Insulin-Like Growth Factor-I Receptor Signaling in Tamoxifen ...

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Endocrinology 146(11):4609 – 4618 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2005-0247

Insulin-Like Growth Factor-I Receptor Signaling in Tamoxifen-Resistant Breast Cancer: A Supporting Role to the Epidermal Growth Factor Receptor J. M. Knowlden, I. R. Hutcheson, D. Barrow, J. M. W. Gee, and R. I. Nicholson Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, United Kingdom There is considerable evidence that the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) cross-talk in breast cancer cells. In the present study, we have examined whether EGFR/IGF-IR cross-talk exists in EGFR-positive tamoxifen-resistant variants of MCF-7 (Tam-R) and T47D (T47D-R) breast cancer cell lines. Although Tam-R cells expressed reduced IGF-IR protein levels compared with their wild-type MCF-7 counterparts, phosphorylated IGF-IR protein levels were equivalent in the two cell lines under basal growth conditions, possibly as a consequence of increased IGF-II expression in Tam-R cells. IGF-II activated both IGF-IR and EGFR in Tam-R cells, whereas only activation of IGF-IR was observed in wild-type cells. In contrast, epidermal growth factor rapidly induced EGFR, but not IGF-IR, phosphorylation in Tam-R cells. IGF-II promoted direct association of cSRC with IGF-IR, phosphorylated c-SRC, and increased EGFR phosphorylation at tyrosine 845, a c-SRC-dependent phos-

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N IMPORTANT GROWTH regulatory pathway active in a variety of cancer types, including breast, uses the IGF-I receptor (IGF-IR) (1–3). This receptor is a member of the type II receptor tyrosine kinase (RTK) family, which also comprises the insulin receptor and exists as a heterotetramer of two ␣- and two ␤-subunits (4). It is activated after ligand binding of insulin, IGF-I, or IGF-II to the ␣-subunit, and, like classical RTKs, receptor stimulation leads to autophosphorylation of tyrosine residues located within the ␤-subunit and recruitment of adaptor proteins such as Src homology collagen and the IR substrate-I (4). These adaptor proteins function, in turn, as intermediates between the receptor and downstream signaling pathways which include MAPK and phosphatidylinositol 3-kinase cascades, which serve to influence key cell survival and proliferation pathways (1, 3, 5, 6). Increased expression and activation of IGF-IR and its associated downstream signaling components have been reported in clinical breast cancer specimens and linked to disease progression and recurrence (7, 8). Furthermore, expression of IGF-IR has been found in most breast cancer cell lines including endocrine-responsive MCF-7 cells (1). In these First Published Online July 21, 2005 Abbreviations: DCCM, Dendritic cell conditioned medium; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FCS, fetal calf serum; IGF-IR, IGF-I receptor; 4-OH-TAM, 4-hydroxytamoxifen; RTK, receptor tyrosine kinase; WT, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

phorylation site. Pretreatment with either AG1024 (IGF-IRspecific inhibitor) or an IGF-II neutralizing antibody inhibited basal IGF-IR, c-SRC, and EGFR phosphorylation, and AG1024 significantly reduced Tam-R basal cell growth. The c-SRC inhibitor SU6656 also inhibited growth, reduced basal and IGF-II-induced c-SRC and EGFR phosphorylation, and blocked EGFR activation by TGF␣. Similarly, in T47D-R cells, AG1024 and SU6656 inhibited basal and IGF-II-induced phosphorylation of c-SRC and EGFR, and SU6656 reduced TGF␣induced EGFR activity. These results suggest the existence of a unidirectional IGF-IR/EGFR cross-talk mechanism whereby IGF-II, acting through the IGF-IR, regulates basal and ligandactivated EGFR signaling and cell proliferation in a c-SRCdependent manner in Tam-R cells. This cross-talk between IGF-IR and EGFR is not unique to Tam-R cells because this mechanism is also active in a tamoxifen-resistant T47D-R cell line. (Endocrinology 146: 4609 – 4618, 2005)

cells, a significant level of productive cross-talk has now been shown to exist with the estrogen receptor (ER). As such, estrogens appear to favor synergistic interactions with IGFs, resulting in an increase in expression of the IGF-IR and growth. Conversely, IGFs prime the activation of several kinases that are able to phosphorylate ER and initiate estrogen response element-mediated gene expression (1, 9 –11). Importantly, the antiestrogen tamoxifen inhibits IGF-I-mediated proliferation in ER-positive breast cancer cells (10 –12). Interestingly, recent evidence suggests a role for IGF-IR signaling in tamoxifen resistance. Increased sensitivity to the proliferative effects of IGF-I/II has been reported in tamoxifen-resistant MCF-7 cell lines after treatment with either estradiol or tamoxifen (13, 14). Furthermore, the selective IGF-IR tyrosine kinase inhibitor AG1024 and the anti-IGF-IR monoclonal antibody ␣IR-3 have both been shown to block growth of tamoxifen-resistant MCF-7 cell variants (13, 15). Epidermal growth factor receptor (EGFR) and c-erbB2, members of the type I RTK family (16), have similarly been implicated in the generation of antihormone resistance in breast cancer. In the clinic, it has been demonstrated that overexpression of EGFR and c-erbB2 is associated with a lack of response to endocrine therapy and a poorer prognosis (17– 20). Furthermore, transfection of either EGFR or c-erbB2 into hormone-sensitive breast tumor cells promotes hormoneindependent growth, and in vitro models of acquired tamoxifen resistance have demonstrated that raised levels of both EGFR and c-erbB2 may contribute to increased proliferative activity (21–24). Indeed, in recent reports, we have demon-

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Endocrinology, November 2005, 146(11):4609 – 4618

strated increased levels of both EGFR and c-erbB2 mRNA and protein expression in two antihormone-resistant MCF-7 breast cancer cell models and have shown that these receptors play a key role in driving resistant cell growth (25, 26). Significantly, there is now growing evidence to suggest that a degree of cross-talk exists between members of the types I and II RTK families and their ligands in several cell models. Studies have highlighted the presence of a direct association between IGF-IR and EGFR in both breast cancer cell lines (27) and normal mammary epithelial cells (28) and between IGF-IR and c-erbB2 in mammary epithelial cancer cells, including MCF-7 cells (29). Support for the presence of indirect mechanisms of cross-talk between IGF-IR and EGFR and their ligands have also been reported in a range of cell lines (30 –35), although evidence for this in breast cancer cell lines remains very limited (36). On the basis that IGF-IR signaling activity is significantly recovered in our in-house tamoxifen-resistant breast cancer MCF-7 cell line (15), and with the knowledge that these cells have increased levels of EGFR protein (15, 25), we attempted, in this study, to further dissect the relative importance of EGFR and IGF-IR signaling in these cells. We have examined potential areas of cross-talk between these two receptors, elucidated the possible mechanisms involved, and assessed whether these mechanisms are also active in another tamoxifen-resistant cell line derived from T47D breast cancer cells. Materials and Methods Development of tamoxifen-resistant MCF-7 and T47D breast cancer cells Human wild-type (WT)-MCF-7 cells were a gift from AstraZeneca Pharmaceuticals (Cheshire, UK), and the tamoxifen-resistant cell line (Tam-R) was developed by continually exposing WT-MCF-7 cells to 4-hydroxytamoxifen (4-OH-TAM; 100 nm) over a period of 6 months (25). WT T47D breast cancer cells, purchased from the American Type Culture Collection (Manassas, VA), were used to generate T47D tamoxifen-resistant cells. After 2 wk of routine maintenance, the T47D cell monolayers were washed with Dulbecco’s PBS, and then 4-OH-TAM (100 nm in ethanol) was added in phenol red-free RPMI medium containing 5% charcoal-stripped steroid-depleted fetal calf serum (FCS), antibiotics, and glutamine (4 mm). Cells were continuously exposed to this treatment regimen for 3 months, during which time the medium was replaced every 4 d, and the cell cultures were passaged by trypsinization after 70% confluency was reached. Cells were seeded into new flasks at a ratio of 1:5 of the confluent cell number. This cell line, designated T47D-R, was cultured for several more months in medium containing 4-OH-TAM before characterization studies.

Cell culture WT-MCF7 cells were routinely cultured in phenol red-free RPMI medium supplemented with 5% FCS plus penicillin-streptomycin (10 IU/ml–10 ␮g/ml) and Fungizone (2.5 ␮g/ml). The Tam-R and T47D-R cell line was grown in phenol red-free RPMI medium containing 5% charcoal-stripped steroid-depleted FCS, antibiotics, glutamine (4 mm), and 4-OH-TAM (100 nm in ethanol). Both cell lines were maintained at 37 C in a humidified 5% CO2 atmosphere. All routine tissue culture medium and constituents were purchased from Life Technologies, Inc. Europe Ltd. (Paisley, UK), and tissue culture plastics were obtained from Nunc (Roskilde, Denmark).

Experimental procedures Western blotting and RT-PCR studies. Each cell line was grown for 4 d to allow the cells to achieve approximately 70% confluence before being

Knowlden et al. • EGFR/IGF-IR Cross-Talk in Tamoxifen Resistance

transferred into phenol red/steroid- and serum-free dendritic cell conditioned medium (DCCM) (Biosynergy Europe, Cambridge, UK) for 24 h. The cells were then lysed to measure basal protein and mRNA expression. To examine the effects of pharmacological agents, cells were lysed after a further incubation in DCCM supplemented with either IGF-II growth factor (100 ng/ml in 10 mm acetic acid/0.1% BSA; R&D Systems, Abingdon, UK), epidermal growth factor (EGF; 10 ng/ml in PBS), TGF␣ (10 ng/ml in PBS), the IGF-IR tyrosine kinase inhibitor AG1024 (20 ␮m in dimethylsulfoxide; Merck Biosciences Ltd., Nottingham, UK) (37), anti-IGF-II neutralizing antibody (0.5–10 ␮g/ml in PBS; R&D Systems, Abingdon, UK), the c-SRC inhibitor SU6656 (1 ␮m in dimethylsulfoxide; Calbiochem) (38), the selective EGFR tyrosine kinase inhibitor, gefitinib (1 ␮m in ethanol; AstraZeneca, Macclesfield, UK) (25), or a combination of these treatments. Controls in all cases were incubated for the same periods of time with or without the appropriate vehicle. All experiments were performed at least three times. Immunocytochemistry studies. Cells were grown on sterile 3-aminopropyltriethoxysilane-coated coverslips at 1 ⫻ 104 cells/cm2 according to the protocol described above. Basal total and phosphorylated IGF-IR expression was assessed by incubation of cell monolayers for 24 h in DCCM before PBS wash and cell fixation according to the immunocytochemical assay being performed.

Growth studies Cell population growth was evaluated by means of trypsin dispersion of the cell monolayers (performed in triplicate) after a 7-d incubation with increasing concentrations of AG1024 (20 ␮m), SU6656 (1 ␮m), gefitinib (1 ␮m), IGF-II (100 ng/ml), or IGF-II in combination with each inhibitor. Controls were incubated for the same period of time with the appropriate vehicle. Cells were then measured using a Coulter counter (Luton, UK). All cell culture experiments were performed at least three times.

Protein cell lysis Cells were washed three times with PBS, and cell lysis was performed as previously described (25). Briefly cells were lysed using 200 ␮l ice-cold lysis buffer [50 mm Tris (pH 7.5), 5 mm EGTA, 150 mm NaCl, and 1% Triton X-100] containing protease inhibitors (2 mm sodium orthovanadate, 200 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 20 ␮m phenylarsine, 10 mm sodium molybdate, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin). The cellular contents were transferred to Eppendorf tubes and clarified by centrifugation at 13,000 rpm for 15 min at 4 C, and supernatant aliquots were stored at ⫺20 C until required. Total protein concentrations were determined using the DC Bio-Rad protein assay kit (Bio-Rad Labs Ltd., Hemel Hempstead, UK).

Immunoprecipitation Cell lysates containing 1 mg protein were immunoprecipitated using 1 ␮g specific antibody, and tubes were incubated on ice for 1 h. Protein A agarose (30 ␮l; Insight Biotechnology Ltd., Wembley, UK) was added to the mixture, and the tubes were placed onto a rotary mixer at 4 C for a further 2 h. The immune complex was centrifuged at 3000 rpm at 4 C for 5 min and washed with ice-cold lysis buffer. This procedure was repeated two more times and the resultant pellet resuspended in 20 ␮l 2⫻ Laemelli sample loading buffer containing 0.01 m dithiothreitol. Samples were boiled at 100 C for 5 min to release the bound proteins before gel loading.

Western blotting Protein samples from either total cell lysates (20 –100 ␮g) or after immunoprecipitation were subjected to electrophoresis separation on a 7.5% polyacrylamide gel and then trans-blotted onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). Blots were blocked at room temperature for 1 h in 5% Western Blocking Reagent (Roche Diagnostics, Mannheim, Germany) made up in TBS-Tween 20 (0.05%) and then incubated for a minimum of 2 h in primary antibody diluted 1/1000 in 5% Western Blocking Reagent/TBS-Tween (0.05%). The membranes were washed three times in TBS-Tween (0.05%) and then incubated for 1 h with secondary IgG horseradish peroxidase-



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labeled donkey antirabbit antibody (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK), diluted 1/10,000 in 5% Western Block Reagent/TBS Tween (0.05%). Detection was performed using West Dura long-duration sensitive chemiluminescent detection reagents (Pierce and Warriner Ltd., Cheshire, UK). Antibodies used were directed against total EGFR SC-03, total IGF-IR SC-712 (Insight Biotechnology Ltd.), phosphorylated EGFR (Y1068), total and phosphorylated ERK1/2 (Cell Signaling Technology, Hertfordshire, UK), phosphorylated IGFIR/IR (Y1158), total and phosphorylated (Y418) c-SRC and phosphorylated EGFR (Y845) (Biosource International, Nivelles, Belgium), ␤-actin (Sigma-Aldrich Co. Ltd.; Poole, Dorset, UK), and specific phosphorylated IGF-IR (Y1316), a kind gift from AstraZeneca.

control) samples were amplified for 40, 37, and 25 cycles using specific primers for IGF-I, IGF-II, and ␤-actin (housekeeping positive control), respectively, and conditions were optimized as described previously (39). Briefly, an initial denaturing step of 95 C for 2 min was followed by a set number of cycles of 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. PCR products were separated on a 3% wt/vol agarose gel, containing ethidium bromide, visualized by UV illumination, scanned, and densitometry values were corrected for ␤-actin. Primers used were ␤-actin, 5⬘ GGA GCA ATG ATC TTG ATC TT and 3⬘ CCT TCC TGG GCA TGG AGT CCT (39); IGF-II, 5⬘ TGG GAA TCC CAA TGG GGA AG and 3⬘ CTT GCC CAC GGG GTA TCT (40); and IGF-I, 5⬘ TGC TCT TCA GTT CGT GTG TG and 3⬘ TGG CAT GTC ACT CTT CAC TC (40).

Immunocytochemical assays

Real-time RT-PCR. Real-time RT-PCR was performed using the Opticon 2 Module (Genetic Research Instrumentations Ltd., Essex, UK). PCRs were set up using a Quantitect SYBR Green PCR Kit (Qiagen Ltd., Crawley, UK) based on the manufacturer’s instructions. Briefly, each reaction mix contained Quantitect SYBR Green PCR Mastermix (HotStartaq DNA polymerase, 1⫻ SYBR Green PCR buffer, SYBR Green 1, ROX, dNTPS, and MgCl2), forward- and reverse-specific primers (as described above, 0.3 ␮m), and sterile pure water. cDNA from experimental samples (equivalent to 0.05 ␮g RNA) or DNA standard was then added to each tube. DNA standards for IGF-II and ␤-actin were prepared from freshly purified PCR-specific products, using a PCR clean up kit (Qiagen Ltd.). Final standard concentrations ranged from 100 – 0.01 pg/␮l and 0.1– 0.00001 fg/␮l for ␤-actin and IGF-II, respectively. All reactions were carried out as described above for semiquantitative PCR except that the initial denaturing step at 95 C was increased to 15 min to allow for the HotStarTaq enzyme to be fully activated. Fluorescence plate readings were collected after each 72 C extension time. For each run, three different samples for both WT-MCF-7 and Tam-R cells were used and amplified in duplicate. The quantities of IGF-II mRNA obtained were then corrected for ␤-actin to normalize the data. After each run, melting curves were also generated to validate product specificity by following the manufacturer’s instructions.

Total IGF-IR. Cover slips were fixed for 5 min in a formal saline/phenol solution. Briefly, coverslips were immersed in formal saline (3.7%) containing phenol (2.5%) at room temperature, rinsed once with ethanol (70%), and then washed two times in PBS-Tween 20 (0.02%) for 5 min. This was followed by incubation overnight at 23 C in total IGF-IR primary antibody (SC-712; Insight Biotechnology Ltd.) diluted 1/125 in PBS. Coverslips were then washed three times for 1 min in PBS, two times for 5 min in PBS-Tween 20 (0.02%) and then incubated for 2 h at room temperature in Rabbit enVision peroxidase-labeled polymer secondary antibody (Dako Ltd., Ely, UK). They were then washed three times for 1 min in PBS, two times for 5 min in PBS-Tween 20 (0.02%), incubated for 6 min at room temperature in enVision DAB Chromagen solution (Dako Ltd.), and then rinsed three times for 3 min in distilled water. The coverslips were then incubated in methyl green (0.5%) for 20 sec as a counterstain, rinsed in distilled water, and allowed to air dry before mountant was applied. Phosphorylated IGF-IR Y1131. Cover slips were fixed in methanol/vanadate/acetone solution. Briefly, coverslips were immersed in methanol containing sodium orthovanadate (2 mm) for 5 min at ⫺10 to ⫺30 C), followed by acetone for 5 min at ⫺10 to ⫺30 C, and then air dried for 20 –30 min. Coverslips were then rinsed once with ethanol (70%), washed two times for 5 min in PBS, and then quickly dipped in PBSTween 20 (0.02%). This was followed by incubation overnight at 37 C in antiphospho IGF-IR/IR (Y1131) primary antibody (Cell Signaling Technology), diluted 1/20 in PBS-Triton (0.4%). Coverslips were then washed three times for 1 min in PBS, two times for 5 min in PBS-Tween 20 (0.02%), and then incubated for 2 h at room temperature in Rabbit enVision peroxidase-labeled polymer secondary antibody. They were then washed three times for 1 min in PBS, two times for 5 min in PBS-Tween 20 (0.02%), incubated for 10 min at room temperature in enVision DAB Chromagen solution, and then rinsed three times for 3 min in distilled water. The coverslips were then counterstained as described above. Phosphorylated IGF-IR Y1316. Cover slips were fixed in paraformaldehyde/vanadate solution. Briefly, coverslips were immersed in paraformaldehyde (2%) containing sodium orthovanadate (2 mm) for 20 min at room temperature, followed by 5-min washes two times in PBS, and then quickly dipped in PBS-Tween 20 (0.02%). This was followed by incubation overnight at 23 C in antiphospho IGF-IR (Y1316) primary antibody (gift from AstraZeneca), diluted 1/50 in PBS-Triton (0.4%). Coverslips were then washed in PBS, incubated in secondary antibody, and detected as described above for IGF-IR Y1131. Evaluation of immunostaining was carried out by two people on an Olympus BH-2 light microscope using a dual-viewing attachment. Membrane and cytoplasmic staining was assessed for total and phosphorylated IGF-IR.

RT-PCR Semiquantitative RT-PCR. Total RNA was isolated from WT-MCF-7, Tam-R, and MDA-231 cells grown under basal conditions, using an RNA isolator kit (Tri Reagent; Sigma Chemical), and 1 ␮g was reverse-transcribed using standard conditions as described previously (39). Sterile water was also used, in place of RNA, as a negative control for RT (⫺RT). Resultant cDNA, ⫺RT negative control, and sterile water (negative PCR

Statistics Overall differences between control and treatment groups were determined by one-way ANOVA. Direct comparisons between control and treatment effects were assessed in Tam-R cells using a post hoc Student’s t test with the Bonferroni adjustment factor. For real-time PCR analysis, an unpaired Student’s t test was performed to compare mRNA levels in both WT-MCF-7 and Tam-R cells. Differences were considered significant at the P ⱕ 0.05 level.

Results IGF-IR expression and activity in WT and tamoxifenresistant MCF-7 cells

RT-PCR analysis of WT-MCF-7 and Tam-R cells revealed that under basal growth conditions, IGF-II, but not IGF-I mRNA, was expressed in each cell line (Fig. 1A), albeit at low levels of expression. When corrected for ␤-actin, however, the levels of IGF-II mRNA appeared slightly higher in the antihormone-resistant cells (Fig. 1B). Quantitative RT-PCR analysis using OPTICON 2 technology confirmed that IGF-II mRNA levels were increased by approximately 3-fold in the Tam-R cells relative to ␤-actin (P ⱕ 0.05; Fig. 1C). Although an examination of the levels of IGF-IR by Western analysis revealed considerably lower levels in the Tam-R cells vs. their endocrine-responsive counterparts, the two cell types showed comparable levels of IGF-IR phosphorylation as shown using both an IGF-IR/IR Y1158 antibody and a specific phospho-IGF-IR Y1316 antibody (Fig. 2A). These data were confirmed by immunohistochemistry, which also showed that the total and activated IGF-IR was membrane associated (Fig. 2B).

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FIG. 1. A, Basal expression levels of IGF-I and IGF-II mRNA expression in WT-MCF-7 and Tam-R cells (MDA-231 cDNA as positive control for IGF-I mRNA expression) assessed after 24 h in serum-free DCCM by semiquantitative RT-PCR analysis. B, After correction for ␤-actin expression. C, Expression of IGF-II mRNA by real-time quantitative RT-PCR, using SyBr Green 1 technology, corrected for ␤-actin expression. Data are representative of three separate mRNA isolations. *, P ⱕ 0.05 vs. WT.

IGF-II activates EGFR in tamoxifen-resistant, but not WT, MCF-7 cells

Incubation of WT-MCF-7 and Tam-R cells with IGF-II (100 ng/ml) induced a rapid increase in IGF-IR Y1158 and Y1316 phosphorylation at 5 min (Fig. 3A), which remained elevated in both cell lines at 30 min. Interestingly, in the Tam-R cells, IGF-IR activation by IGF-II was paralleled by an increase in EGFR Y1068 phosphorylation, with levels peaking at 20 min (Fig. 3A). The effect of IGF-II on EGFR phosphorylation was not observed in the WT-MCF-7 cells (Fig. 3A). Incubation of Tam-R cells with EGF promoted phosphorylation of EGFR at Y1068 but was without effect on IGF-IR Y1158 and Y1316 phosphorylation levels (Fig. 3B). Examination of IGF-IIinduced activation of ERK1/2 revealed an increased phosphorylation at 5 min with levels remaining elevated at 30 min in both cell lines (Fig. 3A). The total levels of IGF-IR, EGFR, and ERK1/2 remained unchanged throughout the time course in all studies (Fig. 3A). IGF-II also promoted growth of Tam-R cells by approximately 15–20% (Fig. 4), and this effect was significantly attenuated in the presence of gefitinib (P ⱕ 0.001, n ⫽ 6) (Fig. 4A). Treatment of Tam-R cells with


FIG. 2. Basal protein expression levels of both phosphorylated and total IGF-IR in WT-MCF-7 and Tam-R cells assessed after 24 h in serum-free DCCM by Western blot (A) and immunocytochemical analysis (B). ␤-Actin levels were assessed to confirm equivalent sample loading. Data are representative of at least three separate experiments for each methodology. Original magnification, ⫻40.

gefitinib alone significantly reduced basal growth by approximately 80% (P ⱕ 0.001, n ⫽ 6) (Fig. 4A). IGF-IR blockade inhibits IGF-IR/EGFR activity in tamoxifen-resistant MCF-7 cells

A 1-h preincubation of Tam-R cells with increasing concentrations of a specific IGF-II neutralizing antibody, progressively reduced the phosphorylation levels of IGF-IR Y1158 and Y1316, EGFR Y1068, and ERK1/2 (Fig. 5A). A time course for this neutralizing antibody, alongside an appropriate isotype-matched control antibody, was initially performed. Optimal inhibition of IGF-IR activity was observed at 1 h, with the neutralizing antibody with no effect of the isotype-matched control being noted at this time point (data not shown). Inhibition of IGF-IR Y1158 and Y1316 phosphorylation by the IGF-II neutralizing antibody could not be overcome by the inclusion of IGF-II (Fig. 5B). The antibody had no effect on the total expression levels of these proteins. Similar effects were observed after the 24-h preincubation of the Tam-R cells with 20 ␮m AG1024. At this concentration, AG1024 markedly inhibited IGF-IR Y1158 and Y1316, EGFR Y1068, and ERK1/2 phosphorylation in both basal and IGFII-primed Tam-R cells (Fig. 5C). Again, a time course to this agent was initially performed with a 24-h incubation with AG1024 being required to observe significant inhibition of IGF-IR phosphorylation (data not shown). Again, total levels of IGF-IR, EGFR, and ERK1/2 remained unchanged for all



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FIG. 3. Western analysis of phosphorylated and total IGF-IR, EGFR, and ERK1/2 protein expression assessed after incubation of WTMCF-7 and Tam-R cells in serum-free DCCM supplemented with IGF-II (100 ng/ml) for 0 –30 min (A) and phosphorylated and total IGF-IR and EGFR protein expression assessed after incubation of Tam-R cells in serum-free DCCM supplemented with either IGF-II (100 ng/ml) for 20 min or EGF (10 ng/ml) for 5 min (B). Data are representative of three separate experiments.

treatment groups. Furthermore, AG1024 significantly reduced basal tumor cell growth by approximately 85% on d 7 (P ⱕ 0.001 vs. control, n ⫽ 5), and this inhibition was maintained in the presence of IGF-II (P ⱕ 0.001 vs. IGF-II alone, n ⫽ 4, Fig. 4B). IGF-II promotes association of c-SRC with both EGFR and IGF-IR in tamoxifen-resistant MCF-7 cells

Under both basal and IGF-II-primed conditions, immunoprecipitation studies provided no evidence of a direct physical association of IGF-IR with EGFR in Tam-R cells (Fig. 6A). Therefore, alternative indirect mechanisms for IGF-IIinduced EGFR phosphorylation were investigated. Immunoprecipitation/Western-blot studies revealed that total c-SRC physically associated with IGF-IR in Tam-R cells, and this association was increased after IGF-II stimulation, as shown in Fig. 6B, whereas total IGF-IR levels remained unchanged. Immunoprecipitation/Western-blot studies also revealed that total c-SRC physically associated with EGFR in Tam-R cells, and this association was increased after IGF-II stimulation, as shown in Fig. 6C, with total levels of EGFR again remaining unchanged.

FIG. 4. The effects of gefitinib (1 ␮M) (A), AG1024 (20 ␮M) (B), and SU6656 (1 ␮M) (C) on both basal and IGF-II-primed (100 ng/ml) growth of Tam-R cells grown in serum-free DCCM on d 7 from initial treatment. The results are expressed as mean (SEM) of triplicate wells and are representative of three separate experiments. ***, P ⱕ 0.001; **, P ⱕ 0.01 vs. control; †††, P ⱕ 0.001; †, P ⱕ 0.05 vs. IGF-II alone.

IGF-II activates c-SRC and promotes phosphorylation of EGFR at Y845 in tamoxifen-resistant MCF-7 cells

IGF-II stimulation increased phosphorylated c-SRC Y418 levels and promoted phosphorylation of EGFR at Y845, a c-SRC-dependent phosphorylation site, in Tam-R cells in a time-dependent manner. Total c-SRC and EGFR levels remained unchanged throughout the time course (Fig. 7A).

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FIG. 5. Western analysis of phosphorylated and total IGF-IR, EGFR, and ERK1/2 protein expression assessed after incubation of Tam-R cells in serum-free DCCM containing increasing concentrations of an IGF-II neutralizing antibody (1–10 ␮g/ml) for 1 h (A), phosphorylated and total IGF-IR protein expression assessed after incubation of Tam-R cells in serum-free DCCM containing anti-IGF-II neutralizing antibody (10 ␮g/ml for 1 h) in either the presence or absence of IGF-II (100 ng/ml) for 20 min (B), and phosphorylated and total IGF-IR, EGFR, and ERK1/2 protein expression assessed after incubation of Tam-R cells in serum-free DCCM supplemented with AG1024 (20 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or the two agents in combination (C). Data are representative of at least three separate experiments.

Furthermore, preincubation of Tam-R cells with increasing concentrations of either IGF-II neutralizing antibody (1–10 ␮g/ml, 1 h) or AG1024 (20 ␮m, 24 h) reduced levels of c-SRC Y418 and EGFR Y845 phosphorylation as shown in Fig. 7, B and C, respectively. These effects of AG1024 could not be overcome by IGF-II stimulation (Fig. 7C). Again, total levels of these proteins remained unchanged for all treatment groups. c-SRC blockade inhibits basal, IGF-II-, and TGFa-induced EGFR activity in tamoxifen-resistant MCF-7 cells

The c-SRC inhibitor, SU6656 (1 ␮m), reduced basal phosphorylation of c-SRC Y418, EGFR Y845, and EGFR Y1068, and this inhibition with SU6656 could not be overcome by IGF-II stimulation (Fig. 7D). There was no change in total


FIG. 6. Western analysis of total IGF-IR levels after immunoprecipitation with total EGFR antibody (A), total c-SRC levels after immunoprecipitation with total IGF-IR antibody (B), and total c-SRC levels after immunoprecipitation with total EGFR antibody in Tam-R cells incubated in serum-free DCCM in either the absence or presence of IGF-II (100 ng/ml) for 20 min (C). Data are representative of three separate experiments.

c-SRC and EGFR levels in response to this inhibitor (Fig. 7D). At this same concentration, SU6656 inhibited Tam-R cell growth by approximately 70% at 7 d (P ⱕ 0.01 vs. control, n ⫽ 4), and this inhibition could not be overcome by the inclusion of IGF-II (P ⱕ 0.05 vs. IGF-II alone, n ⫽ 4) (Fig. 4C). Gefitinib treatment potently inhibited basal and IGF-IIinduced EGFR activity and reduced basal c-SRC activity; however, there was little effect of this agent on IGF-IIinduced phosphorylation of c-SRC (Fig. 8A). There was no effect of gefitinib on either basal or IGF-II-induced IGF-IR activity (Fig. 8A). Interestingly, inhibition of c-SRC and reduction of EGFR Y845 phosphorylation by SU6656 (1 ␮m) was associated with a reduced ability of TGF␣ (1 ng/ml) to phosphorylate EGFR at Y1068 and activate the downstream ERK1/2 MAPK pathway (Fig. 8B). IGF-IR/EGFR cross-talk in tamoxifen-resistant T47D cells

To assess whether this mechanism was unique to Tam-R cells, we examined this novel IGF-IR/EGFR cross-talk mechanism in another tamoxifen-resistant breast cancer cell line, derived from WT T47D cells. Treatment of T47D tamoxifenresistant (T47D-R) cells with IGF-II for 20 min not only resulted in an increase in phosphorylation levels of IGF-IR at sites Y1158 and Y1316 but also an increase in c-SRC and



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FIG. 8. Western analysis of phosphorylated and total c-SRC, EGFR, and IGF-IR protein expression assessed after incubation of Tam-R cells in serum-free DCCM supplemented with gefitinib (1 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or the two agents in combination (A); and phosphorylated and total c-SRC, EGFR, and ERK1/2 after incubation of Tam-R cells in serum-free DCCM supplemented with SU6656 (1 ␮M) for 24 h, TGF␣ (1 ng/ml) for 5 min, or a combination of the two agents (B). Data are representative of at least three separate experiments. FIG. 7. Western analysis of phosphorylated and total c-SRC and EGFR protein expression assessed after incubation of Tam-R cells in serum-free DCCM supplemented with IGF-II (100 ng/ml) for 0 –30 min (A); increasing concentrations of an anti-IGF-II neutralizing antibody (1–10 ␮g/ml) for 1 h (B); AG1024 (20 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or a combination of the two agents (C); and SU6656 (1 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or a combination of the two agents (D). Data are representative of three separate experiments.

EGFR (Y1068 and Y845) phosphorylation (Fig. 9), similar to results found for Tam-R cells. Moreover, a 24-h preincubation of these cells with 20 ␮m AG1024 markedly inhibited basal IGF-IR Y1158 and Y1316, c-SRC, EGFR Y1068, and Y845 phosphorylation levels, and such inhibition could not be overcome after further IGF-II treatment (Fig. 9A). Similar findings were also observed after a 24-h preincubation of the T47D-R cells with SU6656 (1 ␮m). At this concentration, basal phosphorylation levels of c-SRC and EGFR Y1068 and Y845 were inhibited, and such inhibition could not be overcome after additional IGF-II treatment (Fig. 9B). Interestingly, as already found for the Tam-R cells, inhibition of c-SRC and reduction of EGFR Y845 phosphorylation by SU6656 (1 ␮m) was again associated with a reduced ability of TGF␣ (1 ng/ ml) to phosphorylate EGFR at Y1068 in the T47D-R cells (Fig. 9C). Total protein levels remained equivalent for all treatment groups throughout these studies.

Discussion

Previous studies from our group have established that after an initial growth inhibitory phase lasting approximately 3 months, tamoxifen-treated WT-MCF-7 human breast cancer cells are able to reestablish their growth through the generation of an EGFR driven autocrine regulatory loop (25). This mechanism results from a marked increase in both total and phosphorylated EGFR and c-erbB2 levels and activation of downstream MAPK signaling in the Tam-R cells when compared with their WT-MCF-7 counterparts (25). In the present report, we have sought to investigate the role played by the IGF-II/IGF-IR axis in Tam-R cells by examining potential areas of cross-talk between IGF-IR and EGFR. We have also evaluated whether such cross-talk is unique to this cell line by examining IGF-IR signaling in a tamoxifen-resistant T47D breast cancer cell line (T47D-R). Significantly, we now show that Tam-R cells have lower levels of IGF-IR than their WT-MCF-7 counterparts, consistent with previous studies in tamoxifen-resistant MCF-7 and ZR-75–1 cells (41, 42). Despite having lower IGF-IR expression levels, the Tam-R cells nevertheless expressed increased amounts of IGF-II mRNA in accordance with Lee and Yee 1995 (11) and demonstrated an equivalent level of phosphorylated IGF-IR. It should be noted, however, that IGF-I mRNA could not be detected in either cell line, in agreement with

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FIG. 9. Western analysis of phosphorylated and total IGF-IR, c-SRC, and EGFR after incubation of T47D-R cells in serum-free DCCM supplemented with AG1024 (20 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or a combination of the two agents (A); phosphorylated and total c-SRC and EGFR after incubation of T47D-R cells in serum-free DCCM supplemented with SU6656 (1 ␮M) for 24 h, IGF-II (100 ng/ml) for 20 min, or a combination of the two agents (B); and phosphorylated and total c-SRC and EGFR after incubation of T47D-R cells in serumfree DCCM supplemented with SU6656 (1 ␮M) for 24 h, TGF␣ (1 ng/ml) for 5 min, or a combination of the two agents (C). Data are representative of at least three separate experiments.

Quinn et al. (43). Furthermore, phosphorylation of IGF-IR, and not insulin receptor, was confirmed by both Western blotting and immunocytochemistry using an antibody targeting IGF-IR phosphorylated at Y1316. This antibody, unlike those targeting Y1158 and Y1131 also used in this study, does not cross-react with insulin receptor. It is possible that the raised levels of IGF-II mRNA observed in the Tam-R cells may explain why phosphorylated IGF-IR levels are similar in both cell lines despite the reduced IGF-IR total levels seen in the Tam-R cells. However, it remains to be determined whether the increase in IGF-II mRNA observed in the Tam-R cells is translated out into equivalent protein levels. Thus, although numerous studies have previously demonstrated that tamoxifen significantly interferes with IGF-IR signaling in WT-MCF-7 cells as part of its growth inhibitory action (10 –12), such signaling appears to be restored and plays an active role in tamoxifen resistance (13–15). Consistent with the notion that IGF-IR is functional in Tam-R cells, exogenous IGF-II is able to not only promote IGF-IR phos-


phorylation but also increase cell proliferation. Moreover, we also demonstrate that such treatments increase EGFR phosphorylation, a surprising observation not seen in WT-MCF-7 cells. The inability to detect IGF-II-induced increases in EGFR phosphorylation in WT cells would indicate that such a cross-talk mechanism is not functional in these cells. Activation of EGFR with the exogenous growth factor EGF failed to induce IGF-IR basal phosphorylation levels in Tam-R cells, thus establishing that this IGF-IR/EGFR cross-talk mechanism is unidirectional in this cell line. Interestingly, treatment of Tam-R cells with either an IGF-II neutralizing antibody or AG1024 inhibited both basal IGF-IR and EGFR signaling and, in the case of AG1024, significantly inhibited basal cell growth. Similarly, Parisot et al. (13) have demonstrated that growth of a tamoxifen-resistant MCF-7/2-23 cell line can be enhanced by IGF-I and inhibited by the IGF-IR monoclonal antibody, ␣ IR-3. These present findings confirm a role for IGF-IR, through its ability to cross-talk and activate EGFR, in mediating tamoxifen-resistant cell growth. Furthermore, this cross-talk is initiated, under basal growth conditions, by the autocrine release and action of IGF-II. This role for IGF-IR/ EGFR cross-talk in mediating tamoxifen-resistant growth is further supported by the finding that treatment of Tam-R cells with the selective EGFR tyrosine kinase inhibitor gefitinib potently inhibited IGF-II-induced EGFR activity and proliferation while having no effect on either basal or ligandinduced activation of IGF-IR. To date, there appears to be no conclusive clinical data reported in the literature examining the relevance of IGF-IR expression in relation to tamoxifen resistance. However, our group has recently developed immunocytochemical assays for the detection of both total and phospho-specific IGF-IR Y1316 expression in clinical breast cancer specimens. When these assays were applied to a small cohort of tumors, taken from patients who have ER-positive/ EGFR-positive acquired tamoxifen resistance, IGF-IR expression and activity were readily detectable (44). It is therefore feasible that such IGF-IR signaling observed in vivo may indeed be functional and play a role in supporting the growth of such tumors in acquired tamoxifen resistance, complementing our findings in Tam-R cells. There already exists a wealth of evidence that cross-talk occurs between the IGF-IR and EGFR and their ligands, where both direct and indirect interactions have been observed in several different cell types (27, 29 –35). However, only one of these studies was performed using mammary breast cancer cell lines (36). Mechanistically, we found no evidence to suggest that interactions between the IGF-IR and EGFR in the Tam-R cells occur through a direct physical association of the receptors. In contrast to this, however, a recent report successfully identified active heterodimers between these two receptors that could be disrupted by gefitinib in both MCF-7 and T47D breast cancer cell lines (27). However, another study also presented evidence for this, but only in normal mammary epithelial cells and not in their breast cancer cell lines (28). We can only conclude from our own studies that the interaction found between these two receptors in our Tam-R cells occurs indirectly, especially because stimulation of Tam-R cells with IGF-II resulted in a time-delayed phosphorylation of EGFR, indicative of an indirect mechanism. In support of this is the study by Gilmore


et al. (36) who also demonstrated that IGF indirectly transactivated EGFR and its downstream MAPK signaling pathway in mammary epithelial cells, although they failed to identify the mechanism involved. In an attempt to further dissect this indirect cross-talk mechanism observed in Tam-R cells, we examined the potential role of c-SRC as it has previously been implicated in mediating EGFR transactivation in a variety of cell lines (45, 46). IGF-II promoted activation of c-SRC, and this was sensitive to inhibition by AG1024. Activation of c-SRC by EGFR was also evident in Tam-R cells because TGF␣-promoted c-SRC phosphorylation and basal c-SRC activity were sensitive to inhibition by gefitinib. However, gefitinib only partially reduced phosphorylation of c-SRC in response to IGFII, indicating that a proportion of IGF-II-induced c-SRC activity occurred upstream of the EGFR. This was further supported by the finding that IGF-IR and c-SRC physically interacted in Tam-R cells, and this association could be increased after IGF-II stimulation. c-SRC has been shown to mediate EGFR transactivation through direct association with EGFR and phosphorylation of tyrosine residues 845 and 1101 (47, 48). We observed an increase in phosphorylation of EGFR at Y845 after IGF-II stimulation, and this effect could again be blocked by pretreatment with AG1024, confirming the involvement of the IGF-IR in Tam-R cells. We have further demonstrated that this c-SRC-dependent EGFR transactivation mechanism plays a key role in regulating EGFR activity and consequently Tam-R cell growth because SU6656 reduced both basal and IGF-II-primed c-SRC and EGFR signaling and inhibited proliferative activity in this cell line. Indeed, SU6656 inhibited phosphorylation of EGFR at both Y845 and Y1068, suggesting that c-SRC can also regulate EGFR autophosphorylation. A simple explanation for this is that activated c-SRC when bound to EGFR is capable of phosphorylating EGFR on both auto- and nonautophosphorylation sites (48). However, studies have also shown that cells that express a mutant form of Y845 display a decrease in their ability to respond mitogenically to EGF, suggesting that this c-SRCmediated phosphorylation site is important for liganddependent receptor activation (47). Furthermore, crystallographic studies have shown that phosphorylation of Y845 on EGFR homologs helps stabilize the enzyme, thus maintaining it in an active state (47). It has therefore been proposed that ligand activation of EGFR may be under pY845 regulation. Our findings support this proposition because TGF␣induced activation of EGFR/ERK1/2 phosphorylation was considerably abrogated in Tam-R cells pretreated with SU6656. In agreement with our Tam-R data, we also found that under basal growth conditions both AG1024 and SU6656 inhibited phosphorylation of c-SRC and EGFR at both Y845 and Y1068 residues in a tamoxifen-resistant T47D cell line. Furthermore, phosphorylation of IGF-IR, c-SRC, and EGFR (Y845 and Y1068) could be enhanced by IGF-II, and this was again sensitive to the inhibitory actions of both AG1024 and SU6656 in this cell line. The importance of c-SRC-dependent phosphorylation of EGFR at Y845 was also demonstrated in T47D-R cells because inhibition of c-SRC activity with SU6656 reduced the ability of TGF␣ to promote autophosphorylation of EGFR at Y1068.


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In conclusion, our studies strongly suggest that the autocrine release and action of IGF-II mediated through the IGF-IR play a significant and crucial supporting role in regulating basal EGFR/MAPK signaling and cell proliferation in Tam-R cells, and this occurs via a c-SRC-dependent mechanism. Activation of c-SRC by IGF-II results in the phosphorylation of Y845 on EGFR, enhancing ligand-dependent signaling through the EGFR. This mechanism represents a novel and unreported finding to date in tamoxifen-resistant breast cancer cell lines, one which may prove to be critical to the growth and development of acquired steroid-resistant breast tumors. Indeed, the findings from T47D-R cells would indicate that this cross-talk mechanism is not unique to Tam-R cells but may be a more general tamoxifen-resistant phenomenon. Further investigation into the generality of this mechanism is required; however, the present findings would indicate that targeting of IGF-IR and c-SRC through drug therapies may prove valuable for the management and treatment of such cancers. Acknowledgments The authors thank Dave Tonge (AstraZeneca, Macclesfield, UK) for the kind gift of the specific phospho-IGF-IR (Y1316) antibody and Lynne Farrow for performing the statistical analyses. Received March 1, 2005. Accepted July 14, 2005. Address all correspondence and requests for reprints to: I. R. Hutcheson, Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, United Kingdom. E-mail: [email protected]. This work was supported by the Tenovus organization.

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