone induced a modest increase in p38 phosphorylation whereas celecoxib treatment decreased phosphorylated p38. Previous studies have shown that EGFR and HER-2/neu tyrosine kinase inhibitors induced p38-dependent apoptosis in prostate cancer cells (48) and in HER-2-overexpressing breast cancer cells (49). This is the first study to show that the combined treatments synergistically increased p38 phosphorylation, creating an imbalance between ERK and p38 activation. These events correlate with the greater apoptotic effect observed with the combined treatment. Studies are ongoing to determine the role of these signaling molecules in mediating the enhanced apoptotic effects of the combined treatments.

Previous studies have shown that long-term treatment of squamous cell carcinoma of the head and neck with ZD1839 and celecoxib produced additive/synergistic growth inhibitory effects and reduced basal levels of phosphorylated EGFR, ERK1/2, and Akt in comparison with the single agents (30). In contrast to our study, these cell culture experiments were conducted with 10% fetal bovine serum. In this study, the effectiveness of the treatments in blocking EGFR, ERK1/2, and Akt phosphorylation was determined in serum-starved cells that were stimulated with EGF. We found that treatment with celecoxib alone reduced expression of EGF-stimulated phosphorylated EGFR and ERK1/2 in HER-2-overexpressing NMF11.2 cells. In squamous cell carcinoma of the head and neck that were cultured in serum, celecoxib did not have an effect on EGFR and ERK1/2 phosphorylation (30, 42). Serum-containing medium has been shown to block the growth inhibitory effects of celecoxib in pancreatic (50) and prostate cancer cells (51).

In summary, this is the first evidence that ZD1839 in combination with celecoxib promotes a supra-additive apoptotic response in breast cancer cell lines with overexpression of HER-2/neu. The enhanced growth inhibitory effect exhibited by the combined treatment was related to inhibition of EGF-dependent activation of EGFR, ERK1/2, and Akt and induction of p38. Our study provides an experimental basis to suggest that the combination of COX-2 and EGFR tyrosine kinase inhibitors may be useful for protection against HER-2-overexpressing breast cancer.


   Acknowledgments
 
We thank AstraZeneca Pharmaceuticals for providing ZD1839 for these studies.


   Footnotes
 
Grant support: National Cancer Institute grant CA103097 (S. Lanza-Jacoby) and National Cancer Institute training grant CA069277 (to Scott Waldman for J. Little).

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.

Received 1/ 6/06; revised 5/17/06; accepted 5/26/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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