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Endocrinology, doi:10.1210/en.2006-1335
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Endocrinology Vol. 148, No. 5 2532-2541
Copyright © 2007 by The Endocrine Society

Expression of Insulin-Like Growth Factor Binding Protein-2 by MCF-7 Breast Cancer Cells Is Regulated through the Phosphatidylinositol 3-Kinase/AKT/Mammalian Target of Rapamycin Pathway

Janet L. Martin and Robert C. Baxter

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Dr. Janet L. Martin, Kolling Institute of Medical Research, E25, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: janetlm{at}med.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein-2 (IGFBP-2) has been implicated in the development and spread of a number of tumor types, and its abrogation in experimental models of cancer is associated with decreased tumor growth. This suggests that targeted inhibition of IGFBP-2 expression in some cancers may have therapeutic benefit. In this study, we investigated signaling pathways involved in extracellular IGFBP-2 expression in an IGF- and estrogen-responsive breast cancer cell line, MCF-7. IGFBP-2 was present at approximately 150 ng per 106 cells in serum-free MCF-7-conditioned medium and constituted the predominant IGFBP. Inhibition of the phosphatidylinositol 3-kinase signaling pathway using LY294002, or the downstream signaling intermediate mammalian target of rapamycin using rapamycin, markedly reduced IGFBP-2 in conditioned medium to approximately 25% of untreated levels (P < 0.001); there was no effect of inhibition of p38 MAPK, and an inhibitor of p44/42 MAPK activation, PD98059, caused only a slight reduction in extracellular IGFBP-2. IGFBP-2 levels were increased 25–30% by estradiol, whereas IGF-I (100 ng/ml) increased IGFBP-2 levels 2-fold (P < 0.001) by a type 1 IGF receptor (IGFR1)-dependent mechanism. Estradiol enhanced the effect of IGF-I on IGFBP-2 levels, and this was associated with increased phosphorylation of IGFR1. Basal, IGF-, or estradiol-stimulated IGFBP-2 was abrogated by LY294002 and rapamycin and an inhibitor of IGFR1 tyrosine kinase activity, AG1024. Modulation of intracellular hypoxia-inducible factor-1{alpha} had no effect on IGFBP-2 expression. These findings indicate that IGFBP-2 is regulated predominantly through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway, the target of a number of anticancer agents currently in clinical trial and use.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEIN (IGFBP)-2 is a member of the family of six IGFBPs, structurally and functionally related proteins that are important for regulating the potent mitogenic and antiapoptotic effects of IGF-I and IGF-II via their high-affinity binding of these growth factors (1). IGFBP-2 acts as an inhibitor of normal somatic growth in vivo, most likely through interference with the growth-promoting effects of the GH/IGF axis via its regulation of IGF bioactivity (2, 3). Consistent with this, IGFBP-2 has been shown to inhibit the proliferation of normal cells in vitro in a manner that can be overcome by addition of exogenous IGFs (4), suggesting that IGFBP-2’s inhibitory effects are due to modulation of the growth-promoting bioactivity of IGFs.

Cancer cell proliferation, migration, and invasion have also been shown to be inhibited by IGFBP-2 (4, 5, 6). Overexpression of IGFBP-2 in C6 glioblastoma cells resulted in reduced cell proliferation when IGF levels were low (5), and IGF-responsive colon carcinoma cells also showed decreased proliferation in the presence of IGFBP-2 (4). A recent study indicated that IGFBP-2 inhibited IGF-mediated cell migration in vitro, and tumor growth in vivo, via the formation of complexes with {alpha}vß3 integrin. Whereas the mechanism underlying this phenomenon was not specifically addressed, it was proposed that complex formation between IGFBP-2 and {alpha}vß3 interrupted the enhancement of IGF-I signaling normally afforded by vitronectin binding to {alpha}vß3 integrin (6).

In contrast with these studies, however, a number of others indicate that IGFBP-2 may function as a growth promoter in malignant disease. Elevated serum levels of IGFBP-2 have been reported in metastatic disease associated with colorectal cancer, adrenocortical tumors, ovarian tumors, prostate cancer, and leukemias (7, 8, 9, 10, 11, 12). More recently IGFBP-2 was also found to be overexpressed in breast carcinoma in situ and invasive breast cancer, compared with normal glandular or hyperplastic breast tissue (13). Such clinical correlates implicating IGFBP-2 in the development or progression of malignant disease are supported by laboratory studies showing that overexpression of IGFBP-2 increases the tumorigenic potential of adrenocortical tumor, epidermoid carcinoma, and glioma cells (5, 14, 15). Significantly, growth of DU145 prostate cancer cells in vitro (16) or LNCaP prostate cancer cells grown as tumors in vivo is abrogated by IGFBP-2 antisense therapy (17). Collectively these studies suggest that targeting the expression or bioactivity of IGFBP-2 may be of value in the treatment of some malignancies.

A number of factors have been reported to affect IGFBP-2 gene or protein expression in a range of cell types, including IGFs, insulin, steroids, and glucocorticoids (18, 19, 20, 21, 22, 23), but the signaling pathways by which they do so remain essentially unexplored. In the present study, we used the MCF-7 breast cancer cell line as a model in which to examine pathways and regulatory factors involved in the expression of IGFBP-2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture reagents and plasticware were purchased from Trace Biosciences (North Ryde, New South Wales, Australia) and Nunc (Roskilde, Denmark). BSA, CoCl2, bovine insulin, estradiol 17-ß, and {alpha}-tubulin antibody were from Sigma (St. Louis, MO), and recombinant human IGF-I and Leu24Ala31-IGF-I were the generous gifts of Genentech, Inc. (South San Francisco, CA). The IGF-I analog Long Arg3-IGF-I (LR3-IGF-I) was purchased from GroPep (Adelaide, South Australia). The following antibodies were purchased from Cell Signaling (Beverly, MA); phospho-Thr202/Tyr204 and total p44/42 MAPK, phospho-Ser473 and total Akt, phospho-Ser389 p70S6K and total p70S6K, phospho-Tyr1135/1136 type 1 IGF receptor (IGFR1), and total IGFR1. Hypoxia-inducible factor (HIF)-1{alpha} and phosphotyrosine (pY20) antibodies were purchased from BD Biosciences (North Ryde, New South Wales, Australia), and topotecan was from Merck (Kilsyth, Victoria, Australia). Signaling pathway inhibitors PD98059, AG1024, LY294002, rapamycin, and SB203580 and protein A/G Sepharose beads were from Calbiochem. Electrophoresis and enhanced chemiluminescence (ECL) reagents were purchased from Bio-Rad (Hercules, CA), Amrad-Pharmacia (Ryde, New South Wales, Australia), and Pierce (Rockford, IL). IGF-I, IGFBP-2, and IGFBP-6 were radiolabeled with 125I sodium iodide (ICN Biomedicals, Seven Hills, New South Wales, Australia) using chloramine T and purified by size exclusion chromatography; IGFBP-3 cross-linked to radiolabeled IGF-I was prepared as described (24).

Assays
Estrogen receptor (ER) was originally measured in breast tumor cytosols by solid-phase enzyme immunoassay, using the ER-EIA monoclonal assay kit (Abbott, Abbott Park, IL) according to the manufacturer’s protocol. After ER analysis, cytosols were stored at –80 C. Approval to use archived specimens was given by the institutional human research ethics committee. RIA of IGFBP-2 (11), IGFBP-3 (24), and IGFBP-6 (25) in cell culture media or breast tumor cytosols was performed as previously described.

Cell culture
Breast cancer cell lines (ER positive lines MCF-7 and T47D and the ER-negative line Hs578T) were obtained from the American Type Culture Collection (Manassas, VA), and were maintained in RPMI 1640 containing 5% fetal calf serum and 10 µg/ml bovine insulin. For experiments, cells were plated at a density of 5 x 104 cells/well in 48-well plates or 5 x 105 cells/well in 6-well plates in serum- and insulin-containing medium and cultured for 2 d. Media were replaced by phenol-red-free RPMI 1640 without serum (SFM), containing 1 g/liter BSA for 48 h. Media were replaced with SFM containing additives (IGFs, pathway inhibitors, etc.), as indicated for individual experiments. At the desired time point (24 or 48 h after addition), media were collected and stored frozen at –20 C until assay. For analysis of signaling intermediates, treated cells were washed in cold saline, lysed in reducing Laemmli buffer [62.5 mM Tris-HCl (pH 6.8), containing 20 g/liter sodium dodecyl sulfate (SDS), 100 ml/liter glycerol, 1 g/liter bromphenol blue and 50 mM dithiothreitol] at 4 C for 10 min, and transferred to Microfuge tubes for storage at –80 C until immunoblot analysis, as described below.

Immunoprecipitation
For IGFR1 immunoprecipitation, IGF-stimulated or control cells in 25-cm2 flasks were lysed in 250 µl radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ß-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride] for 10 min at 4 C. Cells were scraped into the lysis buffer, transferred to cold Microfuge tubes, sonicated on ice 5 times for 10 sec each, and then microfuged for 10 min at 4 C. Supernatants were transferred to fresh tubes, and 2.5 µg nonimmune rabbit IgG or anti-IGFR1 antibody was added to 250 µl of supernatant and incubated overnight at 4 C. Twenty microliters of a 50% slurry of protein A/G Sepharose beads were added to each tube, and mixtures were incubated with end-over-end stirring for 2 h at 4 C. Beads were washed five times with RIPA buffer, and proteins were eluted into 250 µl 2x Laemmli buffer [125 mM Tris (pH 6.8), 40 g/liter SDS, 100 mM dithiothreitol, 20% glycerol, 0.2 g/liter bromphenol blue] at 95 C for 5 min. Samples were microfuged before electrophoresis.

SDS-PAGE and Western analysis
Cell lysates, media, or immunoprecipitates were resolved on 7.5 or 10% SDS polyacrylamide gels and transferred to Hybond C nitrocellulose for Western analysis, as previously described (26). After transfer, filters were blocked in 50 g/liter skim milk powder in TBS-T (Tris-buffered saline with Tween 20: 10 mM Tris, 150 mM NaCl (pH 7.4) containing 1 ml/liter Tween 20) or 50 g/liter BSA in TBS-T for phosphotyrosine analysis and probed with antibodies (as indicated for individual experiments) diluted in TBS-T containing 50 g/liter BSA at 4 C for 16 h. Filters were washed in cold TBS-T and then incubated with the appropriate horseradish peroxidase-labeled secondary antibody for 1–2 h at room temperature. Washed filters were developed by ECL using Pierce reagents. Total and phosphorylated proteins were analyzed on replicate blots, and filters were also probed with {alpha}-tubulin antibody as loading control. Bands were visualized using a FUJIFILM luminescent image analyzer LAS-300 (Fuji Photofilm Co. Ltd., Tokyo, Japan), and quantified using ImageGauge software (Science Lab, 2004; Fuji).

Statistics
All experiments were performed a minimum of three times, and individual experiments were carried out in triplicate or quadruplicate. Statistical analysis was performed using Statview for Macintosh (SAS Institute, Cary, NC) using ANOVA with Fisher’s protected least significant differences. Differences were considered statistically significant where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The pattern of IGFBP expression in breast cancer cell lines reflects that in breast tumors
It has been suggested that although IGFBP-2 expression is positively associated with expression of ER breast cancer cell lines in vitro (27), a similar relationship is not found in breast tumor tissue (28). The latter study based its finding on ligand blot analysis of tumor cytosols, a relatively insensitive method for quantifying IGFBPs in biological samples. Therefore, we used RIAs specific for IGFBP-2, -3, and -6 to measure these proteins in breast tumor cytosols in which the ER content had previously been assessed as part of clinical diagnosis. As shown in Fig. 1AGo, IGFBP-2 levels in ER-positive (ER+ve) breast tumor cytosols (ER > 15 fmol/mg cytosolic protein) were significantly higher than those in ER-negative tumors (ER–ve) (P < 0.01 by ANOVA). Measurement of IGFBP-3 in these cytosols confirmed earlier findings (29, 30) of a negative correlation between ER and this IGFBP in breast tumors, whereas analysis of IGFBP-6, which has not been reported previously in breast tumors, indicated no difference between ER+ve and ER–ve breast tumors. Analysis of medium conditioned by ER+ve and ER–ve breast cancer cell lines indicated a similar pattern of expression of these IGFBPs (Fig. 1BGo). IGFBP-2 was high in ER+ve lines MCF-7 and T47D and low in the ER–ve line Hs578T, IGFBP-3 was more highly expressed in the ER–ve line, and there was no clear association between the presence of ER and IGFBP-6 expression.


Figure 1
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  		FIG. 1. Expression of IGFBPs in breast tumor cytosols and breast cancer cell lines. IGFBP-2, IGFBP-3 and IGFBP-6 were measured in ER–ve or ER+ve breast tumor cytosols as indicated (A) or 24 h-cell conditioned media from ER+ve MCF-7 and T47D and ER–ve Hs578T, breast cancer cells (B) by RIA as described in the Materials and Methods. The number of cytosols analyzed in A is indicated in parentheses. IGFBP levels in conditioned media were corrected for cell number and are expressed as nanograms per 106 cells.

 
Secreted IGFBP-2 is regulated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway in MCF-7 and T47D cells
To identify pathways involved in the expression of IGFBP-2, MCF-7 and T47D cells were exposed to inhibitors of signaling pathways known to be involved in cell proliferation and survival: PI3-kinase (inhibited by LY294002), p44/42 MAPK/ERK1/2 (inhibited by PD98059), and p38 MAPK (inhibited by SB203580). Cells were treated for 24 h with inhibitors, and conditioned media were collected for assay of IGFBP-2. As shown in Fig. 2AGo, a marked decrease in IGFBP-2 expression by MCF-7 cells was apparent in response to treatment with LY294002, with a significant effect at an inhibitor concentration of 1 µM (78 ± 3% of control, P < 0.05) and levels further decreasing to 26 ± 2% of control at 30 µM LY294002. IGFBP-2 expression was also inhibited when the PI3-kinase pathway was blocked further downstream of Akt/protein kinase B using rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) activation (Fig. 2BGo), with 1 and 100 nM rapamycin reducing IGFBP-2 levels to 76 ± 5 and 32 ± 1% of control levels, respectively. By contrast, inhibition of the p44/42 MAPK pathway using PD98059 had a relatively small effect on IGFBP-2 levels, which were decreased by approximately 20% at the highest dose of PD98059 tested (30 µM) in MCF-7 cells (Fig. 2CGo), whereas the p38 kinase pathway inhibitor SB203580 had no effect on IGFBP-2 at doses up to 10 µM MCF-7 (Fig. 2DGo); higher concentrations of these inhibitors could not be tested due to cytotoxicity. Similarly for ER+ve T47D breast cancer cells, IGFBP-2 expression was inhibited in response to blockade of the PI3-kinase pathway using LY294002 (Fig. 2EGo) or rapamycin (Fig. 2FGo), although reduced sensitivity to rapamycin was apparent in this cell line, and a maximal effect on IGFBP-2 resulted in levels reduced to 60% of control (Fig. 2FGo). There was no effect of PD98059 or SB203580 on IGFBP-2 expression by T47D cells (data not shown).


Figure 2
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  		FIG. 2. PI3-kinase pathway blockade inhibits expression of IGFBP-2 in MCF-7 and T47D breast cancer cells. MCF-7 cells (A–D) or T47D cells (E and F) were incubated with the indicated concentrations of inhibitors of PI3-kinase (LY294002, A and E), mTOR (rapamycin, B and F), MAPK kinase (PD98059, C), or p38 MAPK (SB203580, D) for 24 h. Media were collected and assayed for IGFBP-2 by RIA. Data are expressed relative to IGFBP-2 levels in untreated, SFM (~80 ng/ml for MCF-7, ~40 ng/ml for T47D). Mean ± SE are shown for data pooled from three experiments carried out in quadruplicate. *, P < 0.05 and **, P < 0.001, compared with no inhibitor (by ANOVA).

 
IGFBP-2 expression in MCF-7 cells is modulated by IGFR1 activation
IGF-I is a potent stimulator of the PI3-kinase signaling pathway, which mediates its effects on cell survival, and IGFs have been reported to increase IGFBP-2 expression in other cell systems (18) and in vivo (31). We therefore examined whether IGFs alter the levels of secreted IGFBP-2 in the MCF-7 cell line, using IGF analogs with altered affinity for IGF receptors and IGFBPs to assess whether any observed effects were IGF receptor independent. Treatment of MCF-7 cells for 24 h with 100 ng/ml IGF-I increased IGFBP-2 in media nearly 2-fold (Fig. 3AGo). The IGF analog LR3-IGF-I, which binds with near normal affinity to IGFR1 but markedly reduced affinity to IGFBPs (32), exhibited greater potency for stimulating IGFBP-2, compared with wild-type IGF-I, with a maximal effect at 10-fold lower concentrations of the peptide. By contrast, an IGF analog with near-normal affinity for IGFBPs but markedly reduced receptor binding, Leu24Ala31-IGF-I, had no effect on IGFBP-2 levels (Fig. 3AGo).


Figure 3
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  		FIG. 3. IGF-I stimulated IGFBP-2 expression is associated with IGFRI activation. A, MCF-7 cells were treated for 24 h with IGF-I, LR3-IGF-I, or Leu24Ala31-IGF-I at the indicated concentrations, and then media were collected for analysis of IGFBP-2 by RIA. *, P < 0.001, compared with control (ctl) by ANOVA and Fisher’s protected least significant differences. B, MCF-7 cells were incubated with 100 ng/ml IGF-I, LR3-IGF-I, or Leu24Ala31-IGF-I or control (ctl) medium for 10 min at 37 C, washed, and lysed in reducing Laemmli sample buffer. Samples were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with phospho-IGFR1 (Tyr1135/1136) or total IGFR1 antibodies as indicated and developed by ECL. C, Lysates of MCF-7 cells treated as described for B were immunoprecipitated with IGFR1 antibody (R) or control IgG (N), as described in Materials and Methods. Precipitates were separated by 7.5% reducing SDS-PAGE, probed with antiphosphotyrosine antibody (pY20) or IGFR1 as indicated, and developed by ECL. D, Media conditioned by MCF-7 in the absence or presence of 100 ng/ml IGF-I were ligand blotted with [125I]IGF-I (IGF-I) or immunoblotted (IB) with IGFBP-2 antiserum ({alpha}-BP-2), as described in Materials and Methods. Immunoblots were developed by ECL. The migration distance of molecular mass markers are shown on the right (as kilodaltons).

 
Immunoblot analysis of phosphorylated IGFR1 in lysates from treated cells using a phosphospecific antibody indicated that IGF-I and LR3-IGF-I, but not Leu24Ala31-IGF-I, increased phosphorylation of the IGFR1 (Fig. 3BGo). Because this antibody might also detect insulin receptor phosphorylated at Tyr1150/1151, these results were confirmed by probing IGFR1 immunoprecipitates of control, IGF-I-, LR3-IGF-I-, or Leu24Ala31-IGF-I-treated cells for phosphotyrosine (Fig. 3CGo). Consistent with the data obtained using the phosphospecific antibody shown in Fig. 3BGo, IGF-I and LR3-IGF-I caused a marked induction of phosphorylation of IGFR1, whereas Leu24Ala31 did not (Fig. 3CGo). These results indicate that stimulation of IGFBP-2 expression by IGFs is associated with IGFR1 activation. Interestingly, extended exposure of the blot indicated a very low level of IGFR1 phosphorylation in the control cell lysate (i.e. in the absence of exogenous IGF-I), and a similar low level was also apparent in lysates from Leu24Ala31-treated cells (Fig. 3CGo). Ligand and immunoblot analysis (Fig. 3DGo) revealed that IGFBP-2 is the predominant IGFBP present in MCF-7 conditioned medium in the absence or presence of IGF-I (Fig. 3DGo). The presence of a minor species of 24 kDa (IGFBP-4) in untreated and IGF-treated media was also apparent on ligand blot, whereas an approximately 30-kDa IGFBP (IGFBP-5) was evident only after IGF treatment (Fig. 3DGo).

We then examined whether IGF-stimulated IGFBP-2 expression also involved activation of the PI3-kinase/Akt/mTOR pathway. As shown in Fig. 4Go, LY294002 inhibited IGF-stimulated IGFBP-2 expression in the presence of IGF-I (Fig. 4AGo) with similar potency as in its absence (i.e. a significant inhibitory effect at 1 µM LY294002). Rapamycin also blocked the stimulation of IGFBP-2 expression by IGF-I but with reduced potency, compared with its effect in the absence of IGF-I; inhibition of IGF-stimulated IGFBP-2 expression required 10 nM rapamycin (Fig. 4BGo), but in the absence of IGF-I, 1 nM rapamycin was inhibitory to IGFBP-2 (as shown in Fig. 2BGo). Blockade of the p44/42 MAPK pathway had no effect on IGFBP-2 expression stimulated by IGF-I (Fig. 4CGo).


Figure 4
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  		FIG. 4. IGF-stimulated IGFBP-2 expression is blocked by PI3-kinase pathway inhibition. A–C, Media conditioned by MCF-7 cells treated for 24 h with 100 ng/ml IGF-I with or without the indicated concentration of LY294002 (LY, A), rapamycin (rapa, B) or PD98059 (PD, C) were assayed for IGFBP-2 by RIA. IGFBP-2 levels are expressed relative to control (in the absence of IGF-I or inhibitor), and represent pooled data from four experiments performed in triplicate. **, P < 0.001, compared with IGF-I in the absence of inhibitor by ANOVA. D–F, MCF-7 cells were preincubated for 2 h with inhibitors at the concentrations indicated, and then SFM with or without IGF-I (100 ng/ml) was added for 10 min. Cells were lysed into SDS-PAGE sample buffer on ice and then separated by 10% SDS-PAGE. After transfer, filters were probed with antibodies for phospho-Ser473 Akt (ph-Akt) or total Akt (D), phospho-Ser389 p70S6K (ph-S6K) or total p70S6K (E), or phospho-Thr202/Tyr204 MAPK (ph-MAPK) or total MAPK (F) to confirm inhibition of the targeted pathway. Representative blots from three independent experiments are shown. Ctl, Control.

 
To confirm decreased activity of the targeted pathway in response to these inhibitors, lysates from control and IGF-treated cells were analyzed by Western blot using phospho-specific antibodies against the relevant signaling intermediates. In the absence of IGF-I, there was no evidence of Akt phosphorylation (Fig. 4DGo), whereas a low level of phosphorylation of p70S6K (Fig. 4EGo) and p44/42 MAPK (Fig. 4FGo) was apparent. IGF-I (100 ng/ml) treatment for 10 min resulted in markedly increased phosphorylation of Akt, p70S6K, and p44/42 MAPK, consistent with the known effects of IGF-I on these pathways. PI3-kinase blockade upstream of Akt using LY294002 markedly attenuated basal- and IGF-stimulated Akt phosphorylation (Fig. 4DGo), whereas inhibition of mTOR using rapamycin resulted in reduced phosphorylation of p70S6 kinase under both basal and IGF-stimulated conditions (Fig. 4EGo). PD98059 inhibited both basal and IGF-stimulated p44/42 MAPK phosphorylation (Fig. 4FGo).

To examine whether activation of the PI3-kinase/Akt/mTOR pathway by other growth factors affected IGFBP-2 expression, MCF-7 cells were treated with epithelial growth factor (EGF) or insulin and analyzed for IGFBP-2 expression after 24 h; activation of the PI3-kinase/Akt/mTOR pathway by these agents was confirmed by analysis of p70S6K phosphorylation after acute (15 min) exposure to either growth factor. EGF (1–100 ng/ml) did not significantly alter IGFBP-2 levels in media (Fig. 5AGo), despite eliciting a marked increase in p70S6K phosphorylation over 15 min at 1 and 10 ng/ml (Fig. 5BGo). Insulin increased IGFBP-2 only at the highest concentration tested (1000 ng/ml), although phosphorylation of p70S6K was increased at a 100-fold lower concentration (Fig. 5DGo). These results indicate that activation of the PI3-kinase/Akt/mTOR pathway is necessary but not sufficient for IGFBP-2 expression, and other factors must be involved.


Figure 5
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  		FIG. 5. EGF and insulin do not stimulate extracellular IGFBP-2 in MCF-7 cells. A and C, MCF-7 cells were treated with the indicated concentration of EGF (A) or insulin (C) for 24 h, and then conditioned media were collected and assayed for IGFBP-2. Data are shown as percent of IGFBP-2 relative to control (untreated). B and D, MCF-7 cells were treated for 15 min with the indicated concentration of EGF (B) or insulin (D), and then lysates were electrophoresed and analyzed for phospho p70S6K as described in Materials and Methods. Bands were quantified using ImageGauge software, with densities shown as arbitrary units. *, P < 0.05; **, P < 0.001, compared with control by ANOVA.

 
We next examined the effects of IGFR1 blockade on basal and IGF-stimulated IGFBP-2 expression. AG1024, which inhibits IGFR1 receptor tyrosine kinase activity, inhibited IGFBP-2 expression in the absence of IGF-I and also blocked the increase in IGFBP-2 expression stimulated by IGF-I (Fig. 6AGo). Phosphotyrosine analysis of IGFR1 immunoprecipitates from unstimulated, AG1024-treated cells confirmed that, in the absence of exogenous ligand, there was a low level of IGFR1 phosphorylation, and this was reduced to undetectable levels by 3 and 10 µM AG1024 (Fig. 6BGo). Analysis of cell lysates using the phosphospecific IGFR1 antibody indicated a greater than 90% decrease in IGFR1 phosphorylation in IGF-treated cells preexposed to 10 µM AG1024 (Fig. 6CGo), and basal and IGF-stimulated p70S6 kinase phosphorylation was also reduced, although not completely abolished, by AG1024 (Fig. 6DGo). Taken together, these results suggest that even in the absence of exogenous IGF-I, IGFBP-2 expression is at least partly mediated by IGFR1 activation of the PI3-kinase/Akt/mTOR pathway.


Figure 6
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  		FIG. 6. IGFR1 blockade inhibits basal IGFBP-2 expression and p70S6 kinase phosphorylation. A, MCF-7 cells were treated for 24 h with IGF-I (100 ng/ml, diagonal stripes) or control medium (open bars) with the indicated concentration of the IGFR1 tyrosine kinase inhibitor AG1024. Collected media were assayed for IGFBP-2. *, P < 0.05, compared with no IGF-I or inhibitor; **, P < 0.001, compared with IGF-I. Data were pooled from three experiments performed in triplicate, and IGFBP-2 is shown as percent of control (no IGF-I or inhibitor). B, MCF-7 cells were treated with the indicated concentration of AG1024 for 24 h, and cells were lysed in RIPA buffer before immunoprecipitation with anti-IGFR1 IgG, as described in Materials and Methods. Precipitates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and then probed with antiphosphotyrosine antibody (pY20) or total IGFR1 as indicated and developed by ECL. C and D, Lysates from cells treated as described in A were prepared in reducing sample buffer and separated on 10% SDS-PAGE gels. Blots were probed with antibodies against phospho-IGFR1 (ph-IGFR1) or total IGFR1 (B), phospho-Ser389 p70S6K (ph-S6K), total p70S6K, or {alpha}-tubulin (C) and developed by ECL. Representative blots from three experiments are shown.

 
HIF-1{alpha} does not regulate IGFBP-2 expression in MCF-7 cells
In mouse embryonic fibroblasts, IGFBP-2 gene expression is reported to be dependent on expression of HIF-1{alpha} (33), which is also known to be induced by IGFs in a number of cell types (34, 35, 36). We therefore investigated whether HIF-1{alpha} might be mediating basal and IGF-stimulated expression of IGFBP-2 in MCF-7 cells by examining the effect of topotecan, a topoisomerase-1 inhibitor that decreases HIF-1{alpha} expression by blocking its translation (37). As shown in Fig. 7AGo, topotecan was relatively ineffective at inhibiting either basal or IGF-stimulated IGFBP-2 expression, with a significant effect only apparent at the highest dose tested (1000 nM), which is well above its reported EC50 for HIF-1{alpha} inhibition of approximately 50 nM (37); immunoblot of cell lysates from topotecan-treated cells confirmed that HIF-1{alpha} was decreased in response to topotecan (Fig. 7AGo, right panel). We then examined the effect of CoCl2, which increases intracellular HIF-1{alpha} by blocking its ubiquitination and degradation. No increase in secreted IGFBP-2 was seen in response to CoCl2 at doses up to 100 µM (Fig. 7BGo); in fact, a significant decrease in IGFBP-2 was apparent at 10 and 100 µM CoCl2. Immunoblot of cell lysates from CoCl2-treated cells confirmed that HIF-1{alpha} was increased in response to CoCl2 over this same concentration range (Fig. 7CGo). We then investigated whether the decrease in IGFBP-2 was associated with decreased mTOR activity. Analysis of phosphorylated p70S6K in control and CoCl2-treated cells indicated that this was indeed the case, with a marked decrease in phospho-p70S6K as a result of CoCl2 treatment (Fig. 7CGo). These findings suggest that IGFBP-2 expression is independent of HIF-1{alpha} expression in MCF-7 cells.


Figure 7
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  		FIG. 7. IGFBP-2 expression is not regulated by HIF-1{alpha}. A and B, MCF-7 cells were treated with the indicated concentration of topotecan in the absence (open bars) or presence (diagonal stripes) of 100 ng/ml IGF-I (A), or CoCl2 (B) for 24 h. Media were collected and assayed for IGFBP-2 by RIA. Pooled data from three experiments in triplicate are shown. *, P < 0.05, compared with no topotecan or CoCl2; **, P < 0.05, compared with IGF-I. In the right panel (A) is shown an HIF-1{alpha} immunoblot of lysates from cells treated with 1000 nM topotecan or SFM for 24 h; the blot was reprobed for {alpha}-tubulin as loading control (ctl). C, Lysates from cells treated as described for B were harvested on ice and subjected to 7.5% SDS-PAGE under reducing conditions. Transferred proteins on the filter were immunoblotted for HIF-1{alpha}, phospho-Ser389 p70S6K, and {alpha}-tubulin as loading control. Shown is a blot with duplicate samples for each treatment, representative of three independent experiments.

 
Estradiol synergizes with IGF-I to stimulate IGFBP-2 expression
We have previously shown that IGFBP-2 expression is increased by dihydrotestosterone in LNCaP prostate cancer cells (20), and estrogen regulation of IGFBP-2 expression has been reported in some cell types (19) and breast tumor explants (38). Because the MCF-7 cell line is ER positive, we examined whether IGFBP-2 expression by these cells is estrogen sensitive. As shown in Fig. 8AGo, IGFBP-2 levels in medium conditioned by estradiol-treated cells were modestly but significantly elevated relative to untreated controls, with a 25% increase apparent at a concentration of 10 nM estradiol (P < 0.001). In addition, in the presence of 10 nM estradiol, the stimulatory effect of IGF-I on IGFBP-2 expression was enhanced when each was present at submaximal concentrations (10 ng/ml IGF-I, 10 nM estradiol; Fig. 8BGo), although this synergy was lost as the concentrations of IGF-I and estradiol approached maximal effective doses.


Figure 8
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  		FIG. 8. Estradiol stimulates IGFBP-2 expression and enhances the stimulatory effect of IGF-I. A, MCF-7 cells were treated for 24 h with estradiol at the indicated concentration, and then media were assayed for IGFBP-2 by RIA. Data pooled from five experiments performed in duplicate or triplicate are shown. *, P < 0.01 and **, P < 0.001, compared with 0 nM estradiol. B, IGFBP-2 was measured in media from cells treated with IGF-I alone (open bars) or 10 nM estradiol (shaded bars). Pooled data from four experiments performed in triplicate are shown. *, P < 0.05 and **, P < 0.001 for the effect of estradiol, compared with IGF-I at the same concentration. C, MCF-7 cells were incubated with the indicated concentration of estradiol for 24 h and then AG1024 (10 µM) for 2 h, as indicated. IGF-I (100 ng/ml) was added for 10 min as indicated, and then cells were washed in cold PBS and lysed in reducing sample buffer. Cell lysates were fractionated by 7.5% SDS-PAGE and probed for total and phospho-IGF-I receptor, and {alpha}-tubulin as loading control (ctl). The blots shown are representative of three independent experiments.

 
We then examined whether the combined effect of estradiol and IGF-I was due to increased IGFR1 expression. MCF-7 cells were preincubated with estradiol at 10 or 100 nM for 24 h before stimulation with 100 ng/ml IGF-I, and cell lysates were analyzed for phosphorylated IGFR1 using the phosphospecific IGFR1 antibody and total IGFR1. Total IGFR1 levels were slightly increased as a result of preincubation with estradiol at 10 and 100 nM, respectively, with quantitative analysis of these data indicating a maximal increase of approximately 20% with 10 nM estradiol (Fig. 8CGo). There was no evidence of increased IGFR1 phosphorylation in the absence of IGF-I, either with or without estradiol preincubation, when analyzed using the phosphospecific IGFR1 antibody (Fig. 8CGo), and this was confirmed by IGFR1 immunoprecipitation and phosphotyrosine analysis (data not shown). In the absence of estradiol, IGF-I stimulated IGFR1 phosphorylation, and this was increased a further 2.5- and 3.7-fold in cells preincubated with 10 and 100 nM estradiol, respectively.

Finally, we determined the effects of IGFR1 and mTOR blockade on the effects of estradiol on IGFBP-2 expression in MCF-7 cells. Increasing doses of AG1024 (Fig. 9AGo) reversed the stimulatory effect of estradiol on IGFBP-2 expression, reducing levels to those seen in the absence of estradiol. The effects of estradiol were also blocked by rapamycin (Fig. 9BGo), suggesting that activation of mTOR-p70S6 kinase is involved in the effects of estradiol on IGFBP-2 expression in MCF-7 cells.


Figure 9
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  		FIG. 9. Stimulation of IGFBP-2 expression by estradiol is inhibited by IGFR1 or p70S6 kinase blockade. A, MCF-7 cells were incubated with 10 nM estradiol in the presence of the indicated concentration of AG1024 for 24 h, and then media were collected and analyzed for IGFBP-2 by RIA. *, P < 0.001, compared with no AG1024. B, IGFBP-2 was measured in media conditioned by cells treated with the indicated concentration of rapamycin alone (squares) or in the presence of 10 nM estradiol (circles) for 24 h. For each panel of A and B, pooled data from three experiments performed in triplicate are shown. *, P < 0.001, compared with rapamycin in the absence of estradiol.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A potential role for IGFBP-2 in breast cancer is indicated by a recent study demonstrating overexpression of the protein in breast carcinoma in situ and invasive breast cancer, compared with normal glandular or hyperplastic breast tissue (13). This suggests that in some breast cancers, as in some other malignant diseases, abrogation of IGFBP-2 expression may be of therapeutic benefit. In this study we investigated the regulation of IGFBP-2 expression using the ER-positive breast cancer cell line, MCF-7, which constitutes an excellent model for this because it has high endogenous expression of IGFBP-2 and responds to steroids and IGFs, which have been implicated in both malignant disease and IGFBP-2 expression.

Expression of IGFBP-2 by MCF-7 cells was first described by Clemmons et al. (27), who demonstrated a positive relationship between secreted IGFBP-2 levels and ER status in breast cancer cell lines. It was subsequently reported that in breast tumor tissue, there was no clear relationship between expression of the two markers (28), suggesting that the findings made in vitro did not reflect the pattern of expression of IGFBP-2 in vivo. We have now shown in more than 40 breast tumor cytosols that there is an association between expression of the ER and IGFBP-2. Whereas the cause of the discrepancy between our findings and those of Pekonen et al. (28) is not known, it may be due to the different methods used to analyze IGFBPs in the two studies. We used a highly specific and sensitive RIA to quantify IGFBP-2 in tumor cytosols, whereas the earlier study based its report on RT-PCR analysis of a very small number of tumors (three ER negative and two ER positive) and ligand blot analysis of tissue homogenates. This latter technique assigns IGFBPs according to their apparent molecular mass by SDS-PAGE, and the 34-kDa protein presumed to be IGFBP-2 in that study may have been IGFBP-6, expression of which we have shown does not correlate with ER status. Consistent with earlier studies (27, 29, 30), we have also shown that IGFBP-3 is highly expressed both in ER-negative breast tumor tissue and ER-negative breast cancer cells in vitro.

In MCF-7 breast cancer cells, IGFBP-2 expression was found to be regulated predominantly through the PI3-kinase/Akt/mTOR signaling pathway, an important cell survival pathway, which is dysregulated in many cancers. LY294002, which inhibits PI3-kinase activity upstream of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and Akt/PKB, and rapamycin, which targets mTOR downstream of these molecules, markedly reduced IGFBP-2 levels in MCF-7 cell conditioned medium. By contrast, inhibition of the p38 MAPK pathway had no effect on IGFBP-2 levels, whereas p44/42 MAPK blockade using PD98059 resulted in only a slight decrease in IGFBP-2 expression. However, the PI3-kinase signaling pathway appears to be necessary but not sufficient for maximal IGFBP-2 expression because insulin and EGF failed to stimulate IGFBP-2 expression at concentrations that increased phosphorylation of p70S6K.

Two previous studies addressed the possible involvement of the PI3-kinase pathway in IGFBP-2 expression by overexpressing the tumor suppressor PTEN, a key negative regulator of PI3-kinase signaling, in PTEN-null cell lines (39, 40). In two human gastric carcinoma cell lines, increased expression of PTEN (and therefore reduced activation of the PI3-kinase pathway) did not change expression of IGFBP-2 mRNA (39), whereas in the glioma cell line U251, IGFBP-2 mRNA and protein expression appeared decreased by PTEN induction (40). This latter observation is consistent with the findings of the present study, and considered together, they indicate that manipulation of PI3-kinase signaling either by reexpression of an endogenous antagonist or by pharmacological means can have a marked effect on IGFBP-2 expression. It is interesting to note that no effect of the rapamycin analog CCI-779 on IGFBP-2 expression was apparent in the study of Levitt et al. (40) in U251 cells, whereas we found that rapamycin markedly inhibited IGFBP-2 expression in MCF-7 cells; however, U251 cells are relatively resistant to rapamycin (41), which may explain the finding in those cells.

Consistent with the observation that decreased PI3-kinase signaling reduces IGFBP-2 expression, activation of this pathway by IGF-I increased IGFBP-2 levels in MCF-7 cells. This was abolished by LY294002 and rapamycin but not PD98059, suggesting that like basal IGFBP-2 expression, IGF-stimulated IGFBP-2 expression is mediated by PI3-kinase signaling. Whereas the potency of LY294002 was similar in IGF-unstimulated and -stimulated cells, a 10-fold higher concentration of rapamycin was required to inhibit IGF-stimulated IGFBP-2 expression, compared with IGF-unstimulated cells. Whereas the cause of this is not known, it may be due to IGF-I altering expression of proteins, which can affect cellular sensitivity to rapamycin. For example, IGF-I has been shown to increase expression of members of the family of Bcl-2 proteins, including Bcl-2 itself and Bcl-xL (42, 43, 44), and these antiapoptotic proteins have been shown to confer increased resistance to the effects of rapamycin in animal models in vivo (45).

The effect of IGF-I was dependent on interaction with IGFR1 because an IGF analog with reduced affinity for this receptor, Leu24Ala31-IGF-I, had no effect on IGFBP-2 levels, whereas LR3-IGF-I, which binds with near normal affinity to IGFR1 but not IGFBPs (32), increased IGFBP-2 with greater potency than IGF-I. A similar finding was made in a study of transcriptional regulation of IGFBP-2 by IGFs, in which IGFBP-2 promoter activity was stimulated to a greater level by IGF analogs with reduced affinity for IGFBPs than wild-type IGF-I (21). We also found that AG1024, a highly specific tyrosine kinase inhibitor that exhibits significantly lower IC50 for the IGFR1 than the insulin receptor (46), blocked the effect of IGF-I on IGFBP-2 expression, providing further evidence of the involvement of the IGFR1 in IGF-stimulated IGFBP-2 expression in MCF-7 cells. It was interesting to note that despite completely blocking the stimulatory effect of IGF-I on IGFBP-2 expression, AG1024 did not completely abolish IGFR1 phosphorylation. The reason for this apparent discrepancy is not clear; however, it is quite difficult to relate the degree of IGFR1 phosphorylation with the change in IGFBP-2 expression because there is clearly not a one-to-one relationship between the two. For example, quantification of the phosphorylated IGFR1 immunoblots (shown in Figs. 3Go and 6Go) indicate that receptor phosphorylation is increased greater than 20-fold by IGF-I, whereas the increase in IGFBP-2 in response to IGF-I is closer to 2-fold. It is also possible that receptor phosphorylation and activation needs to be above a threshold level before significant changes in downstream events, such as IGFBP-2 expression, can be brought about.

Phosphotyrosine analysis of IGFR1 immunoprecipitates revealed a low level of IGFR1 phosphorylation in the absence of exogenous ligand, which was not detectable with the phosphospecific IGFR1 antibody, possibly because of insensitivity of this method of analysis or because the receptor was phosphorylated at a site different from the one being probed. The underlying cause of IGFR1 phosphorylation in MCF-7 cells in the absence of exogenous ligand is not clear. Expression of IGFs by MCF-7 breast cancer cells (or other breast epithelial cells in vitro or in vivo) has not been clearly demonstrated, and it is generally thought that the source of mitogenic IGF receptor ligands in the breast is from stromal tissue (47). However, other molecules have been shown to transactivate IGFR1, including the EGF receptor (48), estrogen (49), and angiotensin II (50), and it is possible that such a mechanism is responsible for the basal IGFR1 activation observed in the present study. Phosphorylation of p70S6K was also apparent in the absence of exogenous IGF-I, and this was reduced, although not abolished, by AG1024 over the same concentration range at which IGFR1 phosphorylation was inhibited. This suggests that some, but not all, of the basal phosphorylation of p70S6K is secondary to endogenous IGFR1 activation. Identifying other factors involved in endogenous activation of the PI3-kinase pathway, and their role in IGFBP-2 expression, is an important area for further study.

A link between IGFBP-2 expression and hypoxia was suggested in two earlier studies, with a requirement for HIF-1{alpha} for maximal expression of IGFBP-2 described in mouse embryonic fibroblasts (33), and hypoxia shown to increase IGFBP-2 in neonatal rat retina (51). Furthermore, HIF-1{alpha} is induced by IGFs in a number of cell types (34, 35, 36), raising the possibility that this transcription factor is mediating the effect of IGF-I on IGFBP-2 expression in MCF-7 cells. However, we found no evidence of association between the levels of IGFBP-2 and HIF-1{alpha} in MCF-7 breast cancer cells. Under conditions in which IGFBP-2 was very highly expressed, HIF-1{alpha} was barely detectable by immunoblot, and elevated HIF-1{alpha} in response to CoCl2 was not associated with increased IGFBP-2; in fact, a decrease in IGFBP-2 at the highest doses of CoCl2 was associated with reduced PI3-kinase/Akt/mTOR pathway activity. In addition, topotecan, a potent inhibitor of HIF-1{alpha} expression, was only slightly inhibitory to IGFBP-2 at doses far exceeding the reported EC50 for this agent. Taken together, these findings argue against an important role for HIF-1{alpha} in IGFBP-2 expression in MCF-7 cells. The reason for the discrepancy between our findings and those of the earlier studies is not clear but may reflect cell-specific differences in HIF-1{alpha} regulation of genes, as has been suggested by profiling of hypoxia-induced genes in cells of different origin (52). Clearly, understanding the significance of induction of IGFBP-2 by hypoxia and HIF-1{alpha} in some cell types, but not others, represents an important area for further study.

Immunoblot analysis was previously used to show that estradiol and IGF-I independently increase IGFBP-2 in MCF-7 conditioned medium (27, 53), but a synergistic effect of the two on IGFBP-2 expression has not been examined in other studies. Interaction between the estrogen and IGF systems is a well-recognized phenomenon and occurs on a number of levels. Estrogen modulates expression of IGFR1, its ligands, or regulatory IGFBPs (reviewed in Ref. 47), and ER{alpha} can induce phosphorylation of the IGFR1 (54) apparently through formation of a ternary complex comprising the two receptors and the docking protein Shc (49). Signaling intermediates downstream of activated IGFR1, such as p44/42 MAPK, may also phosphorylate ER (55). In our cell system, there was only a slight (10%) increase in IGFR1 expression in response to estradiol, but IGF-stimulated IGFR1 phosphorylation was increased more than 3-fold by estradiol, suggesting that the mechanism of estradiol’s effect in this instance is not simply through increased IGFR1 expression. Inhibition of IGFR1 tyrosine kinase activity reversed the stimulatory effect of estradiol in the absence of IGF-I, indicating involvement of the receptor in estradiol’s independent effect on IGFBP-2 expression. Whereas delineating how these growth-regulatory systems interact to affect IGFBP-2 expression remains an important goal, the collective findings of this study indicate that inhibition of either IGFR1 or the PI3-kinase/Akt/mTOR pathway can block IGF- and estradiol-stimulated IGFBP-2 expression. The importance of this pathway in many processes central to tumorigenesis, including cell survival, metabolism, cell migration, invasion, and angiogenesis, have already made it an attractive target for the development of anticancer agents. Inhibitors of components of PI3-kinase pathway such as protein-dependent kinase-1, Akt, and mTOR are showing promise in clinical trials for the treatment of a variety of malignancies (56). The possibility that part of their efficacy lies in reducing the expression of growth-stimulatory IGFBP-2 is intriguing and clearly warrants further investigation.


   Acknowledgments
 
The authors thank Dr. Diana Benn (Kolling Institute of Medical Research) for providing the breast tumor cytosols and data regarding their ER status and Dr. Stephen Philcox (Northern Clinical School, University of Sydney) for the analysis of IGFBPs in tumor cytosols.


   Footnotes
 
This work was supported by National Health and Medical Research Council Grant 302153 (to J.L.M. and R.C.B.) and a Cancer Institute New South Wales (NSW) Career Development and Support Fellowship (to J.L.M.). J.L.M. is a Cancer Institute NSW Fellow.

Disclosure Statement: The authors have nothing to declare.

First Published Online February 8, 2007

Abbreviations: ECL, Enhanced chemiluminescence; EGF, epithelial growth factor; ER, estrogen receptor; ER+ve, ER positive; ER–ve, ER negative; HIF, hypoxia-inducible factor; IGFBP, IGF binding protein; IGFR1, type 1 IGF receptor; mTOR, mammalian target of rapamycin; PI3-kinase, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog deleted from chromosome 10; RIPA, radioimmunoprecipitation assay; SDS, sodium dodecyl sulfate; SFM, serum-free medium; TBS-T, Tris-buffered saline with Tween 20.

Received September 28, 2006.

Accepted for publication February 1, 2007.


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 Introduction
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 Results
 Discussion
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