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Mol Cancer Ther. 2006;5:3023-3031
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor–positive human breast cancer cells

Matthew H. Herynk1, Amanda R. Beyer1, Yukun Cui1, Heidi Weiss1, Elizabeth Anderson2, Tim P. Green2 and Suzanne A.W. Fuqua1

1 Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas; and 2 AstraZeneca, Cheshire, United Kingdom

Requests for reprints: Suzanne A.W. Fuqua, Breast Center, Baylor College of Medicine, One Baylor Plaza, Mailstop 600, Houston, TX 77030. Phone: 713-798-1671; Fax: 713-798-1673. E-mail: sfuqua{at}bcm.edu

Abstract

It has long been appreciated that estrogenic signaling contributes to breast cancer progression. c-Src is also required for a number of processes involved in tumor progression and metastasis. We have previously identified the K303R mutant estrogen receptor {alpha} (ER{alpha}) that confers hypersensitivity to low levels of estrogen. Because ER{alpha} and c-Src have been shown to interact in a number of different systems, we wanted to evaluate the role of c-Src kinase in estrogen-stimulated growth and survival of ER{alpha}-positive breast cancer cells. MCF-7 cells stably expressing the mutant receptor showed increased c-Src kinase activity and c-Src tyrosine phosphorylation when compared with wild-type ER{alpha}-expressing cells. A c-Src inhibitor, AZD0530, was used to analyze the biological effects of pharmacologically inhibiting c-Src kinase activity. MCF-7 cells showed an anchorage-dependent growth IC50 of 0.47 µmol/L, which was increased 4-fold in the presence of estrogen. In contrast, cells stably expressing the mutant ER{alpha} had an elevated IC50 that was only increased 1.4-fold by estrogen stimulation. The c-Src inhibitor effectively inhibited the anchorage-independent growth of both of these cells, and estrogen was able to reverse these effects. When cells were treated with suboptimal concentrations of c-Src inhibitor and tamoxifen, synergistic inhibition was observed, suggesting a cooperative interaction between c-Src and ER{alpha}. These data clearly show an important role for ER{alpha} and estrogen signaling in c-Src–mediated breast cancer cell growth and survival. Here, we show that c-Src inhibition is blocked by estrogen signaling; thus, the therapeutic use of c-Src inhibitors may require inhibition of ER{alpha} in estrogen-dependent breast cancer. [Mol Cancer Ther 2006;5(12):3023–31]

Introduction

Estrogen signaling has long been known to stimulate a variety of effects, including cell growth and survival, two important mechanisms in the progression of cancer. These effects are mediated through cognate receptors of estrogen, estrogen receptors (ER) {alpha} and ß. Although ER{alpha} is required for normal mammary gland development (1), work with knockout mice has not shown a requirement for ERß in mammary gland development (2). Interestingly, 70% of breast tumors express ER{alpha} (3, 4), making the estrogen pathway an effective target for a large number of clinical therapeutics.

We have previously identified an ER{alpha} A908G somatic mutation in one third of premalignant breast lesions (5), and others have found the mutation at a low frequency in invasive breast cancers (6). The resulting A908G transition leads to the substitution of the 303 lysine for an arginine (K303R; ref. 5); this lysine is an important acetylation site in ER{alpha} (7) and is involved in regulation of the S305 protein kinase A phosphorylation site (8). Furthermore, this mutated receptor has reduced binding to corepressors and increased binding to coactivators, which may play a role in its hypersensitivity to estrogen signaling (5). Although the K303R-ER{alpha} mutation is one of many receptor variants that have been identified (for a complete review, see ref. 9), it is the only somatic mutation that has been identified in a large number of patient samples.

The nonreceptor tyrosine kinase c-Src has been implicated in a number of different cancers, including breast, colon, and pancreatic cancers (for a complete review, see ref. 10). A number of different groups have shown that c-Src protein is up-regulated in approximately half of breast cancers versus normal mammary epithelium (11, 12). Additionally, transgenic mice expressing a mouse mammary tumor virus v-Src develop mammary hyperplasias (13), whereas knocking out the c-Src gene delays mammary tumorigenesis of polyoma middle T antigen–induced tumors (14). In contrast, knockout of the related c-Yes proto-oncogene did not affect mammary tumorigenesis in this model (14). Collectively, these data show an important and specific role for c-Src in mammary tumor development.

In 1993, Miggliaccio et al. (15) showed that estrogen stimulation of MCF-7 breast cancer cells led to an immediate tyrosine phosphorylation and activation of the c-Src kinase. It was later shown that estrogen stimulation can activate a number of other classic signal transduction pathways, such as phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 (16, 17), and that ligand-induced ER{alpha} activation recruited a c-Src/p85/ER{alpha} complex (16, 18). In addition to ER{alpha} signaling to c-Src, c-Src can phosphorylate ER{alpha} on tyrosine 537 (19). Because of the two-way crosstalk between these two proteins, the aberrant activation of either protein could lead to feedback loops that could significantly influence cellular signaling.

Estrogen signaling stimulates growth and survival in ER{alpha}-positive breast cancer cells, and these mechanisms may involve the non–receptor tyrosine kinase c-Src. We thus sought to determine if blockade of c-Src kinase activity could inhibit estrogen-stimulated growth and survival of ER{alpha}-positive breast cancer cells. AZD0530 is a novel, potent, and highly selective Src/Abl kinase inhibitor (20). Here, we show that MCF-7 breast cancer cells respond to c-Src inhibition with reduced anchorage-dependent growth and reduced ability to form colonies in soft agar. Additionally, in the presence of estrogen, cells are less sensitive to c-Src inhibition. We also show that cells expressing the hypersensitive K303R-ER{alpha} mutation have elevated c-Src kinase activity and are less sensitive to c-Src inhibition both in the presence and absence of estrogen when compared with cells expressing only the wild-type (WT) ER{alpha}. When suboptimal concentrations of AZD0530 and tamoxifen were tested, synergistic inhibition of anchorage-independent growth was observed. Collectively, these data show that inhibition of c-Src may be an effective therapeutic strategy for ER{alpha}-positive breast cancer; however, estrogen signaling may alter the ability of c-Src inhibitors to block cell growth.

Materials and Methods

Chemicals
The c-Src inhibitor AZD0530 was provided by AstraZeneca and dissolved in DMSO (20). AZD0530 was diluted in tissue culture medium, and the corresponding DMSO controls had no effect on the analyses done. G418 and all cell culture reagents were purchased from Life Technologies (Grand Island, NY). Tamoxifen (4-hydroxytamoxifen) was purchased from Sigma (St. Louis, MO) and the stock solution was dissolved in ethanol at 10–3 mol/L.

Cell Culture Conditions
MCF-7 breast cancer cells were maintained on plastic in MEM supplemented with 10% fetal bovine serum (FBS), 0.1 mmol/L nonessential amino acids, 2 mmol/L L-glutamine, 50 units/mL penicillin/streptomycin (Life Technologies), at 37°C with 5% CO2/95% air. Unless otherwise noted, cells were passaged and removed from flasks when 70% to 80% confluent. For cell passage, cells were rinsed with PBS and trypsinized in 0.05% trypsin and 0.02% EDTA for 2 min at 37°C. Trypsin activity was quenched with the addition of medium containing 10% FBS.

Generation of Stable WT ER{alpha} and K303R-ER{alpha}–Expressing Cells
Generation of the yellow-fluorescent protein (YFP)–tagged expression constructs, YFP-WT ER{alpha} and YFP-K303R-ER{alpha}, has been previously described (8). MCF-7 cells were stably transfected using Fugene according to the manufacturer's instructions (Roche, Indianapolis, IN), and individual clones were isolated and expanded with G418 selection. Stably transfected clones were screened for expression of the exogenous ER{alpha} by immunoblot analysis. One clone stably expressing YFP-WT ER{alpha} and one clone stably expressing YFP-K303R-ER{alpha} with similar levels of the exogenous YFP-ER{alpha} and endogenous ER{alpha} were chosen for further analysis.

Immunoprecipitation and Immunoblot Analysis
Cells were plated in six-well dishes for immunoblot analysis (3 x 105 per plate) or in 10-cm plates for immunoprecipitation (3 x 106 per plate) in MEM supplemented with 10% FBS. Cells were then incubated overnight at 37°C in 5% CO2, followed by 48 h starvation in phenol red–free MEM with 5% charcoal-stripped FBS. Moreover, cells were pretreated with varying concentrations of AZD0530 for 30 min and 10–9 mol/L estrogen for 10 min as indicated before lysis [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 2% NP40, 0.25% deoxycholic acid, 1 mmol/L EDTA, 1 mmol/L Na3VO4, and 1:200 protease inhibitor cocktail tablet; Roche]. c-Src immunoprecipitation was done using 300 µg of total cellular protein with 1 µg anti-Src antisera (clone 327, Oncogene Research Products, Cambridge, MA) and protein A/G (Oncogene Research Products) with rotation at 4°C for 3 h. For coimmunoprecipitation experiments, we used 2 mg of total cellular protein and 2 µg of anti-Src antisera (clone 327) or 2 µg of anti-ER{alpha} (H-184, Santa Cruz Biotechnology; San Diego, CA) and protein A/G with rotation at 4°C for 2 h. Immunoprecipitated proteins were washed thrice with lysis buffer.

Proteins, either immunoprecipitated or whole-cell lysate, were heated in Laemmli's sample buffer for 5 min, separated by 7.5% SDS-PAGE, transferred to nitrocellulose membrane (Whatman, Inc., Sanford ME), and probed with the following antisera, as indicated: anti-Src antisera (clone GD11, 1:1,000) and anti-phosphotyrosine antisera (4G10, 1:5,000) from Upstate Biotechnology Inc. (Lake Placid, NY); anti–phospho-Fak antisera (pY861, 1:1,000) and anti–phospho-c-Src antisera (pY418, 1:1,000) from Biosource (Camarillo, CA); anti-Fak antisera (clone 77, 1:1,000) from BD Biosciences (San Jose, CA); anti-tubulin antisera (clone D66, 1:1,000) from Sigma-Aldrich (St. Louis, MO); and anti-ER{alpha} (clone 6F1, 1:1,000) from NovaCastra (Newcastle upon Tyne, United Kingdom). Antisera were diluted in TBST (TBS/0.1% Tween 20, v/v) with 5% dried milk or 5% bovine serum albumin. Peroxidase-conjugated secondary antisera, goat anti-mouse anti-serum (1:3,000; Amersham Biosciences; Piscataway, NJ), and goat anti-rabbit anti-serum (1:3,000; Amersham Biosciences) were used to detect the respective primary antibodies, and immunoreactive proteins were visualized with ECL chemiluminescence technology (Alpha Innotech, San Leandro, CA). Autoradiographic protein levels were quantified in the linear range of the film by scanning the image using a Canon LicoScan scanner and analyzing with the Scion Image software program (Scion Corp., Frederick, MD). Each sample measured was calculated as the ratio of the average area of the phosphorylated protein over the average area of the respective total protein levels.

Cell Proliferation as Measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Analysis
Cells were serum-starved for 48 h in phenol red–free MEM with 5% charcoal-stripped FBS before plating. Cells (3,000 per well) were plated into each well of a 96-well plate and allowed to adhere for 8 h before the addition of increasing concentrations of AZD0530 and estrogen (10–9 mol/L). Seventy-two hours later, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT; thiazolyl blue) was added to each well and incubated at 37°C and 5% CO2 for 2 h followed by medium removal and solubilization in 100 µL DMSO. The resulting color change was read at 570 nm and calculated as absorbance above background. Each experiment contained 12 different doses of AZD0530 in quadruplicate. A minimum of three experiments was combined for IC50 calculations. The absorbance readings were used to determine the IC50 using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA). Briefly, values were log-transformed, then normalized, and nonlinear regression analysis was used to generate a sigmoidal dose-response curve to calculate IC50 values for each cell line.

Agarose Colony-Forming Assay
Cells (5,000 per well) were plated in 1.5 mL of 0.3% agarose, 5% charcoal-stripped FBS in phenol red-free MEM, with a 0.6% agarose base in six-well plates, with various concentrations, as indicated, of AZD0530, tamoxifen, and estrogen. Cells were then incubated at 37°C and 5% CO2 with fresh medium added every 3 days. After 14 days, 150 µL of MTT were added to each well and allowed to incubate at 37°C for 4 h. Plates were then placed in 4°C overnight and colonies >50 µm in diameter were quantitated.

Statistical Analysis Tests for Synergy
To determine if suboptimal concentrations of tamoxifen and AZD0530 synergistically inhibit cell growth, a model-free test for synergy was used based on two simultaneous one-sided t tests comparing each of the single-agent doses of AZD0530 (1 µmol/L) and tamoxifen (10–8 mol/L) with a combined regimen of the two drugs at suboptimal doses (21). Additionally, dose levels of AZD0530 (0, 0.1, and 1 µmol/L) and tamoxifen (0, 10–9, and 10–8 mol/L) were used in an ANOVA model to assess the effect of AZD0530 or tamoxifen alone and the interaction between the two drugs.

c-Src Immunocomplex Kinase Assay
Cells (3 x 106) were plated in 10-cm plates with MEM supplemented with 10% FBS and incubated for 24 h at 37°C and 5% CO2. Cells were then serum-starved for 48 h in phenol red–free MEM supplemented with 5% charcoal-stripped FBS followed by treatment with 10–9 mol/L estrogen for 10 min before lysis in 50 mmol/L HEPES (pH 7.0), 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L NaPPi, 1% glycerol (v/v), 0.1% Triton X-100 (v/v), 1 mmol/L Na3VO4, and 1:200 protease inhibitor cocktail tablet (22). Immunocomplex kinase assays were done on c-Src immunoprecipitated from 500 µg of total cellular protein with 1 µg anti-Src antisera (clone 327) and 50 µL of 10% (w/v) Pansorbin cells (Calbiochem, San Diego, CA) with rotation at 4°C for 2 h. Immunoprecipitated pellets were washed thrice with immunocomplex kinase assay wash buffer [0.1% Triton X-100, 150 mmol/L NaCl, and 10 mmol/L NaPO4 (pH 7.0); ref. 22]. Ten microliters of 100 µg/mL acid–denatured rabbit muscle enolase (Sigma) was added as an exogenous phosphorylation substrate before the addition of kinase reaction buffer containing 20 mmol/L HEPES (pH 7.0), 6 mmol/L magnesium chloride, 20 mmol/L sodium orthovanadate, and 10 µCi per reaction of [{gamma}-32P]ATP (3,000 Ci/mmol; NEN, Boston, MA). Reactions were initiated by addition of 50 µL of kinase reaction buffer and allowed to proceed for 10 min at 23°C. The reaction was stopped by the addition of 3x Laemmli's sample buffer. For analysis of c-Src after kinase assay, samples were subjected to 8% SDS-PAGE followed by 1 h fixation in methanol/water/acetic acid (5:5:1). The gel was dried and radioactive bands were detected by autoradiography and quantitated on a Bio-Rad Personal FX PhosphorImager (Bio-Rad, Hercules, CA).

Results

c-Src Binds Similar Amounts of the K303R Mutant ER{alpha} and WT ER{alpha}
Previous reports have shown that c-Src and the ER{alpha} interact in a protein complex (16, 18). Therefore, we first sought to determine if the K303R-ER{alpha} mutation alters the ability of ER{alpha} to bind with c-Src. MCF-7 cells were treated with or without 10–9 mol/L estrogen for 10 min and then lysed. ER{alpha} (Fig. 1A and B, lanes 1 and 2 ) or c-Src (Fig. 1A and B, lanes 3 and 4) was subjected to immunoprecipitation followed by immunoblot for c-Src (Fig. 1A) and ER{alpha} (Fig. 1B). In the absence of estrogen, ER{alpha} and c-Src are in a protein complex (Fig. 1A and B, lanes 1 and 3). Treatment with estrogen did not increase the amount of protein in the complex (Fig. 1A and B, lanes 2 and 4). Similar amounts of starting protein are shown in the whole-cell lysates in Fig. 1A and B (lanes 5 and 6). These data show that ER{alpha} and c-Src are in a hormone-independent complex that was not altered by short-term treatment with estrogen. Figure 1C compares cells stably expressing YFP-WT ER{alpha} vector or YFP-K303R-ER{alpha} vector. Shown in Fig. 1C (top) is the immunoprecipitation with anti-ER{alpha} antisera followed by immunoblot for c-Src (lanes 1–4) compared with whole-cell lysate input (lanes 5–8). Basal levels of c-Src and ER{alpha} binding were similar in WT ER{alpha}–expressing cells when compared with K303R-ER{alpha}–expressing cells (Fig. 1C; lane 1 versus lane 3). Additionally, short-term estrogen treatment did not alter the binding of either WT ER{alpha} or K303R-ER{alpha} with c-Src (Fig. 1C; lane 2 versus lane 4). The bottom panel shows that similar amounts of ER{alpha} protein were immunoprecipitated under all conditions tested. Cells expressing the YFP-ER{alpha} express a 66 kDa (endogenous ER{alpha}) and an ~96 kDa ER{alpha} representing the exogenously added YFP-ER{alpha} as shown in Fig. 1C (bottom). These results confirm that ER{alpha} forms a complex with c-Src that is independent of hormone treatment and that the K303R-ER{alpha} mutation does not seem to alter this complex.


Figure 1
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  		Figure 1. c-Src binds similar amounts of WT and K303R-ER{alpha}. A and B, MCF-7 cells were serum-starved, then treated with or without 10–9 mol/L estrogen (E2) for 10 min before lysis. ER{alpha} (lanes 1 and 2) or c-Src (lanes 3 and 4) was immunoprecipitated (IP) and immunoblotted (IB) for c-Src (A) or ER{alpha} (B). One representative experiment of three. C, MCF-7 cells stably overexpressing WT ER{alpha} (YFP-WT ER{alpha} vector) or the mutant K303R-ER{alpha} (YFP-K303R-ER{alpha} vector) were serum-starved, then treated with or without 10–9 mol/L estrogen for 10 min before lysis. Immunoprecipitated ER{alpha} (lanes 1–4) and whole-cell lysates (lanes 5–8) that have been immunoblotted for c-Src (top) and ER{alpha} (bottom). One representative experiment of two.

 
c-Src Kinase Activity Is Elevated in K303R-ER{alpha}–Expressing Cells
To determine if cells expressing WT ER{alpha} or the mutant ER{alpha} have similar levels of c-Src kinase activity, we next analyzed the c-Src kinase activity and tyrosine phosphorylation of the c-Src 416 residue. A c-Src–specific in vitro immunocomplex kinase assay was done as previously described (22) and a representative assay is shown in Fig. 2A . In the absence of estrogen, WT ER{alpha}–expressing cells showed a low basal level of c-Src kinase activity as shown by phosphorylation of the exogenous substrate enolase (Fig. 2A, lane 1). When cells were treated with estrogen for 10 min, kinase activity was increased by 15% (Fig. 2A, lane 2). In contrast, K303R-ER{alpha}–expressing cells showed an elevated basal level of c-Src activity that was 30% higher (Fig. 2A, lane 3) when compared with WT ER{alpha}–expressing cells. This elevated kinase activity was not further increased by estrogen treatment. To confirm the elevated c-Src kinase activity in K303R-ER{alpha} versus WT ER{alpha}–expressing cells, c-Src was immunoprecipitated and the levels of pY416 and total pY content of c-Src was examined using immunoblot (Fig. 2B). Treatment with estrogen resulted in a 1.7-fold increase in phosphorylation at Y416, and a 1.7-fold increase in total tyrosine phosphorylation of c-Src in WT ER{alpha}–expressing cells (Fig. 2B; lane 1 versus lane 2). Consistent with its observed increased basal kinase activity, K303R-ER{alpha}–expressing cells exhibited a 1.4-fold increase in basal c-Src pY416 and a 1.7-fold increase in total phosphorylation (Fig. 2B, lane 3). The elevated basal c-Src phosphorylation in mutant cells was not further increased by estrogen treatment (Fig. 2B; lane 3 versus lane 4). Thus, whereas WT ER{alpha}–expressing cells increased c-Src kinase activity following estrogen treatment, K303R-ER{alpha}–expressing cells showed constitutive c-Src kinase activity and phosphorylation.


Figure 2
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  		Figure 2. c-Src kinase activity and phosphorylation in WT and K303R-ER{alpha}–expressing MCF-7 cells. WT ER{alpha} or K303R-ER{alpha}–expressing cells were serum-starved, then treated with or without 10–9 mol/L estrogen for 10 min before analysis. A, c-Src was immunoprecipitated and an immunocomplex kinase assay was done to analyze c-Src–induced phosphorylation of the exogenous substrate enolase. Quantitation of 32P-labeled enolase was done on a Bio-Rad Personal FX PhosphorImager. One representative experiment of three. B, c-Src was immunoprecipitated and immunoblotted for pY416, total tyrosine phosphorylation, and c-Src. Numbers, fold change relative to control (lane 1) as described in Materials and Methods. One representative experiment of two.

 
Inhibition of c-Src Tyrosine Phosphorylation
The pharmacologic inhibitor of c-Src, AZD0530 (20), was used to inhibit c-Src phosphorylation and activity. To determine an effective dose of the c-Src inhibitor, MCF-7 cells were treated with increasing amounts of AZD0530 for 30 min because we wanted to analyze short-term signaling events associated with c-Src inhibition. Whole-cell lysates were analyzed for phosphorylation of Y416 of c-Src and total c-Src. In the absence of estrogen, AZD0530 caused a 30% reduction of phosphorylation at Y416 of c-Src versus controls even at concentrations as low as 10 nmol/L (Fig. 3A ; lane 1 versus lane 2). Higher doses of the c-Src–specific inhibitor caused a complete ablation of phosphorylation at Y416 of c-Src (Fig. 3A, lanes 3–7). Even in the presence of estrogen, AZD0530 caused a similar reduction in c-Src phosphorylation (Fig. 3A, lane 8). Thus, estrogen did not significantly alter the dose of c-Src inhibitor required to inhibit c-Src phosphorylation. To show that the reduced c-Src tyrosine phosphorylation correlated with reduced c-Src kinase activity, a downstream target of c-Src, Fak, was analyzed for Y861 of Fak. Levels of pY861 of Fak correlates with the levels of pY416 of c-Src (Fig. 3A, middle), and doses as low as 10 nmol/L of AZD0530 caused a marked reduction in Fak phosphorylation. Estrogen did not affect the ability of pYFak to be reduced by AZD0530. Levels of tubulin (bottom) show equivalent loading. These data confirm that inhibition of c-Src pY416 levels reduced c-Src kinase activity and that AZD0530 was an effective inhibitor of c-Src kinase activity. Additionally, we show that pharmacologic inhibition of c-Src signaling is not altered by short-term estrogen treatment.


Figure 3
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  		Figure 3. Inhibition of c-Src phosphorylation and downstream signaling. A, MCF-7 cells were serum-starved and pretreated with increasing concentrations of AZD0530, as indicated, for 30 min followed by 10 min with (+E2) or without (–E2) estrogen. B, WT ER{alpha} or K303R-ER{alpha} stably expressing cells were serum-starved and pretreated with AZD0530 for 30 min followed by 10 min with or without estrogen. Whole-cell lysates were immunoblotted for pY416 of c-Src and pY861 of Fak as a surrogate marker for c-Src kinase activity. Quantitation shown is the fold difference in phosphorylation relative to control conditions (lane 1). One representative experiment of two is shown for each panel.

 
K303R-ER{alpha}–expressing cells showed constitutively activated c-Src kinase; thus, we wanted to determine if increased c-Src inhibitor would be required to block c-Src signaling in WT ER{alpha} versus K303R-ER{alpha}–expressing cells. WT ER{alpha}– and WT ER{alpha}–expressing cells were pretreated with 0.01 or 0.1 µmol/L of AZD0530 for 30 min followed by 10 min of estrogen treatment. Cells were lysed, separated by SDS-PAGE, and whole-cell lysates were analyzed by immunoblot. Phosphorylation of c-Src at Y416 was partially blocked with 0.01 µmol/L and completely inhibited with 0.1 µmol/L AZD0530 (Fig. 3B, lanes 1 versus lanes 2 and 3). Even with estrogen treatment, 0.1 µmol/L of the inhibitor blocked 40% of the c-Src phosphorylation (Fig. 3B, lanes 4 versus lanes 5 and 6). K303R-ER{alpha}–expressing cells also showed reduced c-Src pY416 in the presence of AZD0530 (Fig. 3B, lanes 7 versus lanes 8 and 9) that was not affected by estrogen treatment (Fig. 3B, lanes 10 versus lanes 11 and 12). The estrogen-induced increase in phosphorylation of c-Src shown by immunoblot of whole-cell lysates confirms the phosphorylation increase shown by immunoprecipitation and immunoblot analysis in Fig. 2B. Immunoblot of whole-cell lysates allowed us to correlate downstream signaling of c-Src kinase with c-Src tyrosine phosphorylation. Immunoblot of Fak showed reduced tyrosine phosphorylation following treatment with AZD0530 (Fig. 3B) and correlated with reduced pY416 levels showing reduced c-Src kinase activity in WT ER{alpha}– and K303R-ER{alpha}–expressing cells. These data show that c-Src inhibitors can effectively block constitutive c-Src phosphorylation and kinase activity in WT and mutant ER{alpha}–expressing breast cancer cells.

c-Src Inhibition of Anchorage-Dependent Growth
It is well known that estrogen treatment stimulates mitogenesis and cell growth in ER{alpha}-positive breast cancer cells. Because estrogen did not alter the ability of AZD0530 to block c-Src phosphorylation, we sought to examine the effectiveness of c-Src on estrogen-stimulated cell growth. We first tested the ability of AZD0530 to block anchorage-dependent growth; the IC50 values of multiple cell lines treated with estrogen are shown in Table 1 . All cells showed a dose-dependent growth inhibition induced by AZD0530 (data not shown). MCF-7 cells exhibited an IC50 of 0.47 µmol/L, and the IC50 was significantly increased 4.5-fold (P < 0.001, t test) in the presence of 10–9 mol/L estrogen (Table 1). Additionally, WT ER{alpha}–expressing cells also showed an IC50 of 0.45 µmol/L, which was not statistically significant compared with the parental MCF-7 cells (P > 0.1, t test). This IC50 was increased 4-fold in the presence of estrogen (P < 0.001, t test). Thus, estrogen signaling results in the requirement for higher concentrations of the c-Src inhibitor to block cell growth. K303R-ER{alpha}–expressing cells exhibited a higher basal IC50 value that was significantly higher than MCF-7 or WT ER{alpha}–expressing cells (P < 0.01, t test). The addition of estrogen to mutant expressing cells increased the IC50 value by only 1.4-fold (Table 1). Thus, when cells are expressing the mutant ER{alpha}, and hence have increased basal c-Src kinase activity, higher levels of c-Src inhibitor were required to reduce anchorage-dependent growth. In the ER{alpha}-negative MCF-7 subline, C4-12-5 (23), estrogen did not affect the concentration of inhibitor required for growth blockade (P = 0.45, t test; Table 1). This result shows that estrogen signaling through the ER{alpha} is required to increase the amount of AZD0530 to effect cell growth. Higher concentrations of AZD0530 may be needed to affect the K303R-ER{alpha} mutant. These results predict that the presence of estrogen in ER{alpha}-positive cells or expression of the K303R-ER{alpha} mutant may alter the efficacy of c-Src inhibitors.


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  		Table 1. IC50 of AZD0530 for various breast cancer cell lines on anchorage-dependent growth

 
Estrogen Treatment Reduces the Ability of AZD0530 to Inhibit Anchorage-Independent Growth
Increased signaling through ER{alpha} decreases the ability of the AZD0530 c-Src inhibitor to reduce anchorage-dependent growth, and c-Src inhibitors have previously been shown to reduce HER2 and c-Met–mediated anchorage-independent growth (24, 25). We next wanted to determine whether inhibition of c-Src would also reduce estrogen-induced, anchorage-independent growth. Cells were grown in 0.3% soft agar with varying concentrations of AZD0530 added as described in Materials and Methods. Under basal conditions, estrogen treatment stimulated a 1.5-fold increase in the ability of MCF-7 cells to form colonies in soft agar (P < 0.01, t test; Fig. 4A ). Inhibition of c-Src kinase activity caused a dose-dependent reduction in soft-agar colony formation as shown in Fig. 4A. Although 1 µm was effective at reducing both basal and estrogen-stimulated colony formation (P < 0.01, t test), 3 µm of AZD0530 completely abolished the ability of MCF-7 cells to form colonies in soft agar (P < 0.001, t test; Fig. 4A). Thus, in the presence of estrogen, more AZD0530 was required to block soft-agar colony-forming ability.


Figure 4
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  		Figure 4. c-Src inhibition reduces anchorage-independent growth in a dose-dependent manner. A, MCF-7 cells. B, WT ER{alpha} or K303R-ER{alpha} stably expressing cells. One representative experiment of two. Cells (5,000 per well) were seeded in 0.3% agarose with increasing concentrations of AZD0530 with (black columns) or without (white columns) 10–9 mol/L estrogen. Cells were allowed to grow for 14 d and the number of colonies >50 µm were quantitated and graphed. One representative experiment of four (A) and one representative experiment of two (B).

 
WT ER{alpha}– or K303R-ER{alpha}–expressing cells were also tested for their ability to form colonies in soft agar (Fig. 4B). In the absence of AZD0530, basal growth was not statistically different between WT ER{alpha}– and K303R-ER{alpha}–expressing cells. Estrogen stimulated a 2.3-fold increase in colony formation in WT ER{alpha} cells (P < 0.01, t test), but a 4-fold increase in K303R-ER{alpha}–expressing cells (P < 0.01, t test; Fig. 4). Similar to MCF-7 cells, both WT ER{alpha}– and K303R-ER{alpha}–expressing cells showed a dose-dependent reduction in soft-agar colony formation following c-Src inhibition (Fig. 4B), and the presence of estrogen required more inhibitor to block soft-agar colony formation. These data show that c-Src inhibition blocked anchorage-independent growth of breast cancer cells and that more drug was required to block cell growth in the presence of estrogen in K303R-ER{alpha}–expressing breast cancer cells.

Recently, Hiscox et al. (26) reported that AZD0530 blocked invasion and migration of tamoxifen-resistant MCF-7 cells. Therefore, we next determined if c-Src inhibition combined with tamoxifen could synergistically inhibit the anchorage-independent growth of MCF-7 cells. Figure 5 (columns 1–5 ) again shows a dose-dependent reduction in soft-agar colony-forming ability, confirming our previous results. Additionally, tamoxifen treatment also showed a dose-dependent reduction in soft-agar colony-forming ability (Fig. 5, columns 6–8). Because 3 µmol/L AZD0530 and 10–7 mol/L tamoxifen were each >90% effective at inhibiting the anchorage-independent growth of MCF-7 cells, we next tested suboptimal doses of these drugs in combination to determine if additive or synergistic effects could be achieved. Growing cells in the presence of 10–8 mol/L tamoxifen almost completely inhibited basal colony formation but not estrogen-induced colony formation (Fig. 5, column set 7). Tamoxifen (10–9 mol/L) alone only inhibited 34% of basal growth, and 24% of estrogen-induced growth (Fig. 5). Low concentrations (0.1 and 1 µmol/L) of AZD0530 alone gave 36% and 70% inhibition, respectively, when used alone (Figs. 4A and 5), and these doses of AZD0530 completely inhibited basal colony formation in combination with low doses (10–8 or 10–9 mol/L) of tamoxifen (Fig. 5, columns 9–12).


Figure 5
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  		Figure 5. Synergistic cell growth inhibition with AZD0530 and tamoxifen. MCF-7 cells (5,000 per well) were seeded in 0.3% agarose with increasing concentrations of AZD0530 or tamoxifen (Tam) as controls, or suboptimal concentrations of AZD0530 plus tamoxifen with (black columns) or without (white columns) 10–9 mol/L estrogen. Cells were allowed to grow for 14 d and the number of colonies >50 µm were quantitated and graphed. This experiment was repeated twice with similar results.

 
A model-free test for synergy was used based on two simultaneous one-sided t tests comparing each of the single-agent doses of AZD0530 and tamoxifen with a combined regimen of the two drugs at suboptimal doses (21). Specifically, the comparison between AZD0530 at 1 µmol/L and AZD0530 at 0.1 µmol/L plus tamoxifen (10–9 mol/L) was statistically significant (P < 0.0001). Likewise, the second comparison between tamoxifen at 10–8 mol/L was compared with AZD0530 at 0.1 µmol/L plus tamoxifen at 10–9 mol/L was also found to be statistically significant (P = 0.022), indicating synergistic effects between the two drugs. Finally, estimates of the differences between specific drug combinations versus control groups were calculated based on an ANOVA model with interaction terms between the two drugs. The mean difference in the number of colonies between AZD0530 at 0.1 µmol/L plus tamoxifen at 10–9 mol/L versus the control group was 139.3 (95% confidence interval, 124.1–154.5, P < 0.0001). This effect was more than the combined effect of either drug alone compared with the control group. However, when cells were treated with estrogen, the interactions were not significant. Thus, both AZD0530 and tamoxifen were effective at reducing soft-agar colony formation; but here we show that combinations of these inhibitors were more effective than either inhibitor alone and estrogen was able to block this effect.

Discussion

Estrogen-induced activation of ER{alpha} has been shown to activate the c-Src signaling pathway (17). ER{alpha} and c-Src bind in a protein complex that has been proposed to involve other proteins such as modulator of nongenomic activity of ER and phosphatidylinositol 3-kinase (16, 27, 28). Our studies showed hormone-independent interactions between ER{alpha} and c-Src that were not altered by estrogen or the K303R-ER{alpha} mutation. Although their interaction did not change, K303R-ER{alpha}–expressing cells showed a 30% increase in basal c-Src kinase activity, which was correlated with increased phosphorylation of c-Src. We have previously reported that the K303R-ER{alpha} mutant is hypersensitive to very low levels of estrogen (5), and here we showed that expression of the mutant ER{alpha} led to higher basal c-Src kinase activity in breast cancer cells. Thus, although this mutation in ER{alpha} did not seem to alter the ability of the receptor to bind with c-Src, as has been shown for the TIF-2 coactivator (5), it did alter the ability of the receptor to phosphorylate and activate c-Src.

Although c-Src activity was elevated, this increase was very low, and therefore its effects on the cell could be minimal; thus, a c-Src inhibitor was used to address this question. Anchorage-dependent growth analysis showed that estrogen-stimulated growth required higher concentrations of AZD0530 to achieve the same inhibitory effects seen in the nonstimulated systems. This result was also confirmed in anchorage-independent growth assays. From the anchorage-dependent and anchorage-independent growth data, one would have expected that estrogen stimulation would increase c-Src phosphorylation as it did under basal conditions. However, in our model system, estrogen did not affect inhibitor blockade of c-Src phosphorylation. Estrogen signaling has been shown to activate a number of prosurvival pathways, including Bcl-2 (29), caspase regulation (30), and calcium homeostasis (31) to name a few. Estrogen signaling may be activating alternative survival and growth pathways that can bypass the requirement for c-Src activation, hence an estrogen-induced survival effect. Cells expressing the mutant receptor had an elevated basal IC50; thus, more drug may be required to block the basal in vitro growth rate of K303R-ER{alpha}–expressing cells. Additionally, estrogen only stimulated a <1.5-fold increase in the IC50 required to inhibit the K303R-ER{alpha}–expressing cells. Although the moderately increased c-Src activity in K303R-ER{alpha}–expressing cells was enough to alter the basal biology of the cells in serum-free conditions as shown by the growth assays, it may also raise the basal level of signaling to a threshold level, thereby serving to "prime" the cell for other activating stimuli such as estrogen or growth factor signaling. One such example has been shown in PC12 cells in which chemotactic migration is not stimulated until a threshold level of extracellular signal-regulated kinase 1/2 signaling has been reached (32). Thus, the increased c-Src activity observed in the K303R-ER{alpha}–expressing cells may not have crossed the threshold level required to cause other biological effects, but it may be at the threshold and thus primed for a stimulus.

It has recently been shown that tamoxifen-resistant MCF-7 cells were more sensitive to AZD0530 than their nonresistant counterparts (26). We therefore tested the effect of combining suboptimal doses of tamoxifen and AZD0530 in soft-agar growth assays. Our data showed that the addition of AZD0530 made the cells more sensitive to growth inhibition by tamoxifen. Hiscox et al. (26) also showed that in tamoxifen-resistant epidermal growth factor receptor overexpressing MCF-7 cells, the combination of AZD0530 and the epidermal growth factor receptor inhibitor gefitinib blocked migration and invasion in an additive manner. A number of studies have shown that inhibiting c-Src activity in breast cancer cells blocks estrogen-induced cell cycle progression and mitogenesis (33, 34). Additionally, c-Src inhibitors have also been shown to block estrogen-induced migration in endometrial cells (35) and disrupt adherans junctions in endothelial cells (36). Thus, multiple biological phenotypes required for tumor progression may be converging and signaling through c-Src. It remains to be determined if c-Src inhibitors can inhibit or reverse tamoxifen resistance in vivo. Although these data support investigating the potential therapeutic benefit of c-Src kinase inhibition in women with ER{alpha}-positive breast cancer, a rational approach evaluating the c-Src activity, ER{alpha} levels, and perhaps the K303R mutation status is warranted. Although the influence of circulating estrogen still requires further elucidation, the data suggest that strategies combining drugs that lower estrogen signaling with a c-Src kinase inhibitor could provide a rational approach to treating these patients.

Footnotes

Grant support: DAMD17-03-1-0417 (M.H. Herynk), NIH/National Cancer Institute grants CA58183 and CA72038, and an AstraZeneca grant (S.A.W. Fuqua).

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 7/ 7/06; revised 9/29/06; accepted 10/30/06.

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