Affiliations of authors: Departments of Medicine and Molecular and Cellular Biology, Breast Center, Baylor College of Medicine, Houston, TX (CL, CS, YZ, JH, ES, JC, QS, HK, SH, SKM, CKO, PHB); M. D. Anderson Cancer Center, Houston (XX); AstraZeneca, Macclesfield, U.K. (AW).
Correspondence to: Powel H. Brown, MD, PhD, Baylor College of Medicine, One Baylor Plaza, MS 600, Houston, TX 77030 (e-mail: pbrown{at}breastcenter.tmc.edu)
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ABSTRACT |
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INTRODUCTION |
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Several agents prevent ER-positive breast cancer in carcinogen-treated rats, including retinoids, vitamin D analogs, difluo- romethylornithine, cyclooxygenase-2 inhibitors, and antiestrogens. We have recently shown that naturally occurring and synthetic retinoids prevent the development of ER-negative breast cancer in transgenic mice (6,7). However, the toxicity of these agents may limit their use in women. We have therefore investigated the cancer-preventing activity of other novel agents.
Some of the most promising agents for the prevention of ER-negative breast cancer are growth factor receptor tyrosine kinase inhibitors. The epidermal growth factor receptor (EGFR) is one of a family of four closely related receptors (EGFR or erbB1, HER2/neu or erbB2, HER3 or erbB3, and HER4 or erbB4) that use tyrosine kinase activity as the signal transduction trigger. The EGFR pathway contributes to a number of processes involved in tumor survival and growth, including cell proliferation and inhibition of apoptosis, angiogenesis, and metastasis, thus making it an attractive target for cancer prevention and treatment. ZD1839 (gefitinib or Iressa) is an orally active EGFR tyrosine kinase inhibitor that blocks signal transduction processes implicated in the proliferation and survival of cancer cells. Several in vitro and in vivo studies (8,9) have shown that ZD1839 can inhibit the growth of various types of cancer cells including breast cancer cells. ZD1839 also suppresses the growth of xenografts (8,9) and noninvasive human breast cancer or ductal carcinoma in situ (10).
In this study, we investigated the ability of ZD1839 to inhibit the growth of normal, precancerous, and malignant human mammary epithelial cells and to prevent tumorigenesis in a model relevant to human ER-negative breast cancer. We also determined the effect of ZD1839 on signal transduction in ER-negative breast cells.
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MATERIALS AND METHODS |
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ZD1839 was obtained from AstraZeneca (Macclesfield, U.K.).For tissue culture studies, ZD1839 was suspended in dimethyl sulfoxide at a final concentration of 1 µM. For in vivo animal studies, ZD1839 was suspended in distilled water containing 1% Tween 80. Purified human recombinant EGF was obtained from Sigma Chemical Company (Carlsbad, CA).
Cell Lines
Normal human mammary epithelial cells (HMECs) were obtained from Cambrex (East Rutherford, NJ) and from Martha Stampfer (184 cells, Lawrence Berkeley National Laboratory, Berkeley, CA). The immortal cell line MCF10A was obtained from American Type Culture Collection (Manassas, VA), and the immortalized cell line 184B5 was obtained from Martha Stampfer. The breast cancer cell line MDA-MB-468 was obtained from American Type Culture Collection. Cells were grown in the following culture media: MEGM (Clonetics, San Diego, CA) for normal HMECs and 184 and 184B5 cells; DME/F-12 with 5% horse serum and supplements (1.4 x 106 M hydrocortisone, insulin at 10 µg/mL, choleratoxin at 100 ng/mL, EGF at 20 ng/mL, penicillin at 100 U/mL, and streptomycin at 100 µg/mL) for MCF10A cells; and improved minimum essential medium (high-zinc option; Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum and penicillin (100 U/mL)/streptomycin (100 µg/mL) (Life Technologies) for MDA-MB-468 cells. All cell lines used were ER-negative.
Western Blot Analysis
After plating, cells were starved in serum-free medium for 24 hours and were treated with either vehicle (dimethyl sulfoxide) or 1 µM ZD1839 in serum-free medium for 8 hours. The cells were then stimulated with EGF (still in the presence of dimethyl sulfoxide or ZD1839) for the indicated times. Whole-cell protein extracts were collected at 0, 5, 10, 15, and 30 minutes after EGF stimulation (0 time samples were collected immediately before addition of EGF). Protein extracts were electrophoresed on a sodium dodecyl sulfatepolyacrylamide gel and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Life Sciences, Arlington Heights, IL). The following primary antibodies were used for western blot analysis. Phosphorylated EGFR (product 2234, 1 : 1000 dilution), total EGFR (product 2232, 1 : 1000 dilution), and phosphorylated mitogen-activated protein kinase (MAPK; product 9101, 1 : 1000 dilution) were obtained from Cell Signaling Technology (Beverly, MA). Total MAPK (product 06182, 1 : 1000 dilution) antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Actin antibody (product MAB1501, 1 : 100 000 dilution) was obtained from Chemicon (Temecula, CA). Anti-rabbit or anti-mouse antibody (1 : 4000 dilution; Amersham, Piscataway, NJ) was used as the secondary antibody. Blots were developed with the enhanced chemiluminescence system (Amersham), and the expression levels were measured with a densitometer (Alpha-Innotech, San Leandro, CA). Relative expression levels of phosphorylated EGFR and phosphorylated MAPK were determined after normalizing for protein loading by dividing the level of phosphorylated proteins by the level of actin for each sample.
Cell Growth Assays
Proliferation assays to measure the effect of ZD1839 on anchorage-dependent cell growth. Normal or immortal breast cells (1 x 106 cells) were plated in quadruplicate cultures and then treated with ZD1839, as indicated. For extended treatments, cells were incubated with ZD1839 for 012 days with fresh drug and medium replaced every 2 days. Cell proliferation was measured with the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Relative cell growth was then assessed by measuring absorbance at 620 nm with a spectrophotometric plate reader. Cells grown in medium alone were used as controls. Experiments were carried out for 4, 8, 10, and 12 days because of the different growth rates of the different cell lines.
Soft agar assay for anchorage-independent growth. Approximately 5 x 104 MDA-MB-468 cells were suspended in 4 mL of 0.35% SeaPlaque agarose (FMC BioProducts, Rockland, ME) supplemented with complete culture medium. This suspension was layered over 1.5 mL of 0.7% agar/medium base layer in one well of a six-well plate and treated with 1 µM ZD1839 for 14 days; colonies with a diameter larger than 0.05 mm were then counted.
Transgenic Mice
Female MMTV-erbB2 transgenic mice (The Jackson Laboratory, Bar Harbor, ME) were used for this experiment. The MMTV promoter from the mouse mammary tumor virus long terminal repeat causes the erbB2 gene to be expressed in the mammary gland. The mice were housed in the institutional animal facilities, and appropriate animal treatment guidelines were followed. Animals were obtained at 1012 weeks of age, given a pituitary isograft to chronically stimulate the MMTV promoter [as described by Lydon et al. (11)], and treated with ZD1839 as described below. Animals were fed a controlled diet of AIN-76A Purified Diet (Harlan Teklad, Madison, WI).
Treatment and Data Collection
Mice were treated with ZD1839 at 10 mg/kg (n = 18) or 100 mg/kg (n = 19) suspended in distilled water containing 1% Tween 80 or with vehicle for 6 days/week from age 3 months to 12 months. ZD1839 or vehicle (n = 19) was administered in 0.1 mL by gastric gavage with a 20-gauge gavage needle. Tumors were measured twice a week with electronic calipers (Mitutoyo, Utsunomiya, Japan), and tumor volume was determined by multiplying the square of the width (w) by the length (l) and dividing by two (i.e., w2l/2). Individual tumor size and tumor location for each animal were recorded twice a week. Weights of all mice were recorded weekly. Animals were killed when they developed tumors of 1000 mm3 or more or at the end of the experiment. Two hours before killing, the mice were injected intraperitoneally with bromodeoxyuridine (Amersham) (3 mg/mL in phosphate-buffered saline; 100 µL/10 g body weight). The end of the experiment was defined as the time when all vehicle-treated mice had developed a tumor (at 330 days of age or 240 days of treatment). At that time, all remaining mice (vehicle-treated and ZD1839-treated) were killed, and tumors were resected. The primary end point of the study was time to tumor formation. After the mice were killed, each tumor was resected, and separate portions were processed for histologic analysis, explanted into tissue culture to prepare in vitro tumor cell lines, or frozen for future use in biomarker studies. Explanted tumor cells were grown in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin, and 1% Fungizone (Invitrogen, Carlsbad, CA).
Histology and Biomarker Analysis
Histology was performed as previously described (12). Briefly, samples were fixed in neutral-buffered 10% formalin (i.e., phosphate-buffered 10% formaldehyde) overnight and then embedded in paraffin. Tissue sections were then mounted on slides and processed for staining with hematoxylineosin.
Immunohistochemical staining for erbB2, ER-, and p27 was performed by a modified avidinbiotin complex technique as previously described (7). Briefly, tissue sections on glass slides were deparaffinized in xylene and rehydrated. Antigen retrieval was performed by microwaving the samples in 0.01 M citric acid, and endogenous peroxidase activity was blocked by incubation in 30% methanolic hydrogen peroxide. Nonspecific binding was blocked with 20% normal goat serum. Sections were then incubated at 23 °C for 4 hours with one of the following antibodies: rabbit anti-c-erbB2 polyclonal antibody (Neomarkers, Fremont, CA) diluted 1 : 50 in phosphate-buffered saline (PBS), rabbit anti-ER-
polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1 : 2000 in PBS, or rabbit anti-p27 polyclonal antibody (Abcam Limited, Cambridge, MA) diluted 1 : 200 in PBS. After three washes in PBS, sections were incubated with biotinylated goat anti-rabbit immunoglobulin G antibody for 30 minutes at 23 °C and then incubated with the ABC kit (Vector Laboratories, Burlingame, CA) for 30 minutes in the dark. Sections were then incubated with 3-amino-9-ethylcarbazole (2 mg/mL; Sigma Chemical Co., St. Louis, MO) to visualize the peroxidase complex. Levels of p27 were evaluated by visual assessment with a semiquantitative scoring system that rated the staining intensity (from 0 to 3), as previously described (13).
Staining for bromodeoxyuridine was performed with the DAKO ARK (Animal Research Kit; DAKO, Copenhagen, Denmark) system. Briefly, tissue sections were cut, mounted onto slides, and deparaffinized. Endogenous peroxidase was blocked with 30% hydrogen peroxide. Slides were then rinsed, and nonspecific binding was blocked (A/B Blocking Kit; Vector Laboratories, Burlingame, CA). Bromodeoxyuridine was stained with a mouse anti-bromodeoxyuridine monoclonal antibody (clone Bu20a; DAKO). The slides were then incubated with streptavidinhorseradish peroxidase, and peroxidase activity was visualized with diaminobenzidine chromagen intensified with 0.2% osmium tetroxide. Counterstaining was done with Harris acidified hematoxylin. The stained sections were reviewed and scored with an ocular grid. The percentage of positive cells was determined with tissue samples from four mice from each group by counting the numbers of positive and negative cells in 10 high-powered fields (counting approximately 4000 tumor cells or 1000 normal cells per slide) in each treatment group, and results were expressed as an average percentage with 95% confidence intervals (CIs).
Statistical Analysis
Two outcome measures were considered in the animal studies: tumor-free survival and tumor multiplicity. Tumor-free survival was measured from time of initiation of treatment to the time of first appearance of a tumor (defined as a palpable mass of 100 mm3). Tumor-free survival curves were estimated by the KaplanMeier product limit method and compared with the generalized Wilcoxon test (14). Tumor multiplicity was determined by counting the total number of tumors occurring in each animal up to the time they were killed. Multiplicity was summarized by means and 95% confidence intervals, and multiplicity between groups (number of animals = 18 or 19 per group) was compared by one-way analysis of variance (15) and Student's t test. Student's t test was used to determine the statistical significance of the difference in biomarker expression. Relative changes in biomarker levels were backtransforming differences in means (and 95% confidence intervals of differences in means) of log-transformed data. Analyses were performed with SAS, version 8.1 (SAS Institute, Cary, NC). All statistical tests were two-sided.
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RESULTS |
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To investigate the effect of ZD1839 on signal transduction in breast cells, we measured the expression of phosphorylated and total EGFR and MAPK proteins in HMECs and MDA-MB-468 cells by western blot analysis. Cells were treated with ZD1839 for 8 hours and then stimulated with EGF for 5, 10, 15, or 30 minutes before analysis. In both cell lines, EGFR phosphorylation was completely blocked, but the expression of total EGFR did not change compared with that in untreated control cells (Fig. 1). In both cell lines, MAPK phosphorylation was markedly inhibited but the expression of total MAPK did not change (Fig. 2). Although ZD1839 totally blocked phosphorylation of EGFR in these cells, it only partially blocked MAPK phosphorylation. Thus, other signal transduction pathways may still be able to activate MAPK in these cells, even if EGFR kinase activity is completely blocked.
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We next determined the effect of ZD1839 on the growth of normal breast cells (184 cells and HMECs), immortalized human breast epithelial cells (184B5 and MCF10A cells), and breast cancer cells (MDA-MB-468 cells). ZD1839 at 1 µM completely inhibited the proliferation of 184, HMEC, 184B5, and MCF10A cells (Fig. 3, A) and also inhibited the anchorage-independent growth of MDA-MB-468 breast cancer cells (Fig. 3, B). Thus, ZD1839 at 1 µM is able to suppress the growth of ER-negative normal (184 cells and HMECs), immortalized (184B5 and MCF10A cells), and malignant breast cells (MDA-MB-468 cells).
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We next investigated the ability of ZD1839 to inhibit the development of ER-negative mammary tumors in MMTV-erbB2 transgenic mice. These mice carry the unactivated neu/c-erbB2 protooncogene under the transcriptional control of the MMTV promoter and develop focal tumors beginning at 6 months of age, with a median incidence at 230 days old (when the MMTV promoter is stimulated by a pituitary isograft). We have previously shown that the tumors arising in these mice are ER-negative (7).
MMTV-erbB2 mice were treated daily for 6 of 7 days with vehicle or ZD1839 at 10 mg/kg or 100 mg/kg from 3 to 12 months of age. The number and size of all mammary tumors were measured twice a week. The mice were observed daily for any apparent signs of toxicity, and they were weighed weekly. ZD1839 treatment did not affect the histologic appearance of normal mammary glands or breast tumor tissues (Fig. 4, A). As shown in Fig. 4, B, the mammary glands in these mice are ER-negative. Although a few normal mammary duct cells express ER, the tumors that arise in these mice are uniformly ER-negative.
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ZD1839 Treatment and c-erbB2 Transgene Expression
To determine whether the tumor-suppressive effects of ZD1839 were caused by inhibiting the expression of the c-erbB2 transgene, we investigated the expression of c-erbB2 in normal and malignant mammary tissues by use of immunohistochemistry and western blotting. As shown in Fig. 6, c-erbB2 expression was similar in normal and tumor tissue after treatment with vehicle or ZD1839. We obtained the same result by western blot analysis (data not shown). Therefore, ZD1839 did not affect c-erbB2 transgene expression.
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To further investigate the mechanism of the tumor-suppressive effects of ZD1839, we examined the effect of ZD1839 on cell proliferation in normal and cancerous cells from these mice, as measured by bromodeoxyuridine incorporation with a bromodeoxyuridine-specific antibody. As shown in Fig. 7, A, ZD1839 treatment caused a 20.3% (95% CI = -13.7% to 44.2%) relative reduction in the proliferation of normal breast cells, although this difference did not reach statistical significance (P = .16 by Student's t test). In tumors, 10.3% (95% CI = 8.0% to 12.6%) of the cells from vehicle-treated mice stained positive for bromodeoxyuridine, and 6.0% (95% CI = 4.0% to 8.0%) of the cells from mice treated with ZD1839 at 100 mg/kg stained positive for bromodeoxyuridine. Thus, ZD1839 at 100 mg/kg induced a 42.0% (95% CI = 20.2% to 58.2%) relative reduction in proliferation (P = .005, Student's t test). These results suggest that the ability of ZD1839 to prevent breast cancer development was associated with reduced proliferation in normal and malignant breast cells.
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It has previously been shown (16) that ZD1839 treatment leads to increased expression of the cell cycle inhibitor p27 and cell cycle blockade in cancer cells. Therefore, we measured the expression of p27 in mammary tissues by immunohistochemical staining for p27. Treatment with ZD1839 at 100 mg/kg increased the expression of p27 in normal mammary tissue (48.7%, 95% CI = 27% to 74.2%) and tumor tissues (50.3%, 95% CI = 35.8% to 66.7%) (both P<.001, Student's t test; Fig. 7, B). We found the same results by western blotting (data not shown). This increase in p27 is consistent with the reduced proliferation shown in Fig. 7, A. Thus, ZD1839 blocked EGFR and MAPK activation, induced p27, suppressed proliferation, and ultimately inhibited breast tumorigenesis.
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DISCUSSION |
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The mechanism by which ZD1839 suppresses breast cancer development is unclear. ZD1839 has been shown to prevent autophosphorylation of EGFR in a number of cultured tumor cell lines, resulting in an inhibition of the activation of key downstream signaling molecules, such as MAPK and Akt (1719). ZD1839 reduces breast cancer cell proliferation (9,2022) and alters expression of cell cycle regulators, such as p27, thereby causing a cell cycle blockade (9). These previous results are consistent with the results in this article. We observed that ZD1839 completely blocked EGFR autophosphorylation and partially inhibited the downstream MAPK phosphorylation in normal, precancerous, and malignant breast cells. Thus, phosphorylated EGFR and phosphorylated MAPK could be used to assess the cellular response to EGFR blockade by ZD1839. Our study showed that treatment with ZD1839 was also associated with reduced proliferation and increased expression of p27 in normal and malignant breast cells. We observed increased p27 expression in normal mammary gland cells and in tumor cells from mice treated with ZD1839 at 100 mg/kg. Treatment with ZD1839 at 100 mg/kg also caused a decrease in proliferation in normal (by 20.3%, 95% CI = -13.7% to 44.2%) and malignant (by 42%, 95% CI = 20.2% to 58.2%) mammary cells. The modest reduction in proliferation in normal cells likely reflects the low basal level of proliferation seen in normal mammary glands. It is probable that ZD1839 also suppressed the growth of preinvasive breast cells (in hyperplasias or carcinomas in situ), which contributes to delayed development of invasive tumors. We are currently testing this hypothesis by analyzing mammary glands from vehicle- or ZD1839-treated mice at early time points when hyperplasias and carcinomas in situ are present.
Lenferink et al. (23) demonstrated that blockade of the EGFR tyrosine kinase with AG-1478 suppresses tumorigenesis in MMTV-neu + MMTV-TGF- bigenic mice. Their findings are consistent with those in this article. Both studies show that EGFR tyrosine kinase inhibitors suppress mammary tumor development. However, in their study, tumorigenesis was induced by overexpression of both transforming growth factor
(TGF-
, the ligand for EGFR) and c-erbB2. It was not clear whether the preventive effect of the EGFR tyrosine kinase inhibitor was predominantly the result of blocking the activation of EGFR by increasing the expression of TGF-
or by blocking erbB2-dependent signals. Results of this study in mice expressing only one transgene, c-erbB2, demonstrated that ZD1839 was able to inhibit tumorigenesis induced by this single transgene.
Promising agents for prevention of ER-negative breast cancer include retinoids, cyclooxygenase-2 inhibitors, and now an EGFR tyrosine kinase inhibitor. Our previous work has demonstrated that a naturally occurring retinoid, 9-cis-retinoic acid, and the retinoid X receptorselective retinoid LGD1069 prevent ER-negative mammary tumor development in mouse models (6,7,12). Howe et al. (24) showed that a selective cyclooxygenase-2 inhibitor, celecoxib, reduced the incidence of mammary tumors in MMTV-neu mice. In this study, we have shown that, in addition to retinoids and cyclooxygenase-2 inhibitors, the EGFR tyrosine kinase inhibitor ZD1839 may also be effective in preventing ER-negative mammary tumor development in MMTV-erbB2 mice. However, comparison of the activity of each of these agents in MMTV-erbB2 mice suggests that ZD1839 and LGD1069 are more effective than celecoxib at suppressing tumorigenesis.
ZD1839 may be most useful for prevention of breast cancer when combined with other chemoprevention agents such as antiestrogens. Antiestrogens such as tamoxifen and raloxifene have been shown to reduce the risk of ER-positive breast cancer. An increasing body of evidence demonstrates that growth factor networks are highly interactive with the ER signaling that controls breast cancer growth. We (in this article) and others (19,22) have shown that ZD1839 inhibits proliferation in both ER-positive and ER-negative breast cells. Recent studies from Wakeling et al. (25) demonstrated that ZD1839 blocks MAPK activity in tamoxifen-resistant MCF7 cells and that treatment of wild-type MCF7 cells with tamoxifen and ZD1839 prevents development of tamoxifen resistance. These data support the potential clinical utility of ZD1839 in treating tamoxifen-resistant breast cancer and also suggest that the combination of tamoxifen and ZD1839 may be particularly effective in reducing the risk of both ER-positive and ER-negative breast cancer. These studies appear to provide the preclinical rationale for the development of these EGFR tyrosine kinase inhibitors for the prevention of human breast cancer.
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NOTES |
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Supported by the National Institutes of Health Specialized Programs of Research Excellence (SPORE) grant P50 CA58183 (to C. K. Osborne and P. H. Brown).
We thank Dr. Kendall Wu, David Denardo, Dr. Hyesook Seo, and Dr. Sandra Revett for their helpful discussions and critical reading of the manuscript. We also thank Linda Kimbrough for her assistance in preparing the manuscript.
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Manuscript received May 27, 2003; revised October 9, 2003; accepted October 24, 2003.
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