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Endocrinology |
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [M. B., C. M., L. M., E. S.], and Department of Cellular Biology, University of Calabria, Calabria 87036, Italy [M. B., C. M., L. M., S. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we examined IGF-IR signaling and function in ER-negative MDA-MB-231 breast cancer cells and their IGF-IR-overexpressing derivatives. We demonstrated that IGF-I acts as a chemoattractant for these cells. The extent of IGF-I-induced migration reflected IGF-IR levels and required the activation of phosphatidylinositol 3-kinase (PI-3K) and p38 kinases. The same pathways promoted IGF-I-dependent motility in ER-positive MCF-7 cells. In contrast with the positive effects on cell migration, IGF-I was unable to stimulate growth or improve survival in MDA-MB-231 cells, whereas it induced mitogenic and antiapoptotic effects in MCF-7 cells. Moreover, IGF-I partially restored growth in ER-positive cells treated with PI-3K and ERK1/ERK2 inhibitors, whereas it had no protective effects in ER-negative cells. The impaired IGF-I growth response of ER-negative cells was not caused by a low IGF-IR expression, defective IGF-IR tyrosine phosphorylation, or improper tyrosine phosphorylation of IRS-1. Also, the acute (15-min) IGF-I activation of PI-3 and Akt kinases was similar in ER-negative and ER-positive cells. However, a chronic (2-day) IGF-I exposure induced the PI-3K/Akt pathway only in MCF-7 cells. The reactivation of this pathway in ER-negative cells by overexpression of constitutively active Akt mutants was not sufficient to significantly improve proliferation or survival (with or without IGF-I), which indicated that other pathways are also required to support these functions.
Our results suggest that in breast cancer cells, IGF-IR can control nonmitogenic processes regardless of the ER status, whereas IGF-IR growth-related functions may depend on ER expression.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The IGF-IR is necessary for normal breast biology, but recent clinical and experimental data strongly suggest that the same receptor is involved in the development of breast cancer (1 , 3) . The IGF-IR is overexpressed (up to 14-fold) in ER-positive breast cancer cells compared with its levels in normal epithelial cells (1 , 4 , 5) . The elevated expression and hyperactivation of the IGF-IR has been linked with increased radioresistance and cancer recurrence at the primary site (4) . Similarly, high levels of IRS-1, a major signaling molecule of the IGF-IR, correlated with tumor size and shorter disease-free survival in ER-positive tumors (6 , 7) .
IGF-IR ligands, IGF-I and IGF-II, are strong mitogens for many hormone-dependent breast cancer cell lines and have been found in the epithelial and/or stromal component of breast tumors (1) . Importantly, higher levels of circulating IGF-I predict increased breast cancer risk in premenopausal women (8) . In vitro, activation of the IGF-IR, especially the IGF-IR/IRS-1/PI-3K pathway in ER-positive breast cancer cells, counteracts apoptosis induced by different anticancer treatments or low concentrations of hormones (1 , 9, 10, 11) . On the other hand, overexpression of either the IGF-IR or IRS-1 in ER-positive breast cancer cells improves responsiveness to IGF and, in consequence, results in estrogen-independent proliferation (1 , 12 , 13) . In agreement with these observations, blockade of IGF-IR activity with various reagents targeting the IGF-IR or its signaling through IRS-1/PI-3K reduced the growth of breast cancer cells in vitro and/or in vivo (1 , 12 , 14, 15, 16, 17) .
The requirement for the IGF-IR/IRS-1 pathway for growth and survival appears to be a characteristic of ER-positive, more differentiated, breast cancer cells. By contrast, ER-negative tumors and cell lines, often exhibiting less differentiated, mesenchymal phenotypes, express low levels of the IGF-IR and often decreased levels of IRS-1 (1 , 17 , 18) . Notably, these cells do not respond to IGF-I with growth (1 , 19, 20, 21, 22) . Despite the lack of IGF-I mitogenic response, the metastatic potential of ER-negative breast cancer cells can be effectively inhibited by different compounds targeting the IGF-IR. For instance, blockade of the IGF-IR in MDA-MB-231 cells by an anti-IGF-IR antibody reduced migration in vitro and tumorigenesis in vivo, and expression of a soluble IGF-IR in MDA-MB-435 cells inhibited adhesion on the extracellular matrix and impaired metastasis in animals (14 , 16 , 23) . These observations suggested that in ER-negative cells, some functions of the IGF-IR must be critical for metastatic cell spread. Here we addressed the possibility that in ER-negative cells, the IGF-IR selectively promotes growth-unrelated processes, such as migration and invasion, but is not engaged in the transmission of growth and survival signals. Using ER-negative MDA-MB-231 breast cancer cells, we studied IGF-I-dependent pathways involved in migration and the defects in IGF mitogenic signal. For comparison, relevant IGF-I responses were analyzed in ER-positive MCF-7 cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
MDA-MB-231 cells were obtained from American Type Culture Collection. MDA-MB-231/IGF-IR clones were generated by stable transfection of MDA-MB-231 cells with the plasmid pcDNA3-IGF-IR using a standard calcium phosphate precipitate procedure (13)
. Transfectants resistant to 1 mg/ml G418 were screened for IGF-IR expression by fluorescence-assisted cell sorting analysis using an anti-IGF-IR mouse mAb 
-IR3 (10 µg/ml; Calbiochem) and a fluorescein-conjugated goat antimouse IgG2 (1 µg/ml; Calbiochem). Cells stained with the secondary antibody alone were used as a control. Additionally, the parental MDA-MB-231 cells and MCF-7/IGF-IR clones 12 and 15 (13)
, all expressing known levels of the IGF-IR, were analyzed in parallel. IGF-IR expression in MDA-MB-213-derived clones was then confirmed by WB with specific antibodies (listed below). In growth and migration experiments, we used control MCF-7/pc2 and MDA-MB-231/5 M cell lines, which have been developed by transfection of MCF-7 and MDA-MB-231 cells with the pcDNA3 vector. MCF-7, MCF-7/pc2, and MCF-7/IGF-IR cells were described in detail previously (13)
.
Transient Transfection.
Seventy % confluent cultures of MDA-MB-231 and MCF-7 cells were transiently cotransfected with an Akt expression plasmid and a plasmid pCMS encoding GFP (Akt:GFP ratio, 20:1) using Fugene 6 (Roche). Transfection was carried out for 6 h in phenol red-free DMEM containing 0.5 mg/ml BSA, 1 µM FeSO4, and 2 mM L-glutamine (referred to as PRF-SFM; Ref. 13
); the optimal DNA:Fugene 6 ratio was 1 µg:3 µl. Upon transfection, the cells were shifted to fresh PRF-SFM, and the expression of total and active Akt kinase at 0 (media shift), 2, and 4 days was assessed by WB with specific antibodies (see below). In parallel, the efficiency of transfection was evaluated by scoring GFP-positive cells. In all experiments, at least 40% of transfected cells expressed GFP, which indicated a high transfection efficiency. In addition, the expression of Akt plasmids was monitored by measuring the cellular levels of HA-tag and Akt proteins by WB.
Cell Culture.
MDA-MB-231 and MCF-7 cells were grown in DMEM:F12 (1:1) containing 5% CS. MDA-MB-231- and MCF-7-derived clones overexpressing the IGF-IR or expressing vector alone were maintained in DMEM:F12 plus 5% CS plus 200 µg/ml G418. In the experiments requiring 17ß-estradiol- and serum-free conditions, the cells were cultured in PRF-SFM (13)
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Growth Curves.
To analyze the growth in serum-containing medium, the cells were plated in six-well plates in DMEM:F12 (1:1) containing 5% CS at a concentration of 1.5–2.0 x 105 cells/plate; the number of cells was then assessed by direct counting at 1, 2, and 4 days after plating. To study IGF-I-dependent proliferation, the cells were plated in six-well plates in the growth medium as above. The following day (day 0), the cells at 
50% confluence were shifted to PRF-SFM containing 20 ng/ml IGF-I. Cell number was determined at days 1, 2, and 4.
Apoptosis Assay.
The cells grown on coverslips in normal growth medium were shifted to PRF-SFM at 70% confluence and then cultured in the presence or absence of 20 ng/ml IGF-I for 0, 12, 24, 48, and 96 h. Apoptosis in the cultures was determined with the In Situ Cell Death Detection kit, Fluorescein (Roche), following the manufacturer’s instructions. The cells containing DNA strand breaks were stained with fluorescein-dUTP and detected by fluorescence microscopy. Cells that detached during the experiment were spun on glass slides using cytospin and processed as above. Apoptotic index (the percentage of apoptotic cells/total cell number in a sample field) was determined for adherent and floating cell populations, and the indices were combined.
Immunoprecipitation and Western Blotting.
Seventy % cultures were shifted to PRF-SFM for 24 h and then stimulated with 20 ng/ml IGF-I for 15 min, 1 h, 1 day, or 2 days. Proteins were obtained by lysing the cells in a buffer composed of 50 mM HEPES (pH 7.5), 150 mM 1% Triton X-100, 1.5 mM MgCl2, 1 mM CaCl2, 5 mM EGTA, 10% glycerin, 0.2 mM Na3VO4, 1% phenylmethylsulfonyl fluoride, and 1% aprotinin. The IGF-IR was immunoprecipitated from 500 µg of protein lysate with anti-IGF-IR mAb (Calbiochem) and subsequently detected by WB with anti-IGF-IR pAb (Santa Cruz Biotechnology). IRS-1 was precipitated from 500 µg of lysate with anti-IRS-1 pAb (UBI) and detected by WB using the same antibody. Tyrosine phosphorylation (PY) of immunoprecipitated IRS-1 or IGF-IR was assessed by WB with anti-phosphotyrosine mAb PY20 (Transduction Laboratories). Akt, ERK1/ERK2, and p38 MAPKs (active and total), and active GSK-3 were measured by WB in 50 µg of whole cell lysates with appropriate antibodies from New England Biolabs. The expression of HA-tag was probed by WB in 50 µg of protein lysate with anti-HA mAb (Babco). The intensity of bands representing relevant proteins was measured by laser densitometry scanning.
IRS-1-associated PI-3K Activity.
PI-3K activity was determined in vitro, as described by us before (25)
. Briefly, 70% cultures were synchronized in PRF-SFM for 24 h and then stimulated with 20 ng of IGF-I for 15 min or 2 days. Untreated cells were used as controls. IRS-1 was precipitated from 500 µg of cell lysates; IRS-1 IPs were then incubated in the presence of inositol and [32P]ATP for 30 min at room temperature. The products of the kinase reaction were analyzed by TLC using TLC plates (Eastman Kodak). Radioactive spots representing phosphatidylinositol-3-phosphates were visualized by autoradiography, quantified by laser densitometry (ULTRO Scan XL, Pharmacia), and then excised from the plates and counted in a beta counter.
Motility Assay.
Chemotaxis and chemokinesis were tested in modified Boyden chambers containing porous (8-mm), polycarbonate membranes. The membranes were not coated with extracellular matrix. Briefly, 2 x 104 cells (synchronized in PRF-SFM for 24 h) were suspended in 200 µl of PRF-SFM and plated into upper wells. Lower wells contained 500 µl of PRF-SFM. To study chemotaxis, IGF-I (20 ng/ml) was added to lower wells only; to assess chemokinesis, IGF-I was placed in either upper wells only, or in both wells. After 24 h, the cells in the upper wells were removed, whereas the cells that migrated to the lower wells were fixed and stained in Coomassie Blue solution (0.25 g of Coomassie blue:45 ml water:45 ml methanol:10 ml glacial acetic acid) for 5 min. After that, the chambers were washed three times with H2O. The cells that migrated to the lower wells were counted under the microscope (10
, 26)
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Inhibitors of PI-3K and MAPK.
LY294002 (Biomol Research Labs) was used to specifically inhibit PI-3K (27)
. UO126 (Calbiochem), a specific inhibitor of MEK1/2, was used to block ERK1 and ERK2 kinases (28)
, and SB203580 (Calbiochem) was used to down-regulate p38 MAPK (29)
. To determine optimal concentrations of the compounds, different doses (1–100 µM) of the inhibitors were tested in cells treated for 1, 8, 12, and 24 h in PRF-SFM. Additionally, the efficacy of all inhibitors in blocking the phosphorylation of relevant downstream targets (Akt, ERK1/ERK2, and p38 kinases) was determined by WB. In this experiment, the cells were stimulated with IGF-I (20 ng/ml) for 15 min. LY294002 and UO126 were supplemented simultaneously with IGF-I, whereas SB203580 was added 30 min before IGF-I treatment. Ultimately, for both growth and migration experiments, LY294002 was used at the concentration 50 µM, UO126 at 5 µM, and SB203580 at 10 µM. At these doses, the inhibitors did not affect cell proliferation and survival at 24 h, with the exemption of LY294002, which inhibited (by 20%) the proliferation of MCF-7/IGF-IR clone 12 in PRF-SFM. A shorter treatment (12 h) with LY294002 had no impact on the growth and survival of the cells (evaluated by cell proliferation and In Situ Cell Death Detection assays, as described above). Thus, the effects of LY294002 on migration were assessed at 12 h, whereas the actions of UO126 and SB203580 were assessed at 24 h of treatment.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.5 x 104 IGF-IRs; Refs. 30
, 31
), our first goal was to test whether increasing IGF-IR expression would induce IGF-I-dependent growth in MDA-MB-231 cells. To this end, several MDA-MB-231 clones overexpressing the IGF-IR (MDA-MB-231/IGF-IR cells) were generated by stable transfection, and the receptor content was analyzed by binding assay, fluorescence-assisted cell sorting analysis (data not shown), and WB (Fig. 1)
. We determined that MDA-MB-231 clones 2, 21, and 31 express approximately 3 x 104, 1.5 x 104, and 2.5 x 105 IGF-IRs/cell, respectively, whereas the parental MDA-MB-231 cells express approximately 7 x 103 IGF-IRs/cell (19)
. For comparison, 6 x 104 IGF-IRs were found in ER-positive MCF-7 cells (Fig. 1
; Ref. 13
).
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2.5 x 105 IGF-IRs/cell), an evident growth retardation at days 2 and 4 (P < 0.05) was observed (Fig. 2A)
. In contrast, similar overexpression of the IGF-IR in ER-positive MCF-7 cells significantly augmented proliferation (Fig. 2B)
. The growth of control clones MDA-MB-231/5 M and MCF-7/pc2 was comparable with that of the corresponding parental cell lines (Fig. 2)
.
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. In all ER-negative cell lines, the rate of cell death was significantly increased at days 2 and 4 of the experiment. Notably, at these later time points, MDA-MB-231/IGF-IR clone 31 was dying faster in the presence of IGF-I than in PRF-SFM and more rapidly than the parental cells (Fig. 3A
and data not shown). Conversely, in ER-positive cells, the stimulation of the IGF-IR always induced proliferation. In addition, at later time points, especially at day 4, the growth rate in IGF-I was significantly (P < 0.05) increased in MCF-7/IGF-IR cells relative to that in MCF-7 or MCF-7/pc2 cells (Fig. 3B)
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3-fold, in ER-positive cells, but it was totally ineffective in MDA-MB-231 and MDA-MB-231/IGF-IR cells (Table 1)
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Because both acute and chronic effects of growth factors are important for biological response (36)
, we studied IGF-IR signaling at different times after stimulation: 15 min, 1 h, 2 days, and 4 days. In both ER-positive and ER-negative cell types, IGF-I signaling seen at 15 min was identical to that at 1 h, whereas IGF-I response at 2 days was similar to that at 4 days. Thus, Fig. 4
demonstrates the representative results obtained with cells stimulated for 15 min and 2 days.
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. Analogous IGF-I effects were seen in MCF-7 cells and their IGF-IR-overexpressing derivatives (Fig. 4B)
. A basal level of IGF-IR and IRS-1 tyrosine phosphorylation was observed in cells expressing high receptor levels. This effect most likely can be attributed to the autocrine stimulation of the IGF-IR by IGF-I-like factors (12)
.
One of the major growth/survival pathways initiated at IRS-1 is the PI-3K pathway (32
, 37)
. The repeated measurements of IRS-1-associated PI-3K activity in vitro demonstrated that at 15 min after IGF-I addition, PI-3K activity was similar in both cell types, but at 2 days, in MDA-MB-231 and MDA-MB-231/IGF-IR cells, IGF-I did not stimulate PI-3K through IRS-1, or induced it very weakly, whereas in MCF-7 and MCF-7/IGF-IR cells, a significant level of PI-3K activation was observed (Fig. 5)
.
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. Akt is known to phosphorylate (on Ser-9) and down-regulate GSK-3ß (23
, 32
, 34)
. We found that in both cell types, the phosphorylation of GSK-3ß reflected the dynamics of Akt activity, with no induction of phosphorylation observed at 2 days in ER-negative cells (Fig. 4C)
and IGF-I-stimulated phosphorylation in MCF-7 and MCF-7/IGF-IR cells (by 40 and 120%, respectively; Fig. 4D
).
Another IGF-IR growth/survival pathway involves ERK1 and ERK2 kinases (1
, 36
, 38)
. This pathway was strongly up-regulated at 15 min and weakly induced at 2 days in MCF-7 and MCF-7/IGF-IR cells. In MDA-MB-231 and MDA-MB-231/IGF-IR cells, the basal activation of ERK1/2 kinases was always high, and the addition of IGF-I only minimally (10–20%) induced the enzymes at 15 min, with no effects seen at 2 days (Fig. 4, E and F)
.
p38, a stress-induced MAPK and a known mediator of nongrowth responses in breast cancer cells (35)
, was strongly stimulated by IGF-I in ER-negative cells at 15 min (Fig. 4E)
. By contrast, in ER-positive cells, the enzyme was much stronger when induced at 2 days than at 15 min (Fig. 4F)
. The stimulation of SHC, a substrate of the IGF-IR involved in migration and growth in ER-positive cells (10
, 26)
, was weak in all cell types, and no differences in the activation patterns were observed (data not shown).
Reactivation of Akt Kinase in MDA-MB-231 Cells.
Previous results indicated that MDA-MB-231 and MDA-MB-231/IGF-IR cells are unable to sustain IGF-I-dependent activation of the PI-3K/Akt survival pathway when cultured in the absence of serum for 2–4 days. Consequently, we tested whether cell death under PRF-SFM conditions can be reversed by a forced overexpression of the Akt kinase. Two different expression plasmids encoding constitutively active forms of Akt, Myr-Akt, and Akt/E40K (24)
were transiently transfected into MDA-MB-231 cells. The efficiency of transfection was at least 40% (by scoring GFP-positive cells); correspondingly, the transfected cells expressed elevated (by 40%) levels of the Akt protein and exhibited enhanced Akt phosphorylation (Fig. 6A)
. The improved biological activity of Akt in the transfected cells was indicated by down-regulation of the prolonged ERK1/2 stimulation (39
, 40)
, which was noticeable at day 2 (data not shown) and most pronounced at day 4 (50 and 40% for Myr-Akt and Akt/E40K, respectively; Fig. 6B
) The expression of constitutively active Akt mutants was reflected by a tendency of MDA-MB-231 cells to survive better at 2 days after transfection (at the time of the greatest Akt activity), but the differences did not reach statistical significance (P > 0.05; Fig. 6C
and data not shown).
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. Table 2
demonstrates the impact of the compounds on cell growth/survival at 2 days of treatment. The inhibition of PI-3K with LY294002 reduced the growth of MCF-7 and MCF-7/IGF-IR cells, but it did not influence or had only minimal effects on MDA-MB-231 and MDA-MB-231/IGF-IR cells. Furthermore, the action of LY294002 was counteracted by IGF-I in ER-positive, but not in ER-negative, cells. The inhibition of MEK1/2 and ERK1/ERK2 with UO126 reduced the growth and/or survival in both cell types, but only in MCF-7 and MCF-7/IGF-IR cells was IGF-I able to oppose this effect. Down-regulation of p38 kinase with SB203580 reduced the survival of MDA-MB-231 and MDA-MB-231/IGF-IR cells and to a lesser extent the growth and survival of MCF-7 and MCF-7/IGF-IR cells. IGF-I did not reverse the antimitogenic action of the p38 kinase inhibitor in either of the cell lines studied (Table 2)
. Cumulatively, these results suggested that in ER-positive cells, IGF-I transmits mitogenic signals through PI-3K and ERK1/ERK2 pathways. By contrast, IGF-I does not induce growth or survival signal through these pathways in ER-negative cells.
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. The addition of IGF-I to the upper well or both upper and lower wells always suppressed chemotaxis of all cell lines (Table 3)
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, down-regulation of PI-3K with LY294002 inhibited basal migration of both cell types, with a more pronounced effect in ER-negative cells. Similarly, blockade of p38 kinase inhibited motility of all cell lines studied. The inhibition of MEK1/2 and ERK1/2 with UO126 never suppressed the migration of ER-positive and ER-negative cells; in fact, the compound stimulated cell motility. The addition of IGF-I as a chemoattractant significantly counteracted the effects of all three inhibitors; however, no clear association between the cellular levels of the IGF-IR and this competing action of IGF-I was noted (Table 4)
. These results suggested that IGF-I-dependent motility in both types of cells requires the PI-3K and p38 pathways but does not rely on the activity of ERK1/ERK2.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In breast cancer cell lines, a hormone-dependent and less aggressive phenotype correlates with a good IGF-IR expression (1 , 19 , 42) . By contrast, different ER-negative, breast cancer cell lines express low levels of the IGF-IR and generally do not respond to IGF-I with growth (1 , 18, 19, 20, 21, 22) . However, many ER-negative cell lines appear to depend on the IGF-IR for tumorigenesis and metastasis. For instance, blockade of the IGF-IR in MDA-MB-231 cells by anti-IGF-IR antibody reduced migration in vitro and tumorigenesis in vivo, and expression of a soluble IGF-IR in MDA-MB-435 cells impaired growth, tumorigenesis, and metastasis in animal models (1 , 14 , 16 , 23) . These observations suggest that some growth-unrelated pathways of the IGF-IR may be operative in the context of ER-negative cells.
Here we studied whether this particular IGF-I dependence of ER-negative breast cancer cells relates to the nonmitogenic function of the IGF-IR, such as cell migration. Our experiments indicated that the IGF-IR is an effective mediator of cell motility. Furthermore, IGF-I-induced migration was proportional to IGF-IR content. We demonstrated, for the first time, that in MDA-MB-231 ER-negative cells, IGF-IR signaling pathways responsible for cell movement include PI-3K and p38 kinases. Indeed, an acute IGF-I stimulation of MDA-MB-231 and MDA-MB-231/IGF-IR cells appears to induce both PI-3K and p38 kinases, suggesting that this short-time activation may be involved in migration. Both of these pathways have been shown previously to regulate cell motility in breast cancer cells and other cell types (35
, 44)
. Interestingly, the migration of both ER-negative and ER-positive cells was enhanced by a specific MEK1/MEK2 inhibitor UO126. We observed this effect over a broad range of UO126 doses (1–10 µM) and in several MDA-MB-231- and MCF-7-derived clones; the same doses always suppressed cell proliferation in serum-containing medium and PRF-SFM (data not shown and Table 2
). These peculiar effects suggest that MEK1/2 may represent a regulatory point balancing mitogenic and nonmitogenic cell responses.
In contrast with the positive effects of IGF-I on cell motility in ER-negative and ER-positive breast cancer cells, this growth factor never stimulated the proliferation of MDA-MB-231 cells, whereas it induced the growth of MCF-7 cells and MCF-7-derived clones overexpressing the IGF-IR. It is has been established by Rubini et al. (30)
and Reiss et al. (31)
that mitogenic response to IGF-I requires a threshold level of IGF-IR expression (in fibroblasts, 1.5 x 104). Here, we demonstrated that increasing the levels of the IGF-IR from 7 x 103 up to 2.5 x 105 and subsequent up-regulation of IGF-IR tyrosine phosphorylation was not sufficient to induce IGF-I-dependent growth of MDA-MB-213 cells. Similar results were obtained by Jackson and Yee (21)
, who showed that overexpression of IRS-1 in ER-negative MDA-MB-435A and MDA-MB-468 breast cancer cells did not stimulate IGF-I-dependent mitogenicity. These authors suggested that the lack of IGF-I response, even in IRS-1-overexpressing ER-negative cells, was related to insufficient stimulation of ERK1/ERK2 and PI-3K pathways (21)
. Defective insulin response in ER-negative cell lines has also been described by Costantino et al. (45)
and linked with an increased tyrosine phosphatase activity.
Our experiments suggested that the lack of IGF-I mitogenicity in MDA-MB-231 and MDA-MB-231/IGF-IR cells was not related to the impaired IGF-IR or IRS-1 tyrosine phosphorylation. The cells were also able to respond to an acute IGF-I stimulation with a marked activation of the PI-3K/Akt and ERK-1/ERK2 pathways. We hypothesize that this transient stimulation could be sufficient to induce some IGF-I response, such as cell motility. Mitogenic response, on the other hand, may rely on a more sustained activation of critical IGF-IR signals, as demonstrated before with mouse embryo fibroblasts (36)
. Indeed, the most significant difference in IGF-I signal between ER-negative and ER-positive cells rested in the impaired long-term stimulation of the PI-3K/Akt pathway; MDA-MB-231 and MDA-MB-231/IGF-IR cells were unable to sustain this IGF-I-induced signal for 1 or 2 days, whereas in MCF-7 and MCF-7/IGF-IR cells, the PI-3K/Akt pathway was still active at this time. The subsequent experiments with MDA-MB-231 cells transfected with constitutively active Akt mutants demonstrated that the increased biological activity of Akt was not sufficient to completely reverse cell death in PRF-SFM (Fig. 6C
and data not shown). This suggested that although a sustained Akt activity could be important in the survival of breast cancer cells, other pathways, or a proper equilibrium between Akt and other pathways (such as ERK1/2), are also critical. The latter possibility could be supported by our finding that hyperactivation of Akt down-regulates the ERK1/2 pathway. Normally, this pathway appears to play a role in the survival of ER-negative cells (Table 2)
.
In summary, our data suggest that IGF-IR signaling and function may be different in hormone-dependent and -independent breast cancer cells. In ER-positive MCF-7 cells, IGF-IR transmits various signals, such as growth, survival, migration, and adhesion. In ER-negative MDA-MB-231 cells, the growth-related functions of the IGF-IR become attenuated, but the receptor is still able to control nonmitogenic processes, such as migration. It is likely that this kind of evolution is also involved with the response to other growth factors. Epidermal growth factor, for instance, is an effective mitogen for ER-positive breast cancer cells but does not stimulate the proliferation or survival in MDA-MB-231 cells, despite high EGF-R expression (46)
. However, as demonstrated recently by Price et al. (46)
, EGF is a potent chemoattractant for MDA-MB-231 cells. EGF-induced migration in MDA-MB-231 cells requires PI-3K and phospholipase C
and is not inhibited by antagonists of ERK1/ERK2.
In conclusion, mitogenic and nonmitogenic pathways induced by growth factors in breast cancer cells may be dissociated, and attenuation of one is not necessarily linked with the cessation of the other. Delineating the nonmitogenic responses will be critical for the development of drugs specifically targeting metastatic cells.
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FOOTNOTES |
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1 This work was supported by Department of Defense Breast Cancer Research Program Grants DAMD17-96-1-6250, DAMD17-97-1-7211, and DAMD-17-99-1-9407 and by the American-Italian Cancer Foundation. 
2 To whom requests for reprints should addressed, at Kimmel Cancer Center, Thomas Jefferson University, 233 South Tenth Street, BLSB 631, Philadelphia, PA 19107. Phone: (215) 503-4512; Fax: (215) 923-0249; E-mail: surmacz1{at}jeflin.tju.edu
3 The abbreviations used are: IGF-IR, insulin-like growth factor I receptor; IRS-1, insulin-receptor substrate 1; ER, estrogen receptor; GFP, green fluorescent protein; mAb, monoclonal antibody; WB, Western blot; CS, calf serum; IP, immunoprecipitation; pAb, polyclonal antibody; MAPK, mitogen-activated protein kinase; PI-3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; GSK, glycogen synthase kinase; MEK, MAPK kinase; PRF-SFM, phenol red-free serum-free medium.
Received 8/18/00. Accepted 7/17/01.
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 B. G. Hollier, J. A. Kricker, D. R. Van Lonkhuyzen, D. I. Leavesley, and Z. Upton Substrate-Bound Insulin-Like Growth Factor (IGF)-I-IGF Binding Protein-Vitronectin-Stimulated Breast Cell Migration Is Enhanced by Coactivation of the Phosphatidylinositide 3-Kinase/AKT Pathway by {alpha}v-Integrins and the IGF-I Receptor Endocrinology, March 1, 2008; 149(3): 1075 - 1090. [Abstract] [Full Text] [PDF]
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 H. Gong, P. Guo, Y. Zhai, J. Zhou, H. Uppal, M. J. Jarzynka, W.-C. Song, S.-Y. Cheng, and W. Xie Estrogen Deprivation and Inhibition of Breast Cancer Growth in Vivo through Activation of the Orphan Nuclear Receptor Liver X Receptor Mol. Endocrinol., August 1, 2007; 21(8): 1781 - 1790. [Abstract] [Full Text] [PDF]
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R. X.-D. Song, Z. Zhang, Y. Chen, Y. Bao, and R. J. Santen Estrogen Signaling via a Linear Pathway Involving Insulin-Like Growth Factor I Receptor, Matrix Metalloproteinases, and Epidermal Growth Factor Receptor to Activate Mitogen-Activated Protein Kinase in MCF-7 Breast Cancer Cells Endocrinology, August 1, 2007; 148(8): 4091 - 4101. [Abstract] [Full Text] [PDF]
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 D. Sisci, C. Morelli, C. Garofalo, F. Romeo, L. Morabito, F. Casaburi, E. Middea, S. Cascio, E. Brunelli, S. Ando, et al. Expression of nuclear insulin receptor substrate 1 in breast cancer J. Clin. Pathol., June 1, 2007; 60(6): 633 - 641. [Abstract] [Full Text] [PDF]
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K. Dhar, S. Banerjee, G. Dhar, K. Sengupta, and S. K. Banerjee Insulin-like Growth Factor-1 (IGF-1) Induces WISP-2/CCN5 via Multiple Molecular Cross-talks and Is Essential for Mitogenic Switch by IGF-1 Axis in Estrogen Receptor-Positive Breast Tumor Cells Cancer Res., February 15, 2007; 67(4): 1520 - 1526. [Abstract] [Full Text] [PDF]
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 Z. Ma, S. L. Gibson, M. A. Byrne, J. Zhang, M. F. White, and L. M. Shaw Suppression of Insulin Receptor Substrate 1 (IRS-1) Promotes Mammary Tumor Metastasis Mol. Cell. Biol., December 15, 2006; 26(24): 9338 - 9351. [Abstract] [Full Text] [PDF]
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 S. Maor, D. Mayer, R. I Yarden, A. V Lee, R. Sarfstein, H. Werner, and M. Z Papa Estrogen receptor regulates insulin-like growth factor-I receptor gene expression in breast tumor cells: involvement of transcription factor Sp1 J. Endocrinol., December 1, 2006; 191(3): 605 - 612. [Abstract] [Full Text] [PDF]
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 A. Srirangam, R. Mitra, M. Wang, J. C. Gorski, S. Badve, L. Baldridge, J. Hamilton, H. Kishimoto, J. Hawes, L. Li, et al. Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer Clin. Cancer Res., March 15, 2006; 12(6): 1883 - 1896. [Abstract] [Full Text] [PDF]
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J. Lin, P. M. Ridker, N. Rifai, I-M. Lee, J. E. Manson, J. E. Buring, and S. M. Zhang A Prospective Study of Hemoglobin A1c Concentrations and Risk of Breast Cancer in Women. Cancer Res., March 1, 2006; 66(5): 2869 - 2875. [Abstract] [Full Text] [PDF]
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C. Garofalo, M. Koda, S. Cascio, M. Sulkowska, L. Kanczuga-Koda, J. Golaszewska, A. Russo, S. Sulkowski, and E. Surmacz Increased Expression of Leptin and the Leptin Receptor as a Marker of Breast Cancer Progression: Possible Role of Obesity-Related Stimuli Clin. Cancer Res., March 1, 2006; 12(5): 1447 - 1453. [Abstract] [Full Text] [PDF]
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B. S. Miller and D. Yee Type I Insulin-like Growth Factor Receptor as a Therapeutic Target in Cancer Cancer Res., November 15, 2005; 65(22): 10123 - 10127. [Abstract] [Full Text] [PDF]
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 M. Remacle-Bonnet, F. Garrouste, G. Baillat, F. Andre, J. Marvaldi, and G. Pommier Membrane Rafts Segregate Pro- from Anti-Apoptotic Insulin-Like Growth Factor-I Receptor Signaling in Colon Carcinoma Cells Stimulated by Members of the Tumor Necrosis Factor Superfamily Am. J. Pathol., September 1, 2005; 167(3): 761 - 773. [Abstract] [Full Text] [PDF]
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E. R. Levin Integration of the Extranuclear and Nuclear Actions of Estrogen Mol. Endocrinol., August 1, 2005; 19(8): 1951 - 1959. [Abstract] [Full Text] [PDF]
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J M Gee, J F Robertson, E Gutteridge, I O Ellis, S E Pinder, M Rubini, and R I Nicholson Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S99 - S111. [Abstract] [Full Text] [PDF]
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 Y. Ling, L. A. Maile, J. Lieskovska, J. Badley-Clarke, and D. R. Clemmons Role of SHPS-1 in the Regulation of Insulin-like Growth Factor I-stimulated Shc and Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells Mol. Biol. Cell, July 1, 2005; 16(7): 3353 - 3364. [Abstract] [Full Text] [PDF]
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M Koda, M Sulkowska, L Kanczuga-Koda, and S Sulkowski Expression of insulin receptor substrate 1 in primary breast cancer and lymph node metastases J. Clin. Pathol., June 1, 2005; 58(6): 645 - 649. [Abstract] [Full Text] [PDF]
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 S. C.J.P. Gielen, E. E. Hanekamp, L. J. Blok, F. J. Huikeshoven, and C. W. Burger Steroid-Modulated Proliferation of Human Endometrial Carcinoma Cell Lines: Any Role for Insulin-like Growth Factor Signaling? Reproductive Sciences, January 1, 2005; 12(1): 58 - 64. [Abstract] [PDF]
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 X. Tian, M. R. Aruva, W. Qin, W. Zhu, K. T. Duffy, E. R. Sauter, M. L. Thakur, and E. Wickstrom External Imaging of CCND1 Cancer Gene Activity in Experimental Human Breast Cancer Xenografts with 99mTc-Peptide-Peptide Nucleic Acid-Peptide Chimeras J. Nucl. Med., December 1, 2004; 45(12): 2070 - 2082. [Abstract] [Full Text] [PDF]
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A. Oh, H.-J. List, R. Reiter, A. Mani, Y. Zhang, E. Gehan, A. Wellstein, and A. T. Riegel The Nuclear Receptor Coactivator AIB1 Mediates Insulin-like Growth Factor I-induced Phenotypic Changes in Human Breast Cancer Cells Cancer Res., November 15, 2004; 64(22): 8299 - 8308. [Abstract] [Full Text] [PDF]
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C. Garofalo, D. Sisci, and E. Surmacz Leptin Interferes with the Effects of the Antiestrogen ICI 182,780 in MCF-7 Breast Cancer Cells Clin. Cancer Res., October 1, 2004; 10(19): 6466 - 6475. [Abstract] [Full Text] [PDF]
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 V. Vella, R. Mineo, F. Frasca, E. Mazzon, G. Pandini, R. Vigneri, and A. Belfiore Interleukin-4 Stimulates Papillary Thyroid Cancer Cell Survival: Implications in Patients with Thyroid Cancer and Concomitant Graves' Disease J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2880 - 2889. [Abstract] [Full Text] [PDF]
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 R. X. Song, C. J. Barnes, Z. Zhang, Y. Bao, R. Kumar, and R. J. Santen The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor {alpha} to the plasma membrane PNAS, February 17, 2004; 101(7): 2076 - 2081. [Abstract] [Full Text] [PDF]
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 D. Sachdev, J. S. Hartell, A. V. Lee, X. Zhang, and D. Yee A Dominant Negative Type I Insulin-like Growth Factor Receptor Inhibits Metastasis of Human Cancer Cells J. Biol. Chem., February 6, 2004; 279(6): 5017 - 5024. [Abstract] [Full Text] [PDF]
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K. Moelling, K. Schad, M. Bosse, S. Zimmermann, and M. Schweneker Regulation of Raf-Akt Cross-talk J. Biol. Chem., August 16, 2002; 277(34): 31099 - 31106. [Abstract] [Full Text] [PDF]
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X. Zhang and D. Yee Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) Inhibits Breast Cancer Cell Motility Cancer Res., August 1, 2002; 62(15): 4369 - 4375. [Abstract] [Full Text] [PDF]
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 J. Albanell and J. Baselga Unraveling Resistance to Trastuzumab (Herceptin): Insulin-Like Growth Factor-I Receptor, a New Suspect J Natl Cancer Inst, December 19, 2001; 93(24): 1830 - 1832. [Full Text] [PDF]
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