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Tenovus Centre for Cancer Research (J.M.W.G., M.E.H., I.R.H., T.A.M., D.B., J.M.K., R.A.M., N.J., R.I.N.), Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, United Kingdom; and AstraZeneca (A.E.W.), Macclesfield SK10 4TG, United Kingdom
Address all correspondence and requests for reprints to: J. M. W. Gee, Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, Wales, United Kingdom. E-mail: gee{at}cardiff.ac.uk.
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Introduction |
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(1, 2). However, their clinical application has highlighted significant limitations for many initially responsive patients. Responses are commonly incomplete (3), indicating that a proportion of cancer cells survive the inhibitory activity of antihormonal challenge. Moreover, the inhibitory activity of current agents can be relatively short-lived, with resistant growth commonly emerging during therapy resulting in disease relapse and ultimately death (4). If the resistant state is to be effectively compromised, it is imperative that the compensatory signal transduction mechanisms that initially allow tumor cells to evade the antiproliferative and proapoptotic activity of antihormonal challenge and ultimately support resistant growth are elucidated. Such knowledge would be valuable in the design of future treatment strategies for antihormonal refractory disease. Equally, however, strategic targeting in responsive disease of these compensatory elements simultaneously with antihormonal strategies would be expected to delay, and perhaps even prevent, resistance.
In this light, the epidermal growth factor receptor (EGFR) is particularly worthy of attention. This receptor signals via ERK1/2 MAPK and phosphatidylinositol-3'-kinase/AKT (protein kinase B) to drive proliferation and cell survival (5, 6, 7, 8, 9, 10). EGFR dysregulation has been implicated in initiation, growth, and progression of many human epithelial cancers, in which it commonly associates with poor prognosis (6, 11). In breast cancer, EGFR overexpression occurs in about 50% of patients (12). Increased EGFR, its cognate growth factor ligands (e.g. TGF), or hyperactivation of its signaling elements have invariably been associated with antihormonal resistance, aggressive clinicopathology, disease metastasis, and poor prognosis (11, 13, 14, 15, 16). Transfection studies suggest a causative relationship between EGFR and antihormone resistance (17, 18). Furthermore, elevated EGFR and its signaling has frequently been demonstrated within in vitro breast cancer models that have acquired resistance to antihormonal strategies (19, 20, 21, 22), including our own sublines resistant to the antiestrogens tamoxifen (23, 24) or fulvestrant (Faslodex) (25). In such cells, the EGFR-selective tyrosine kinase inhibitor gefitinib (4-[3-chloro-4-fluoroanilino]-7-methoxy-6-[3-morpholinopropoxy] quinazoline; ZD1839/Iressa) (26) and EGFR-directed antibodies (21) are growth inhibitory. Importantly, therefore, EGFR increases in antihormone-resistant cells are paralleled by growth dependency on EGFR-mediated signaling. Thus, EGFR targeting may have considerable potential for treating antihormonal resistance.
The present investigation extends our in vitro studies to chart the evolution of increased EGFR signaling during tamoxifen challenge of antihormone responsive MCF-7 cells, determining whether increased EGFR provides a compensatory cell survival mechanism that limits antihormonal efficacy and ultimately allows emergence of resistant growth. We examine whether cotreatment of antihormone responsive cells with the EGFR-selective agent gefitinib in anticipation of emergence of EGFR is a more effective antitumor strategy than challenge with the antihormones tamoxifen or fulvestrant alone and address whether antihormone plus gefitinib cotreatment also abrogates development of resistance.
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Materials and Methods |
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For experimentation, MCF-7 cells were seeded at 1 x 106 cells/80-cm2 flask into basal medium comprising RPMI 1640 lacking phenol red supplemented with 5% dextran-coated charcoal-stripped fetal calf serum, antibiotics (as above), and L-glutamine (4 mM). For wk 1 microscopical studies, cells were seeded in basal medium onto sterile 22-mm 3-aminopropyltriethoxysilane-coated glass coverslips (1 x 105 cells/coverslip) and for fluorescence-activated cell sorting (FACS) at 2.5 x 105 cells/25-cm2 flask. After 48 h, preparations were replenished with fresh basal medium containing 1) 4-hydroxytamoxifen (TAM; 0.1 µM, Sigma Chemical Co. Ltd., Dorset, UK), 2) the EGFR selective tyrosine kinase inhibitor gefitinib (TKI; 1 µM; a gift from AstraZeneca Pharmaceuticals), 3) TAM (0.1 µM) plus TKI (1 µM) cotreatment (TAMTKI), or 4) vehicle control (CON; ethanol 0.1 µl/ml). Parallel preparations were made with fulvestrant (FAS; 0.1 µM; a gift from AstraZeneca Pharmaceuticals) or FAS (0.1 µM) plus TKI (1 µM) cotreatment (FASTKI) for a single time point (i.e. wk 4) microscopical analysis of proliferation and apoptosis. The antiestrogen concentration used is growth inhibitory in MCF-7 and an effective inhibitor of estradiol-induced ER signaling and growth, but 10-fold dosage increases fail to improve antitumor activity (24, 25). Similarly, 10-6 M TKI has previously been shown to block EGFR phosphorylation and specifically inhibit EGFR-mediated growth in common with other 4-anilinoquinazolines (24, 25, 26, 27, 28, 29). After a further 5 d, all preparations for the wk 1 time point were harvested for the various analysis techniques described below. For CON or TKI alone, an 80-cm2 flask was subsequently passaged to generate flasks for later time-point seeding and maintain a stock culture, also setting up for wk 2 microscopy and FACS. This regimen of coverslip and flask preparation was continued until wk 5, where 2.5 x 105 cells/60-mm dish were also seeded for Western blotting. For all other groups in which diminished growth was associated with treatment, equivalent preparations for all subsequent time-point analyses were set up at wk 1. To detail evolution of TAM resistance, culture of CON vs. TAM was extended over wk 6–12, obtaining preparations for microscopical and Western analysis at fortnightly intervals.
Growth analysis
MCF-7 cells were seeded into 24-well plates at 4 x 104 cells/well. After 48 h, TAM, TKI, TAMTKI or CON vehicle was added in basal medium. After a further 5 d, cell growth counts were made in triplicate for each group at wk 1 by Coulter counting (Beckman, Luton, UK). Assessment was repeated at wk 2, performing fresh media changes every 3 d. Thereafter, triplicate counts through to wk 5 were possible only for TAM and TAMTKI groups in which diminished growth resulted from treatment because CON or TKI required subculturing after wk 2. Growth was also monitored for all groups including FAS and FASTKI by phase contrast microscopy. Although growth counts were not performed, a further seven experiments were visually monitored over an equivalent time course to address reproducibility of the antitumor effects of TAMTKI vs. the single agents and confirm its impact on resistance.
FACS analysis
Cell cycle.
Reagents for FACS and all subsequent assay procedures were obtained from Sigma unless stated otherwise. The 25-cm2 flask preparations for the various MCF-7 treatment groups and time points were collected by trypsin dispersion of adherent cells pooling with nonadherent cells in the medium. Single-cell suspensions (5 x 105 cells/ml) were prepared and cell cycle progression was examined using an isolated nuclei method and propidium iodide incorporation as described previously (30). All samples were analyzed by flow cytometry using a FACS (FACSCalibur; Becton Dickinson Immunocytometry Systems, Mansfield, MA) with a 15-mW, 488-nM argon-iron laser. Where possible, 50,000 events/sample were recorded. Analysis of cell cycle and measurements of diploid (2N) DNA content of propidium iodide (PI)-stained nuclei were performed using WinMDi and Cylchred programs (version 2.8 and version 1.0.2, respectively; Cytonet UK, Cardiff, UK).
Early apoptosis.
Single-cell suspensions comprising adherent and nonadherent cells were prepared from 25-cm2 flask preparations for each treatment group and time point as described for cell cycle analysis. The 5 x 105 cells/ml were stained using annexin V-fluorescein isothiocyanate (FITC) and PI according to the manufacturer’s protocol (Annexin V-FITC kit, BMS306FI, Bender MedSystems, Vienna, Austria). Analysis of green (annexin V-FITC) and red (propidium iodide uptake) fluorescence was measured by FACS (FACSCalibur; Becton Dickinson Immunocytometry Systems) using standard optics and CellQuest software (Becton Dickinson). Electronic compensation was used to exclude overlapping of the two emission spectra. Where possible, 2 x 104 cells were analyzed. Because the necrotic or late apoptotic fractions demonstrated no obvious changes, only the early apoptotic fraction (annexin V-FITC positive/PI negative) was compared between treatments.
Fluorescence microscopy.
Early apoptosis for each MCF-7 treatment group was further monitored by fluorescence microscopy using an ApoAlert mitochondrial membrane sensor kit (MMS; Clontech Laboratories UK Ltd., Basingstoke, UK). MitoSensor reagent forms red fluorescent mitochondrial aggregates in nonapoptotic cells, but in early apoptosis altered mitochondrial membrane permeability results in MitoSensor remaining in a monomeric green fluorescent cytoplasmic form (31). For each treatment group and time point, duplicate coverslips were incubated with MitoSensor according to the MMS kit protocol. Coverslips were then viewed using fluorescence microscopy, and apoptotic cell percentage was estimated by counting nine fields/coverslip.
Immunocytochemistry and light microscopy
Previously described immunocytochemical fixation and assay procedures (24, 25, 32, 33, 34) were used to monitor ER and progesterone receptor (PgR), EGFR, phosphorylated (activated) EGFR and ERK1/2 MAPK, Ki-67 proliferation antigen, bcl-2, and bax. Appropriately fixed coverslips were analyzed at least in duplicate for each marker. Sensitive immunocytochemical (and Western blotting) procedures were essential throughout this study to reproducibly detect EGFR signaling in MCF-7 before and after treatment. Primary antibodies comprised rat ER or PgR monoclonals (0.1 µg/ml H222 and KD68; Abbott Laboratories, North Chicago, IL), mouse monoclonals to EGFR (1/125 clone 111.6; Stratech Scientific Ltd., Luton, UK), tyrosine-phosphorylated EGFR (1/5 MAB3052; Chemicon International Inc., Temecula, CA), Ki-67 (1/50 clone MIB1; Dako A/S Ltd., Glostrup, Denmark), or bcl-2 (1/100 clone 124; Dako) and rabbit polyclonals to dually phosphorylated (Thr202/Tyr204) ERK1/2 MAPK (1/25; Cell Signaling Technology, New England Biolabs UK Ltd., Hertfordshire, UK) and bax (1/200; Dako). Species-specific PAP-conjugated antibody systems (Abbott Laboratories; Dako) as well as biotinylated antiimmunoglobulins with streptavidin/peroxidase (Biogenex, San Ramon, CA) were employed for detection as appropriate. Diaminobenzidine tetrahydrochloride chromogen-substrate solution (Dako) was used to demonstrate immunopositivity, counterstaining to reveal negativity. Assay controls comprised omission of primary antibody. Staining was evaluated using a BH-2 microscope (Olympus Optical Co., Hamburg, Germany) and was nuclear for ER, PgR, and Ki-67; cytoplasmic for bcl-2 and bax; nuclear and cytoplasmic for phospho-ERK1/2 MAPK; and plasma membrane and cytoplasmic for EGFR and phospho-EGFR. Cell percentages of low, moderate, or high staining intensity were recorded for at least three fields/coverslip so that an H-score could be assigned, where the H-score is an established immunostaining index on a 0–300 scale (32). For Ki-67 (MIB1) immunostaining, counting of percentage positivity indicated cells in cycle vs. negative (G0) cells. For conventional light microscopy, coverslips were 3.7% formaldehyde fixed and hematoxylin/eosin counterstained. Representative photomicroscopy was performed using a Camedia C-200 digital camera (Olympus) and DP-software (Olympus).
Protein cell lysis and Western blotting
Protein cell lysis and Western blotting procedures were as previously described (24). Briefly, MCF-7 cells for each treatment group were lysed (35) and preparations (20 µg protein) resuspended in sample loading buffer under reducing conditions. Samples were then electrophoretically separated on a 7.5% PAGE gel and transblotted onto nitrocellulose membrane. Following blocking, blots were incubated for 1 h in primary antibody diluted 1/1000 for total or phosphorylated EGFR, ERK1/2 MAPK, and AKT, and 1/5000 for ß-actin reference control. Primary antibodies employed were rabbit polyclonal anti-EGFR (1005 SC-03; Insight Biotechnology Ltd., Wembley, UK) and mouse monoclonal antiphospho-EGFR (05–483 clone 9H2 specific for phospho-Y1173; TCS/Upstate Biotechnology, Buckinghamshire, UK). Anti-ERK1/2 MAPK (no. 9102), antiphospho-ERK1/2 MAPK (no. 9101, specific for dually phosphorylated Thr202/Tyr204 in ERK1/2 MAPK), anti-AKT (no. 9272) and antiphospho-AKT (no. 9271; specific for phosphorylated Ser473 in AKT) were rabbit polyclonals (Cell Signaling Technology). Anti-ß-actin mouse monoclonal antibody was from Sigma (A5441 clone AC-15). All total and phospho-antibodies employed in this study have previously been demonstrated to be specific. Membranes were incubated with 1/20,000 horseradish peroxidase-labeled secondary antibody (Amersham Life Sciences, Buckinghamshire, UK) and signal detection was performed using standard chemiluminescent reagents (Pierce and Warriner Ltd, Cheshire, UK) with extended exposure time. Equivalent loading was confirmed by monitoring ß-actin.
Statistical analysis
Differences between cell growth counts for the treatment groups were evaluated using one-way ANOVA, employing the post hoc multiple comparison Dunnett t test. Marker analysis was performed using the nonparametric Kruskal-Wallis and Mann-Whitney U tests. The P values represent two-sided tests of statistical significance, where differences were considered significant at P < 0.05. All analysis was performed using SPSS (version10.0.5 for Windows, SPSS Inc., Chicago, IL).
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). There was only a small, nonsignificant inhibitory effect of TKI (13%), with growth essentially equivalent to CON at wk 2 (Fig. 1A). In contrast, TAM significantly (55%) reduced growth at wk 2, with the resultant low cell number maintained through to wk 5 (Fig. 1A). Although the TAM culture was generally sparse at this latter time point, several small foci of very densely packed cells were also apparent (Fig. 1B). Continued visual monitoring of TAM cultures to wk 12 revealed that the foci progressively expanded in size and frequency, indicating TAM-resistant growth. A small reduction (26%) in growth was apparent by wk 2 with TAMTKI cotreatment vs. TAM (Fig. 1A). Thereafter, there was a progressive and substantial cell loss with TAMTKI vs. TAM that was significant at wk 3–5. Thus, by wk 5, there was a further 78% growth inhibition with cotreatment vs. TAM alone, cell numbers frequently declining below the seeding density. At wk 5, cotreated cultures consisted entirely of sparsely distributed cells, with no foci of very densely packed cells (Fig. 1C). Continued visual monitoring of TAMTKI cultures revealed a further decline in cell number through to wk 12, in which no viable cells remained.
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, A and B; P = 0.004), with cotreated cultures reduced to a single cell population that could not be maintained for later time-point analysis.
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Proliferation
In accordance with substantial CON growth, maximal MIB1 immunopositivity was observed over wk 3–5 for CON and thereafter up to wk 12 (Figs. 3A and 4A), with a substantial S-phase fraction maintained in parallel (Fig. 4, B and C). There were no significant effects of TKI on these parameters over wk 3–5 (Figs. 3B and 4, A–C). In accordance with its growth-inhibitory effects, TAM significantly reduced MIB1, compared with CON, over wk 3–5 (Fig. 3C), staining decreasing to 40% at the latter time point (Fig. 4A). Cell cycle analysis similarly revealed a substantially reduced S-phase fraction, with a parallel increase in G0/G1 (Fig. 4, B and C). No marked change in G2/M fraction was noted with TAM. Although cell cycle profile did not alter at wk 5 vs. wk 3–4 in a manner that might evidence early emergence of TAM resistance, the occasional foci observed in TAM cultures from wk 5 were intensely MIB1 positive in most of their cells, with prominent mitotic figures. This markedly contrasted the diminished proliferative capacity of the majority of the TAM culture at this time point. Extended monitoring of TAM revealed that although still significantly below CON, MIB1 was incrementally increased to about 70% positivity over wk 5–12 (Fig. 4A), paralleling the increasing frequency and size of emergent MIB1-positive foci with TAM (Fig. 3U).
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and 4A). In parallel there was a further reduction in S-phase fraction with cotreatment vs. TAM and a small additional increase in G0/G1 fraction (Fig. 4, B and C). No marked change in G2/M was noted, compared with TAM. No foci of highly MIB1-positive cells were apparent with cotreatment at wk 5. Parallel microscopical studies performed at wk 4 revealed that inhibition of proliferation was superior with FASTKI cotreatment vs. FAS alone (20% vs. 46% MIB1, respectively, P = 0.001; Fig. 2, C and D).
Apoptosis
Few cells were in early apoptosis in CON cultures as detected over wk 3–5 by FACS annexin V-FITC binding and MMS microscopy (Figs. 3E and 5, A–C). Apoptotic fraction was also minimal with TKI, in general equivalent to CON (Figs. 3F and 5A–C). There was no significant effect of TKI on bcl-2 (mean H-score TKI = 88 vs. CON = 100, P = 0.11; Figs. 3I, and J) or bax (mean H-score TKI = 64 vs. CON = 59, P = 0.37; not illustrated), with cytoplasmic staining noted for both markers. A modest increase in apoptotic fraction (albeit variable in magnitude at each time point) was observed over wk 3–5 with TAM vs. CON (Figs. 3G and 5, A–C). Apoptosis did not alter with TAM at wk 5, compared with wk 3–4 in any consistent manner that would evidence the initial emergence of TAM resistance. bcl-2 was significantly reduced with TAM vs. CON, although some cells retained substantial immunopositivity (Fig. 3K; mean H-score = 32, P = 0.009). There was no significant TAM effect on bax expression or localization (mean H-score = 68; P = 0.26, not illustrated).
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, A and B), particularly using MMS fluorescence microscopy (Figs. 3H and 5C). MMS also revealed an increased apoptotic fraction (32%) at wk 4/5 vs. wk 3 (21%; P = 0.008). In parallel, superior bcl-2 suppression (65%) was apparent with TAMTKI vs. TAM (P = 0.015), with bcl-2 immunostaining barely detectable following cotreatment (mean H-score = 11; Fig. 3L). There was no significant effect of cotreatment on bax expression or localization (mean H-score = 55, P = 0.1; not illustrated). Parallel microscopical studies at wk 4 revealed FASTKI was also significantly superior in promoting apoptosis (50% MMS, P < 0.001), compared with FAS alone (27%; Fig. 2, E and F).
EGFR signaling
EGFR expression.
Immunocytochemistry demonstrated that EGFR was weakly expressed in CON (Fig. 3M), with no obvious change in expression over the time course (Fig. 6A). EGFR positivity was found in 12% of cells, with predominantly cytoplasmic and less than 10% plasma membrane staining (Fig. 3M). There was no consistent change in EGFR staining with TKI (Figs. 3N and 6A). However, although only small cytoplasmic EGFR increases occurred over wk 1–2 (not illustrated), treatment with TAM vs. CON over wk 3–5 substantially and significantly increased EGFR staining (Figs. 3O and 6A). Thus, 33% of cells were EGFR positive by wk 5. EGFR staining was significantly increased by TAM at every subsequent time point, with increases being roughly incremental over the time course. By wk 12, 55% of cells were EGFR positive, with a 250% increase in staining with TAM vs. CON (Fig. 6A). Both cytoplasmic and particularly plasma membrane staining were substantially increased by TAM (Fig. 3O), with the latter comprising at least 30% of staining. Western blotting confirmed that EGFR was increased with TAM vs. CON at wk 5 and all subsequent time points, but ß-actin remained constant (Fig. 7A). Immunostaining was most prominent within the multiple resistant foci that evolved with TAM over wk 5–12. Indeed by wk 12, 70% of cells in these foci were strongly EGFR positive, particularly at the plasma membranes (Fig. 3V).
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and 6A), with only 14% of cells being EGFR positive. At wk 5 there was an 80% decline in EGFR immunostaining with TAMTKI vs. TAM, and Western blotting confirmed this marked EGFR reduction (Fig. 7B). TAMTKI substantially decreased cytoplasmic and plasma membrane EGFR staining (wk 5: P < 0.001), with the latter comprising only 18% of total expression. Decreases in EGFR were more marked with cotreatment at wk 4–5 (87% fall) vs. wk 3 (35% fall; Fig. 6A). The highly EGFR-positive foci noted with TAM at wk 5 were absent following cotreatment.
EGFR phosphorylation (phospho-EGFR).
Phospho-EGFR was barely detectable by immunocytochemistry in CON cultures, and no marked differences could be distinguished with TKI or TAM over wk 3–5 (Fig. 6B). Interestingly, however, the lowest staining was consistently achieved using TAMTKI cotreatment (Fig. 6B). Western blotting did not detect phospho-EGFR in any group at wk 5. However, extended monitoring of TAM cultures revealed that from wk 8, phospho-EGFR immunostaining exceeded CON, and by wk 12 significantly increased phospho-EGFR staining was readily detectable both by immunocytochemistry (Fig. 6B) and Western blotting (Fig. 7C). At wk 12, 60% of TAM-treated cells demonstrated weak phospho-EGFR immunopositivity. This was largely granular cytoplasmic, with 30% plasma membrane staining within the TAM-resistant foci (Fig. 3W).
ERK1/2 MAPK and AKT phosphorylation.
Phospho-ERK1/2 MAPK was immunocytochemically detected in 60% of CON cells throughout the time course (Fig. 6C). Immunostaining was predominantly cytoplasmic, with only 3% nuclear positivity (Fig. 3Q). TKI significantly reduced phospho-ERK1/2 MAPK immunostaining in comparison with CON, with 40% cells remaining positive (Figs. 3R and 6C). TAM also significantly reduced phospho-ERK1/2 MAPK staining over wk 3–5, with 27% of cells remaining positive (Figs. 3S and 6C). Western blotting at wk 5 confirmed decreased phospho-ERK1/2 MAPK with TAM or TKI vs. CON, with parallel decreases in phospho-AKT (Fig. 7B). Continued monitoring of TAM cultures after wk 5 revealed progressive increases in phospho-ERK1/2 MAPK immunostaining (Fig. 6C), and by wk 12 62% positivity was observed, a level equivalent to CON. Western blotting confirmed this recovery with TAM after wk 5 and revealed a parallel increase in phospho-AKT (Fig. 7A). Incremental recovery of these signaling elements coincided with an increasing frequency and size of emergent-resistant foci. Considerable phospho-ERK1/2 MAPK staining was observed in these foci (Fig. 3X), in which H-scores greater than 100 with 25% nuclear staining were commonly achieved by wk 12.
TAMTKI was superior in depleting phospho-ERK1/2 MAPK immunostaining over wk 3–5, with only extremely weak positive staining remaining in 11% cells (Figs. 3T and 6C). There was thus a substantial further decline with cotreatment of 66% and 75%, respectively, vs. TAM or TKI alone over wk 3–5, although this decrease was less obvious at earlier time points (not illustrated). Both cytoplasmic and nuclear immunostaining were markedly reduced by TAMTKI cotreatment (wk 5: P = 0.004 and P = 0.002, respectively). Moreover, no phospho-ERK1/2 MAPK-positive foci were apparent at wk 5 following cotreatment. Western blotting performed at this time point confirmed a superior depletion of phospho-ERK1/2 MAPK and also that phospho-AKT was undetectable with cotreatment (Fig. 7B). No equivalent changes in MAPK or AKT expression were observed (Fig. 7B).
ERs and PgRs
Immunocytochemistry demonstrated substantial ER expression in CON over wk 3–5 (mean H-score = 105), with some PgR also detected (mean H-score = 30). TKI did not significantly change ER or PgR expression (mean H-scores = 108 and 26, respectively). TAM slightly increased ER (P = 0.009; mean H-score = 130) but decreased PgR (P = 0.02; mean H-score = 2). Neither ER (mean H-score = 127) nor PgR (mean H-score = 2) were further altered by TAMTKI vs. TAM (not illustrated).
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Discussion |
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We used MCF-7 as an in vitro model of ER-positive, antihormonal-responsive human breast cancer. MCF-7 cells were growth inhibited (
50%) by tamoxifen from wk 2 through to wk 5, with parallel decreases in Ki-67/MIB1 proliferation marker and S-phase, modest apoptotic increases, and partial suppression of the cell survival protein bcl-2. ER accumulation and reduced expression of the ER-regulated protein PgR were also observed. Comparable results have been previously described during tamoxifen response (36, 37, 38, 39), confirming the key importance of ER signaling in MCF-7. In contrast, EGFR signaling does not contribute significantly to MCF-7 growth before tamoxifen treatment. MCF-7 lacked significant plasma membrane EGFR, and receptor phosphorylation was barely detectable. Furthermore, growth inhibition with the anti-EGFR agent gefitinib was nonsignificant, with no marked effects on proliferation, apoptosis, bcl-2, bax, or EGFR expression and only incomplete suppression of ERK1/2 MAPK and AKT phosphorylation. The ineffectiveness of EGFR-directed antibodies against basal MCF-7 growth is supportive of these data (40).
Importantly, tamoxifen inhibition of MCF-7 was incomplete. Interestingly, EGFR was significantly increased in the cells persisting during tamoxifen treatment, with prominent plasma membrane staining from wk 3. Particularly marked EGFR induction was associated with emergence of highly proliferative, tamoxifen-resistant foci from wk 5. Although there was initially some tamoxifen suppression of ERK1/2 MAPK and AKT phosphorylation [probably because of antihormonal inhibition of IGF receptor signaling (41)], there was recovery in activation after wk 5, again particularly within tamoxifen-resistant foci. This recovery was paralleled by further induction of EGFR, readily detectable receptor phosphorylation, and progressive emergence and expansion in size of resistant foci. ER suppression has been shown to up-regulate EGFR in MCF-7, T47D, and BT474 cells (42, 43). Yarden et al. (43) and Wilson and Chrysogelos (44) demonstrated that EGFR transcription is blocked by long-term estrogen exposure, but antihormonal strategies de-repress this event and enhance growth sensitivity to EGFR ligands (43). Subsequently, increased EGFR signaling is observed in many established models of acquired antihormone resistance in which it is clearly growth promoting (19, 20, 21, 22, 23, 24, 25), with transfection studies further supportive of a role for EGFR signaling in antihormone resistance (17, 18, 45, 46). Thus, although EGFR is of minimal importance to ER-positive, antihormone-responsive cells before treatment, adaptive EGFR increases occur during tamoxifen challenge that may initially allow cells to escape antihormone inhibition and ultimately drive resistant growth. We confirmed this hypothesis by cotreatment with tamoxifen plus the EGFR-specific inhibitor gefitinib (TAMTKI).
From wk 3, TAMTKI effectively blocked tamoxifen-induced EGFR. TAMTKI was associated with the lowest EGFR activation and eliminated ERK1/2 MAPK and AKT phosphorylation. In contrast, there was no further change in ER or PgR with TAMTKI vs. tamoxifen. In parallel with its profound inhibition of EGFR signaling, there was superior growth inhibition with TAMTKI vs. tamoxifen alone (e.g. 78% improvement by wk 5). Importantly, TAMTKI also prevented emergence of resistant foci, with cultures being sparsely distributed and cell number frequently declining below the seeding density. Indeed, continued visual monitoring of TAMTKI cultures revealed that cell numbers further declined, with no viable cells by wk 12. The superior antigrowth effects of TAMTKI were noted in seven equivalent experiments, and resistance again failed to emerge in six of these over the examined time course. The data thus appear highly reproducible and are confirmatory of our preliminary observation that gefitinib/antiestrogen cotreatment improved antitumor activity (23, 47).
Superior growth inhibition by TAMTKI was associated with a further decrease in proliferative activity in the present study. Thus, MIB1 staining was barely detectable with an absence of MIB1-positive, tamoxifen-resistant foci, although S-phase was further reduced with increased G0/G1 accumulation. Additionally, however, there was a superior and progressive promotion of apoptosis vs. tamoxifen alone, confirming a survival role for EGFR signaling during tamoxifen challenge. ERK1/2 MAPK and AKT activation, elements obliterated by TAMTKI cotreatment, can converge on survival as well as proliferative events (9, 48, 49), and AKT overexpression is protective against tamoxifen-induced apoptosis (45). However, profound bcl-2 suppression may also contribute to the superior apoptotic activity of TAMTKI. bcl-2 has previously been linked to growth factor-mediated survival pathways, with its suppression and apoptosis reported during EGFR blockade of several cell types (50, 51, 52, 53). The bax expression and localization was unchanged with TAMTKI in the present study. Nevertheless, the superior bcl-2 decreases observed may allow bax to more efficiently promote apoptosis (54).
Cotreatment with fulvestrant plus gefitinib also demonstrated superior growth-inhibitory effects vs. antihormone alone, indicating the EGFR survival/resistance mechanism is shared by nonsteroidal and steroidal antiestrogens. Thus, fulvestrant plus gefitinib cotreatment exhibited improved antiproliferative and proapoptotic activity and prevented emergence of resistance, with no viable cells after wk 4. The EGFR survival mechanism also appears to be used by multiple antihormone-responsive models. We recently noted improved antitumor activity of tamoxifen plus gefitinib in T47D in vitro, whereas cotreatment with an anti-EGFR antibody and estrogen deprivation is superior in BT474 (43). Supportive in vivo model data are also emerging with anti-EGFR plus antihormone cotreatment strategies (55, 56). Interestingly, EGFR signaling may also comprise a compensatory mechanism employed by tumor cells to limit the efficacy of chemotherapy or radiotherapy, in which anti-EGFR agents again enhance antitumor activity and delay resistance (50, 52, 53, 57, 58).
The present studies were performed in vitro, and hence, it is feasible that EGFR may not be the sole signaling mechanism limiting antihormonal efficacy in the clinic. However, elevated EGFR signaling does associate with tamoxifen resistance, advanced clinical stage, and shortened relapse-free survival in breast cancer patients (11, 13). Importantly, this study provides striking experimental evidence that EGFR inhibitors such as gefitinib could improve antihormonal response in ER-positive breast cancer and combat development of resistance. The value of gefitinib monotherapy in clinical breast cancer is currently being investigated through phase II studies in ER-negative/EGFR-positive patients and recurrent tamoxifen-resistant disease (26, 59, 60). Because gefitinib has an acceptable tolerability profile in cancer patients (61), our data indicate that gefitinib plus antihormone cotreatment should now be assessed as a matter of priority in antihormone-responsive breast cancer clinical trials.
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Acknowledgments |
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Footnotes |
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Iressa and Faslodex are trademarks of the AstraZeneca group of companies.
Abbreviations: AKT, Protein kinase B; CON, vehicle control; EGFR, epidermal growth factor receptor; ER, estrogen target receptor; FACS, fluorescence-activated cell sorting; FAS, fulvestrant; FASTKI, FAS plus TKI; FITC, fluorescein isothiocyanate; MMS, mitochondrial membrane sensor; PgR, progesterone receptor; PI, propidium iodide; TAM, 4-hydroxytamoxifen; TAMTKI, TAM plus TKI; TKI, gefitinib.
Received June 5, 2003.
Accepted for publication July 29, 2003.
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 J.-M. Renoir, C. Bouclier, A. Seguin, V. Marsaud, and B. Sola Antioestrogen-mediated cell cycle arrest and apoptosis induction in breast cancer and multiple myeloma cells J. Mol. Endocrinol., March 1, 2008; 40(3): 101 - 112. [Abstract] [Full Text] [PDF]
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 S. Massarweh, C. K. Osborne, C. J. Creighton, L. Qin, A. Tsimelzon, S. Huang, H. Weiss, M. Rimawi, and R. Schiff Tamoxifen Resistance in Breast Tumors Is Driven by Growth Factor Receptor Signaling with Repression of Classic Estrogen Receptor Genomic Function Cancer Res., February 1, 2008; 68(3): 826 - 833. [Abstract] [Full Text] [PDF]
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 H. S. Rugo The breast cancer continuum in hormone-receptor positive breast cancer in postmenopausal women: evolving management options focusing on aromatase inhibitors Ann. Onc., January 1, 2008; 19(1): 16 - 27. [Abstract] [Full Text] [PDF]
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S. Massarweh and R. Schiff Unraveling the Mechanisms of Endocrine Resistance in Breast Cancer: New Therapeutic Opportunities Clin. Cancer Res., April 1, 2007; 13(7): 1950 - 1954. [Abstract] [Full Text] [PDF]
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 S. Massarweh and R. Schiff Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S15 - S24. [Abstract] [Full Text] [PDF]
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J M W Gee, V E Shaw, S E Hiscox, R A McClelland, N K Rushmere, and R I Nicholson Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S77 - S88. [Abstract] [Full Text] [PDF]
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I. R Hutcheson, J. M Knowlden, H. E Jones, R. S Burmi, R. A McClelland, D. Barrow, J. M W Gee, and R. I Nicholson Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S89 - S97. [Abstract] [Full Text] [PDF]
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R. B. Riggins, K. S. Thomas, H. Q. Ta, J. Wen, R. J. Davis, N. R. Schuh, S. S. Donelan, K. A. Owen, M. A. Gibson, M. A. Shupnik, et al. Physical and Functional Interactions between Cas and c-Src Induce Tamoxifen Resistance of Breast Cancer Cells through Pathways Involving Epidermal Growth Factor Receptor and Signal Transducer and Activator of Transcription 5b. Cancer Res., July 15, 2006; 66(14): 7007 - 7015. [Abstract] [Full Text] [PDF]
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 S. Glaros, N. Atanaskova, C. Zhao, D. F. Skafar, and K. B. Reddy Activation Function-1 Domain of Estrogen Receptor Regulates the Agonistic and Antagonistic Actions of Tamoxifen Mol. Endocrinol., May 1, 2006; 20(5): 996 - 1008. [Abstract] [Full Text] [PDF]
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S. R.D. Johnston Clinical Efforts to Combine Endocrine Agents with Targeted Therapies against Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2 and Mammalian Target of Rapamycin in Breast Cancer Clin. Cancer Res., February 1, 2006; 12(3): 1061s - 1068s. [Abstract] [Full Text] [PDF]
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N. Normanno, M. Di Maio, E. De Maio, A. De Luca, A. de Matteis, A. Giordano, F. Perrone, and on behalf of the NCI-Naples Breast Cancer Group Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer Endocr. Relat. Cancer, December 1, 2005; 12(4): 721 - 747. [Abstract] [Full Text] [PDF]
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 A. R Gunthert, C. Grundker, A. Olota, J. Lasche, N. Eicke, and G. Emons Analogs of GnRH-I and GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen Eur. J. Endocrinol., October 1, 2005; 153(4): 613 - 625. [Abstract] [Full Text] [PDF]
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R I Nicholson, I R Hutcheson, S E Hiscox, J M Knowlden, M Giles, D Barrow, and J M W Gee Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S29 - S36. [Abstract] [Full Text] [PDF]
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A. J Butt, C. M McNeil, E. A Musgrove, and R. L Sutherland Downstream targets of growth factor and oestrogen signalling and endocrine resistance: the potential roles of c-Myc, cyclin D1 and cyclin E Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S47 - S59. [Abstract] [Full Text] [PDF]
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L.-A. Martin, I. Farmer, S. R D Johnston, S. Ali, and M. Dowsett Elevated ERK1/ERK2/estrogen receptor cross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S75 - S84. [Abstract] [Full Text] [PDF]
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A Agrawal, E Gutteridge, J M W Gee, R I Nicholson, and J F R Robertson Overview of tyrosine kinase inhibitors in clinical breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S135 - S144. [Abstract] [Full Text] [PDF]
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S R D Johnston Clinical trials of intracellular signal transductions inhibitors for breast cancer -- a strategy to overcome endocrine resistance Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S145 - S157. [Abstract] [Full Text] [PDF]
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H E Jones, J M W Gee, K M Taylor, D Barrow, H D Williams, M Rubini, and R I Nicholson Development of strategies for the use of anti-growth factor treatments Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S173 - S182. [Abstract] [Full Text] [PDF]
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T Nagahata, T Sato, A Tomura, M Onda, K Nishikawa, and M Emi Identification of RAI3 as a therapeutic target for breast cancer Endocr. Relat. Cancer, March 1, 2005; 12(1): 65 - 73. [Abstract] [Full Text] [PDF]
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S. R. Johnston Combinations of Endocrine and Biological Agents: Present Status of Therapeutic and Presurgical Investigations Clin. Cancer Res., January 15, 2005; 11(2): 889s - 899s. [Abstract] [Full Text] [PDF]
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R I Nicholson, C Staka, F Boyns, I R Hutcheson, and J M W Gee Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy Endocr. Relat. Cancer, December 1, 2004; 11(4): 623 - 641. [Abstract] [Full Text] [PDF]
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 C. Bernard-Marty, F. Cardoso, and M. J. Piccart Facts and Controversies in Systemic Treatment of Metastatic Breast Cancer Oncologist, November 1, 2004; 9(6): 617 - 632. [Abstract] [Full Text] [PDF]
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 S. Guo and G. E. Sonenshein Forkhead Box Transcription Factor FOXO3a Regulates Estrogen Receptor Alpha Expression and Is Repressed by the Her-2/neu/Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Mol. Cell. Biol., October 1, 2004; 24(19): 8681 - 8690. [Abstract] [Full Text] [PDF]
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R. I. Nicholson, I. R. Hutcheson, J. M. Knowlden, H. E. Jones, M. E. Harper, N. Jordan, S. E. Hiscox, D. Barrow, and J. M. W. Gee Nonendocrine Pathways and Endocrine Resistance: Observations with Antiestrogens and Signal Transduction Inhibitors in Combination Clin. Cancer Res., January 1, 2004; 10(1): 346S - 354S. [Abstract] [Full Text] [PDF]
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), TKI (1 µM, 
), TAM (0.1 µM, 
), or TAMTKI cotreatment (0.1 µM plus 1 µM, respectively, x). Results are expressed as mean (± SD) of triplicate cell counts. *, P < 0.001 vs. CON; **, P = 0.039 and ***, P < 0.001 vs. TAM. B, Emergence of resistant growth with TAM at wk 5. C, Absence of resistant growth with TAMTKI at wk 5. Scale bar, 40 µm.
