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Endocrinology, doi:10.1210/en.2007-0740
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Endocrinology Vol. 149, No. 3 1075-1090
Copyright © 2008 by The Endocrine Society

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

Brett G. Hollier, Jennifer A. Kricker, Derek R. Van Lonkhuyzen, David I. Leavesley and Zee Upton

Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland 4059, Australia

Address all correspondence and requests for reprints to: Brett Hollier, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland 4059, Australia. E-mail: b.hollier{at}qut.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BREAST CANCER is the most common form of cancer in women, with one in nine women developing breast cancer in their lifetime (1). Significantly, one in four women diagnosed will die from their disease. The primary tumor is rarely the cause for the high mortality associated with breast cancer, which, rather, arises from the metastatic dissemination of malignant cells and their establishment in critical sites in the body. Unrestrained cancer cell proliferation does not by itself appear to lead to metastasis. Metastatic dissemination depends on the ability of tumor cells to invade tissue boundaries, migrate and survive in secondary target tissues (2). Two factors thought to be pivotal in breast cancer metastasis are exposure to elevated levels of mitogenic hormones and growth factors and altered cellular interactions with the extracellular matrix (ECM). IGF-I is one such mitogenic growth factor that has been shown to play critical roles in breast cancer biology and is a target for novel therapeutic applications (3).

IGF-I is a potent mitogen involved in normal growth and development and carries out its diverse biological actions through endocrine, paracrine, and autocrine mechanisms (4). In addition to its role in normal development, IGF-I can also mediate growth, metastasis and apoptosis in cancer (5). The actions of IGF-I are mediated primarily through the type I IGF receptor (IGF-IR), and activation of this receptor can modulate processes such as DNA synthesis, cell cycle progression, differentiation, angiogenesis, apoptosis, invasion, and migration (6). Through the IGF-IR, IGF-I can activate multiple signaling pathways, including the phosphatidylinositide 3-kinase (PI3-K) and MAPK pathways, and play an important role in the establishment and maintenance of the transformed phenotype (7). IGF-I also interacts with six IGF-binding proteins (IGFBPs 1–6), which bind IGF-I with a higher affinity than the IGF-IR, increasing the in vivo half-life of IGF-I and also modulating its specificity and availability to bind the IGF-IR (8). IGFBPs have also been shown to have both IGF-dependent and -independent effects in modulating breast cell function (9).

In addition to IGFBPs, ECM proteins have also been demonstrated to modulate cellular responses to IGF-I (10, 11). Indeed, growth factor-ECM interactions are critical for most processes essential to cancer cell metastasis, namely cell attachment, proliferation, migration, differentiation, cell survival, and angiogenesis (12). Nevertheless, studies within our laboratory are among the few that have investigated the functional effects of growth factors and ECM proteins when presented to cells as a substrate-bound complex (13, 14, 15, 16). After the initial discovery that IGF-II binds to the ECM protein vitronectin (VN) (17), novel links between IGF-I, IGFBPs, and VN have also been identified. Specifically, we have shown that whereas IGF-II can bind directly to VN, IGF-I associates with VN indirectly through IGFBP-2, -3, -4, and -5 (15). Moreover, the interaction of IGF-II with VN significantly enhances the migration of the poorly metastatic MCF-7 breast carcinoma cell line (14), and more recently IGF-II-VN and IGF-I-IGFBP-VN complexes were demonstrated to enhance the proliferation and migration of HaCAT human skin keratinocytes (13, 16).

The interaction of IGF-I with VN is of relevance to cancer metastasis, as increased expression of VN and VN-binding integrins has been reported at the leading edge of migrating tumor cells (18, 19, 20). VN has also been shown to be associated with the peritumoral stroma and blood vessels and as deposits in the connective tissue matrix around breast tumors (21, 22, 23). VN contains an arginine-glycine-aspartate recognition sequence by which it binds a variety of integrin receptors, including {alpha}vβ1, {alpha}vβ3, {alpha}vβ5, {alpha}vβ6, {alpha}vβ8, and {alpha}IIbβ3 (24), thereby mediating attachment, spreading, and migration of cells within the ECM. This suggests that the interactions between IGF-I and VN will be of particular importance in tumors in which components of the IGF system are overexpressed, by either the tumor itself or the surrounding stroma.

This study aimed to describe the effects of substrate-bound IGF-I-IGFBP-VN complexes on breast cancer cell migration and dissect the mechanisms underlying these responses. The responses of normal nontumorgenic breast cells were also examined because IGF-I has an important role in normal mammary gland development, with many of these processes reflected in breast cancer progression (25). The data reported herein indicate that the IGF-I-IGFBP-VN complexes enhance cellular migration through increased and sustained activation of the PI3-K/AKT pathway via cooperation between the IGF-IR and the VN-binding {alpha}-v integrins ({alpha}v-integrins). We also demonstrate a pivotal role for AKT-1 (AKT) in IGF-I-IGFBP-VN-stimulated migration as pharmacological inhibition of PI3-K/AKT pathway activation attenuated this response. Furthermore, the overexpression of wild-type and activated AKT further increased the migratory response to these complexes which was still dependent on the involvement of the IGF-IR and {alpha}v-integrins. An increased understanding of the interactions between growth factors and the ECM on modulating cellular functions and in this instance, IGF-I with VN, will be of particular importance to the metastasis of breast cancer in vivo because both of these proteins have been demonstrated to be associated with the dissemination of tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and proteins
Human IGF-I, epidermal growth factor, long R3 (LR3) IGF-I, Des (1–3)-IGF-I, and [Leu24][Ala31]IGF-I ([L24][A31]IGF-I) were purchased from Novozymes (Adelaide, South Australia, Australia). Human VN and anti-ERK 1/2 polyclonal antibody were from Promega (Annandale, New South Wales, Australia), with IGFBP-3 (N109D) and anti-AKT polyclonal antibody from Upstate Biotech (Lake Placid, NY). IGFBP-5 was purchased from Dr. Sue Firth (Kolling Institute of Medical Research, University of Sydney, New South Wales, Australia). Mouse monoclonal antibodies directed against the {alpha}v-integrin subunit (AV1), {alpha}vβ5 (P1F6), {alpha}vβ6 (10D5), β1-subunit (P4C10), and the IgG-matched control antibody were purchased from Chemicon (Temecula, CA) and a monoclonal IGF-IR antibody ({alpha}IR3) was purchased from Merck Biosciences (Kilsyth, Victoria, Australia). For detection of phosphorylated signaling intermediates, antiphospho-ERK1/2 MAPK (Thr 202/Tyr 204) (E10), antiphospho-AKT (S473) (587F11), antiphospho-AKT (T308),and antiphospho-p70S6K (T389) monoclonal antibodies were from Cell Signaling Technology (Beverly, MA). All other reagents were purchased from Sigma-Aldrich (Castle Hill, New South Wales, Australia) unless otherwise stated.

Cell culture
The MCF-7 human breast carcinoma cell line (HTB-22, American Type Culture Collection, Manassas, VA) was obtained from Dr. Steven Meyers (Science Research Centre, Queensland University of Technology, Brisbane, Australia), and MDA-MB-231 cells (HTB-26, American Type Culture Collection) were a kind gift from Dr. Chris Schmidt (Queensland Institute of Medical Research, Brisbane, Australia). MCF-7 cells stably overexpressing the β3 integrin (MCF-7-β3) were a gift from Dr. John Price (St. Vincents Institute of Medical Research, Victoria, Australia). All three breast cancer cell lines were grown in DMEM/Ham’s F-12 (DMEM/F12) media (1:1) (Invitrogen, Mulgrave, Victoria, Australia) containing 10% fetal bovine serum (HyClone, South Logan, UT). The MCF-10A cells, a spontaneously immortalized phenotypically normal breast epithelial cell line (26), were a kind gift from Dr. Robert Pauley (Karmanos Cancer Institute, Detroit, MI) and Dr. Janet Martin (Kolling Institute of Medical Research) and were maintained routinely in DMEM/F12 (1:1) containing 15 mM HEPES, 5% horse serum (Invitrogen), 10 µg/ml bovine insulin, 20 ng/ml epidermal growth factor, 50 ng/ml cholera enterotoxin, and 0.5 µg/ml hydrocortisone. All cultures were passaged every 2–3 d by trypsin/EDTA detachment.

Treatment strategy for in vitro assays
In vivo it is thought that cells within organized tissues respond to growth factors bound within the ECM. Therefore, in an effort to more accurately mimic the in vivo cellular environment this study adopted the strategy of prebinding growth factors and VN to polystyrene cultureware (Nalge Nunc International, Roskilde, Denmark) and the lower chamber and undermembrane surface of 12-µm-pore Transwells (Costar, New York, NY). Hence, tissue culture wells and lower Transwell chambers were precoated with select combinations of VN, IGFBP-3 or -5, and IGF-I using procedures previously reported by our laboratory (13, 14, 15, 16).

Transwell migration assays
Migration assays were performed as previously described by our laboratory (13, 14, 16). Briefly, cells that had been serum starved for 4 h were harvested and seeded at a density of 2 x 105 cells/well in serum-free medium + 0.05% BSA into the upper chamber of Transwell inserts that had the undersurface of the 12-µm-pore membranes and lower chamber wells coated with IGF-I, IGFBP, and VN combinations. In some experiments, the cells were preincubated with the indicated concentrations of inhibitory antibodies for 30 min at room temperature or with pharmacological inhibitors (LY294002 and U0126) for 60 min at 37 C with 5% CO2 before seeding into the Transwell. Plates containing the Transwell inserts were then incubated for 5 h at 37 C with 5% CO2. Cells that had migrated to the lower surface of the membrane were fixed in 3.7% paraformaldehyde and stained with 0.01% Crystal Violet in PBS. The number of cells that had migrated to the lower surface of the porous membrane was then quantified by extracting the crystal violet stain in 10% acetic acid and determining the OD of these extracts at 595 nm (27).

Assessment of cell viability
The assessment of cell viability was performed using the CellTiter 96 AQueous one solution cell proliferation assay [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS); Promega], which is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity, or chemosensitivity assays. To assess cell viability, serum-starved cells that had been pretreated with pharmacological inhibitors LY294002 (20 µM) and U0126 (10 µM) for 60 min at 37 C with 5% CO2, were seeded into 96-well plates at a density of 1 x 104 cells/well. Cells were then incubated for 5 h at 37 C with 5% CO2 (i.e. the same time used for migration assays) before adding 20 µl MTS/phenazine ethosulfate solution to each well. Plates were then incubated for 2 h at 37 C with 5% CO2 to allow for color development and the absorbance was then recorded at 490 nm using a 96-well plate reader (Benchmark Plus; Bio-Rad Laboratories, Gladesville, New South Wales, Australia). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of viable cells in culture.

Western immunodetection of signaling intermediates
Subconfluent cells were serum starved for 4 h and detached using a nonenzymatic cell dissociation buffer containing proprietary concentrations of EDTA, glycerol, and sodium citrate designed for the removal of cells without the need for trypsin (Sigma). The cells were then pelleted and resuspended in DMEM/F12-serum-free medium + 0.05% BSA and allowed to recover for 30 min at 37 C. Cells were then seeded onto precoated 6-well plates and incubated for the times indicated at 37 C with 5% CO2. After each incubation, the medium was removed and the cell monolayer washed with PBS containing 2 mM Na3VO4 and 10 mM NaF. Cells were then extracted in lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, and 10 mM NaF with a complete protease inhibitor cocktail (Roche Diagnostics, Castle Hill, New South Wales, Australia). Lysates were centrifuged (14,000 x g, 20 min), the supernatants collected, and their protein concentrations determined using the bicinchoninic acid assay kit (Pierce, Rockford, IL). Samples (10 µg total protein) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. Membranes were then incubated with antiphospho-ERK1/2 MAPK (1:2000), antiphospho-AKT (1:2000), antiphospho-p70S6K (1:2000), or anti-Myc (1:5000) mouse monoclonal antibodies overnight at 4 C in blocking buffer. Membranes were then washed six times for 5 min each in Tris-buffered saline with 0.1% Tween 20 before incubation with horseradish peroxidase-conjugated goat antimouse secondary antibody. After further washes, protein bands were then visualized using enhanced chemiluminescence following the manufacturer’s instructions (GE Healthcare Biosciences, Rydalmere, New South Wales, Australia). The same membranes were subsequently stripped and total levels of ERK 1/2, AKT, or glyceraldehyde-3-phosphate dehydrogenase detected as outlined above to validate equal loading. In select experiments the intensity of phosphorylated ERK 1/2 and AKT bands was determined by densitometric analysis and normalized to total ERK 1/2 and AKT levels.

Transient transfection of MCF-10A cells
The AKT1/PKB{alpha} cDNA allelic pack was purchased from Upstate and used for transient transfection of MCF-10A cells. This contained pUSEamp eukaryotic expression vectors containing wild-type and activated Myc-His tagged mouse AKT1 under the control of the cytomegalovirus promoter and the empty vector control. Two micrograms of each construct were transfected into MCF-10A cells (3 x 105/well) using GeneJuice transfection reagent (Novagen, Madison, WI) for 7 h in serum-free Opti-MEM (Invitrogen). Opti-MEM was then replaced with normal growth media and cells incubated at 37 C with 5% CO2 for 24 h before harvesting.

Statistical analysis
Data are expressed as a percentage of the response observed in wells containing VN alone, unless otherwise stated. The data were pooled from multiple experiments as indicated in the figure legends, with each treatment tested in at least duplicate wells. Data analysis was performed using ANOVA with Dunnett’s and Tukey’s post hoc tests performed where appropriate. Statistically significant differences were considered to be present at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I-IGFBP-VN complexes increase breast cell migration
To determine whether IGF-I bound to VN in the presence of IGFBPs could modulate breast cell migration, the lower well and undersurface of 12-µm-pore membranes of Transwell inserts were coated with combinations of IGF-I and IGFBP-3 or -5 prebound to VN. Cells were allowed to migrate over 5 h, after which the number of cells that had migrated to the undersurface of the membrane was quantitated. Only treatments prebound to VN were tested because all cell lines migrated poorly in the absence of serum and VN (data not shown). Whereas the VN control wells were observed to stimulate substantial cellular migration in MCF-7 cells, the addition of IGF-I in combination with either IGFBP-3 or -5, (forming IGF-I-IGFBP-3-VN and IGF-I-IGFBP-5-VN complexes, respectively) significantly enhanced the migration of MCF-7 cells on VN, (P < 0.05). The responses ranged from 143.6 ± 4.7 to 173.6 ± 4.3% of VN controls for IGF-I-IGFBP-3-VN complexes and 164.6 ± 6.3 to 184.8 ± 6% of VN controls for VN-IGFBP-5-IGF-I complexes (P < 0.05) (Fig. 1AGo). Importantly, these responses were substantially greater than those obtained with either IGFBP or IGF-I alone with VN (Fig. 1AGo), indicating that all three components of the complex are required for optimally enhanced cell migration. IGF-I-IGFBP-3-VN complexes also stimulated comparable results in the T47D breast carcinoma cell line (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org).


Figure 1
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  		FIG. 1. Effect of substrate-bound IGF-I-IGFBP-VN complexes on cellular migration. MCF-7 (A and D), MCF-10A (B and D), MDA-MB-231 (C and D), and MCF-7-β3 cells (E) were seeded into Transwell inserts that had the lower well and membrane surface coated with VN and increasing concentrations of IGF-I prebound in the presence or absence of IGFBP-3 or -5 and allowed to migrate for 5 h (A–C and E). The number of cells transversing the membrane in response to each treatment was then expressed as a percentage of those that migrated on VN only. D, Comparison of MCF-10A, MCF7, and MDA-MB-231 cellular migration in response to VN alone. The data are pooled from three experiments with treatments tested in at least triplicate wells (A–C, and E) or from two duplicate experiments with each cell line tested in 6 wells per experiment (D). The asterisk indicates treatments that significantly increased migration above the VN-only wells (P < 0.05). IGF-I-IGFBP-VN complexes were also observed to increase migration above the individual components of the complex ({dagger}, P < 0.05). Error bars indicate SEM.

 
To assess whether similar responses occur in normal breast cells, migration assays were undertaken using the phenotypically normal MCF-10A cell line. Whereas the overall level of migration in terms of cell numbers was reduced in MCF-10A cells, the responses were similar to those observed in MCF-7 cells. The complex of IGF-I-IGFBP-3-VN induced significant increases in cell migration ranging from 175.8 ± 8.6 to 194.9 ± 4.5% of the VN containing control wells (P < 0.05) (Fig. 1BGo). Complexes containing IGFBP-5 also significantly increased cell migration on VN in MCF-10A cells with responses of 145.6 ± 6.2 to 183.0 ± 10% (P < 0.05) (Fig. 1BGo). As seen for MCF-7 cells, these responses were also significantly greater than the migration induced by IGFBP-3, IGFBP-5, or IGF-I alone with VN (P < 0.05). Increased migration of MCF-7 and MCF-10A cell lines in response to substrate-bound IGF-I-IGFBP-VN complexes was not solely a result of IGF-I-IGFBP increasing attachment of cells to VN because separate assays revealed that any changes in cell attachment were far less than the observed increase in cell migration (supplemental Fig. 2).

To determine whether the enhanced migration observed in MCF-7 and MCF-10A cells would occur in highly metastatic breast cancer cells, parallel migration assays were performed using the MDA-MB-231 cell line (Fig. 1CGo). MDA-MB-231 cells were found to be less responsive to IGF-I, with only the two highest concentrations of IGF-I-IGFBP-3 or IGF-I-IGFBP-5 in combination with VN able to increase migration over that induced by VN alone (responses of 119.2 ± 2.1 and 137.1 ± 5.67% for IGF-I-IGFBP-3-VN and 123.0 ± 4.9 and 125.2 ± 6.1% for IGF-I-IGFBP-5-VN) (Fig. 1CGo). The magnitude of the IGF-I-stimulated increase in migration above that induced by VN alone was substantially less than observed in MCF-7 and MCF-10A cells. However, this is likely due to the fact that very high levels of MDA-MB-231 cell migration are observed in response to VN alone. Indeed, MDA-MB-231 cells are substantially more motile than MCF-7 cells on VN with at least 4 times greater migration on VN than observed for MCF-10A cells (Fig. 1DGo). Thus, due to the higher basal level of migration stimulated by VN, the magnitude by which IGF-I can further increase migration in MDA-MB-231 cells is reduced in comparison with the effect of IGF-I in stimulating MCF-10A and MCF-7 cell migration.

The responses of the MDA-MB-231 cells to these complexes was hypothesized to be due to the expression of the {alpha}vβ3 integrin, which is not expressed by MCF-7 or MCF-10A cells. To determine whether this was the case, migration assays were performed using MCF-7-β3 cells, which overexpress the {alpha}vβ3 integrin and have been fully described previously (28). The MCF-7-β3 cells were also found to have a high level of basal migration on VN with substantially reduced induction of migration by IGF-I-containing complexes, compared with the wild-type {alpha}vβ3-deficient MCF-7 cells. Indeed the responses obtained with the MCF-7-β3 cells were similar to those observed for MDA-MB-231 cells (Fig. 1EGo).

IGF-I-IGFBP-VN-stimulated breast cell migration requires heterotrimeric complex formation and the IGF-IR
To examine whether the observed increases in MCF-7 and MCF-10A cell migration were a result of heterotrimeric complex formation between IGF-I, IGFBPs, and VN, the migration responses of MCF-7 cells obtained using native IGF-I and the IGF-I analogs Long R3-IGF-I (LR3-IGF-I) and Des (1–3)-IGF-I were compared. Both of the IGF-I analogs have a reduced affinity for IGFBPs but retain their ability to activate the IGF-IR (29, 30). Native IGF-I + VN was able to stimulate migration to 126.8 ± 4.8% of the VN control wells, and the addition of IGFBP-3 or IGFBP-5 significantly increased this response to 173.6 ± 4.3 and 184.8 ± 6.0%, respectively (P < 0.05) (Fig. 2Go). Whereas LR3-IGF-I and Des (1–3)-IGF-I induced similar levels of migration on VN as found with native IGF-I (132.4 ± 5.0 and 132.3 ± 2.0%, respectively), the addition of either IGFBP-3 or -5 did not result in any further increase in migration above that of LR3-IGF-I or Des (1–3)-IGF-I alone with VN (Fig. 2Go). This therefore indicates that heterotrimeric complex formation (i.e. the binding of IGF-I to VN via IGFBP-3 or IGFBP-5) is required for IGF-I-IGFBP-VN complex-stimulated migration. In a like manner, the involvement of the IGF-IR in IGF-I-IGFBP-VN-stimulated migration was assessed using the IGF-I analog [L24][A31]IGF-I. This analog binds to IGFBPs but has a reduced affinity for the IGF-IR (31, 32). [L24][A31]IGF-I + VN did not enhance migration above that of the VN alone control wells (103.9 ± 2.8%) or in the presence of IGFBP-3 or IGFBP-5 (102.9 ± 5.1 and 98.5 ± 7.0%, respectively) (Fig. 2Go). These responses were significantly lower than those induced by native IGF-I alone with VN or IGF-I-IGFBP-VN complexes (P < 0.05), thus demonstrating that activation of the IGF-1R is crucial in IGF-I-IGFBP-VN-stimulated migration.


Figure 2
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  		FIG. 2. Substrate-bound IGF-I-IGFBP-VN-stimulated migration requires heterotrimeric complex formation. MCF-7 cells were seeded into Transwell inserts that had the lower well and membrane surface coated with either native IGF-I or IGF-I analogs with reduced binding to IGFBPs (LR3-IGF-I and Des (1–3)-IGF-I) or reduced affinity for the IGF-IR ([L24][A31]IGF-I) in the presence and absence of IGFBP-3 and -5. The number of cells transversing the membrane in response to each treatment was then expressed as a percentage of those that migrated on VN only. The data are pooled from two experiments with treatments tested in 6 wells for each. The asterisk indicates treatments that significantly increased migration above the VN-only wells (P < 0.05). A cross indicates IGF-I analogs that significantly reduced migration in response to the same treatment using native IGF-I (P < 0.05). Transwells were precoated with native IGF-I or IGF-I analogs (30 ng/ml), IGFBP-3/-5 (90 ng/ml), and VN (1 µg/ml). Error bars indicate SEM.

 
Blocking VN-binding integrins and the IGF-IR inhibits IGF-I-IGFBP-VN-stimulated migration
Because enhanced MCF-7 and MCF-10A migration requires IGF-I binding to VN via IGFBPs, we sought to elucidate the role of integrin receptors in this response and further confirm the importance of IGF-IR activation in this process. Meyer et al. (33) have shown that MCF-7 cells express the {alpha}v and β1 subunits and the {alpha}vβ5 heterodimer. In addition to these integrins, MCF-10A cells also express the {alpha}vβ6 integrin (33). We investigated the contributions of these integrins, as well as the IGF-1R, to the cell migration responses by preincubating the cells with monoclonal antibodies with established function blocking properties. As shown in Fig. 3AGo, preincubation of MCF-7 cells with antibodies directed against the {alpha}v integrin subunit, IGF-IR, a combination of both {alpha}v + IGF-1R antibodies, and {alpha}vβ5, caused significant inhibition of IGF-I-IGFBP-3-VN complex-stimulated migration, with responses of 27.5 ± 2.7% ({alpha}v), 65.1 ± 3.2% (IGF-IR), 15.8 ± 0.8% ({alpha}v + IGF-1R), and 44.4 ± 5.1% ({alpha}vβ5) of responses observed in wells containing isotype matched control IgG antibodies, respectively (P < 0.05) (Fig. 3AGo). Similar effects were also observed for complexes containing IGFBP-5. Thus, IGF-I-IGFBP-5-VN complex-induced migration was inhibited to 22.2 ± 2.8% ({alpha}v), 61.6 ± 5.0% (IGF-1R), 14.3 ± 0.6% ({alpha}v + IGF-1R) and 42.1 ± 5.1% ({alpha}vβ5) of the control wells (Fig. 3AGo).


Figure 3
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  		FIG. 3. Involvement of VN-binding integrins and the IGF-IR in IGF-I-IGFBP-VN-stimulated migration. IGF-I-IGFBP-VN-stimulated migration of MCF-7 (A) and MCF-10A (B) cells was determined in the presence of monoclonal anti-integrin blocking antibodies against {alpha}v (1:10), {alpha}vβ5 (25 µg/ml), {alpha}vβ6 (25 µg/ml), β1 (10 µg/ml), and the IGF-IR (10 µg/ml) or control mouse IgG (25 µg/ml). Cells were allowed to migrate for 5 h, after which the migration in the presence of control IgG antibodies was taken as 100%. The asterisk indicates antibody treatments that significantly inhibited IGF-I-IGFBP-VN stimulated migration (P < 0.05). The data are pooled from three experiments with treatments tested in duplicate wells. The lower well and membrane surface of Transwell inserts were precoated with IGF-I (30 ng/ml), IGFBP-3/-5 (90 ng/ml), and VN (1 µg/ml). Error bars indicate SEM.

 
When MCF-10A cells were similarly preincubated with the above-mentioned antibodies, IGF-I-IGFBP-3-VN-stimulated migration was decreased to 37.6 ± 3.0% ({alpha}v), 42.8 ± 3.1% (IGF-1R), 31.1 ± 3.0% ({alpha}v + IGF-IR), and 58.6 ± 4.4% ({alpha}vβ5) of responses observed in wells containing isotype matched control IgG antibodies, respectively (P < 0.05) (Fig. 3BGo). As seen in MCF-7 cells, similar effects were also observed for complexes containing IGFBP-5, with IGF-I-IGFBP-5-VN complex-induced migration inhibited to 45.5 ± 3.0% ({alpha}v), 40.5 ± 2.6% (IGF-1R), 38.8 ± 4.3% ({alpha}v + IGF-IR), and 70.4 ± 6.6% ({alpha}vβ5) of control wells, respectively (Fig. 3BGo). In addition, preincubation of MCF-10A cells with anti-β1 integrin subunit and anti-{alpha}vβ6 integrin antibodies also significantly inhibited both IGF-I-IGFBP-3-VN-stimulated migration, with responses of 74.1 ± 3.6% (β1) and 66.1 ± 3.5% ({alpha}vβ6) of control wells, respectively (P < 0.05). These two antibodies also decreased IGF-I-IGFBP-5-VN-stimulated migration, however not to statistically significant levels. It should be noted that incubation of cells with control IgG antibodies had no effect on cell migration, compared with that observed in the total absence of antibody (data not shown). Whereas the main focus of this study was responses to IGF-I-IGFBP-VN complexes, similar inhibitory effects of specific antibodies, as seen in Fig. 3Go, A and B, were also observed for each cell line in response to VN alone, IGFBP-3+VN, IGFBP-5+VN, and IGF-I + VN treatments (supplemental Fig. 3).

Enhanced cell signaling by IGF-I-IGFBP-VN complexes occurs via MAPK and PI3-K/AKT signal transduction pathways
To investigate possible mechanisms underlying the enhanced cell migration in response to IGF-I-IGFBP-VN complexes, we examined the activation of key intracellular signaling proteins. The ERK/MAPK and PI3-K/AKT pathways were targeted because they are known to be activated downstream of both integrins and the IGF-IR. MCF-7 and MCF-10A cells were serum starved and then seeded into 6-well culture plates that had been coated with VN and combinations of IGF-I and IGFBP-3 or IGFBP-5 (Figs. 4Go and 5Go). After incubation for the indicated times, cell lysates were then analyzed by Western immunoblotting using phospho-specific antibodies against either dually phosphorylated ERK 1/2 (Thr202 and Tyr204) or AKT-1 (AKT) when phosphorylated on Thr308 and Ser473 residues. Activation of ERK 1/2 and AKT were both substantially increased by the IGF-I-IGFBP-3-VN and IGF-I-IGFBP-5-VN complexes in MCF-7 cells (Fig. 4AGo). This effect was greater than that induced by VN alone and IGF-I or IGFBP-3/-5 with VN at all times tested. IGF-I-IGFBP-VN complexes could also activate the PI3-K/AKT axis downstream of AKT, as observed by the phosphorylation of p70S6K in a similar pattern to AKT activation (Fig. 4AGo). The IGF-I-IGFBP-VN complex-induced activation of ERK 1/2 was rapid and transient, attaining maximal activation after 10 min and returning to near basal levels after 30 min. In contrast, increased AKT activation induced by both IGFBP-3 and IGFBP-5-containing complexes was sustained over 5 h (Fig. 4BGo).


Figure 4
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  		FIG. 4. Activation of ERK/MAPK and PI3-K/AKT pathways by IGF-I-IGFBP-VN complexes in MCF-7 cells. MCF-7 cells were seeded on wells precoated with IGF-I (30 ng/ml), IGFBP-3/-5 (90 ng/ml), and VN (1 µg/ml) combinations for the indicated times. Cells were then lysed and levels of phosphorylated ERK 1/2 and AKT determined by immunoblot analysis. Membranes were subsequently stripped and reprobed to determine total levels of ERK 1/2, AKT, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative results of multiple experiments are shown. SFM, Serum-free media.

 

Figure 5
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  		FIG. 5. Activation of ERK/MAPK and PI3-K/AKT pathways by IGF-I-IGFBP-VN complexes in MCF-10A cells. MCF-10A cells were seeded on wells precoated with IGF-I (30 ng/ml), IGFBP-3/-5 (90 ng/ml), and VN (1 µg/ml) combinations for the indicated times. Cells were then lysed and levels of phosphorylated ERK 1/2 and AKT determined by immunoblot analysis. Membranes were subsequently stripped and reprobed to determine total levels of ERK 1/2 and AKT. Representative results of multiple experiments are shown. SFM, Serum-free media.

 
Unlike MCF-7 cells, MCF-10A cells were found to have relatively low total levels of ERK 1 and a higher basal level of ERK 2 phosphorylation (Fig. 5AGo). Maximal ERK 2 activation also occurred by 10 min in MCF-10A cells, returning to near basal levels after 30 min (Fig. 5Go, A and B). In contrast to MCF-7 cells, IGF-I-IGFBP-VN complexes did not induce synergistic increases in ERK 1/2 activation over VN alone and IGF-I or IGFBP-3/-5 with VN. However, similar to that observed in MCF-7 cells, AKT activation was substantially increased by the combination of IGF-I, IGFBP-3/-5, and VN with responses greater than those induced by either VN alone and IGF-I or IGFBP-3/-5 alone with VN at all time points tested in MCF-10A cells (Fig. 5AGo). Furthermore, the activation of AKT was increased and sustained over 5 h (Fig. 5BGo). In all cases, similar effects on cell signaling were found in response to IGF-I-IGFBP-3-VN and IGF-I-IGFBP-5-VN complexes in both cell types (Figs. 4Go and 5Go).

IGF-I-IGFBP-VN stimulated cell signaling involves both {alpha}v-integrins and the IGF-IR
To determine whether VN/integrin interactions, in addition to the IGF-IR, were playing an active role in substrate-bound IGF-I-IGFBP stimulated cell signaling, serum starved MCF-10A cells were harvested and preincubated with monoclonal function blocking antibodies directed against the {alpha}v-integrin subunit and the IGF-IR before seeding into culture wells coated with IGF-I-IGFBP-5-VN complexes (Fig. 6Go, A and B). As can be seen in Fig. 6AGo, inhibition of the {alpha}v-integrin subunit reduced ERK 1/2 and AKT activation to approximately 62% and 59%, respectively, of the activation stimulated in cells preincubated with control IgG antibodies (Fig. 6Go, A and B). Surprisingly, blocking the IGF-IR resulted in only a modest reduction in ERK activation to approximately 74% of control cells. However, IGF-IR inhibition led to a more prominent reduction in AKT activation (~17% of control) in response to substrate-bound complexes. This therefore provides the first evidence that interactions with both VN-binding integrins and the IGF-IR can modulate signaling stimulated by substrate-bound IGF-I-IGFBP-VN complexes.


Figure 6
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  		FIG. 6. Involvement of {alpha}v-subunit containing integrins and the IGF-IR on IGF-I-IGFBP-VN-stimulated ERK/MAPK and PI3-K/AKT pathway activation. A, Serum-starved MCF-10A cells were harvested and preincubated with monoclonal blocking antibodies against the {alpha}v-integrin subunit (1:10), the IGF-IR (10 µg/ml), or control (Con) mouse IgG (25 µg/ml) for 30 min at room temperature before seeding into 6-well culture dishes precoated with IGF-I-IGFBP-5-VN complexes. Cells were then incubated for 30 min at 37 C with 5% CO2, lysed, and levels of phosphorylated ERK 1/2 and AKT determined by immunoblot analysis. B, MCF-10A cells were grown to confluency in 12-well plates, serum starved, and incubated with monoclonal blocking antibodies against the {alpha}v-integrin subunit (1:10), the IGF-IR (10 µg/ml), or control mouse IgG (25 µg/ml) for 2 h at 37 C with 5% CO2. Cells were then stimulated with IGF-I-IGFBP-5 added into the culture medium for 30 min, then lysed, and levels of phosphorylated ERK 1/2 and AKT determined by immunoblot analysis. In A and B, membranes were subsequently stripped and reprobed to determine total levels of ERK 1/2 and AKT. Histograms represent results from densitometric analysis of immunoblots, with the ratio of ERK and AKT activation in the presence of function blocking antibodies expressed relative to the activation in cells incubated with control IgG. Representative blot from a single experiment is shown in A and B. In all cases, IGF-I (30 ng/ml), IGFBP-5 (90 ng/ml), and VN (1 µg/ml) were used.

 
To further confirm the involvement of VN-binding integrins in IGF-I-stimulated cell signaling, an alternate approach was also adopted based on studies undertaken in smooth muscle cells in which blockade of the {alpha}vβ3 integrin has been extensively demonstrated to inhibit IGF-I signaling (34, 35). In this approach, preplated MCF-10A cells were allowed to grow to confluency before being serum starved and incubated with {alpha}v-integrin subunit and IGF-IR antibodies. The adherent cells were then stimulated with IGF-I-IGFBP-5 added into the culture medium for 30 min before cell lysis and protein isolation. As expected, blockade of the IGF-IR led to substantial inhibition of IGF-I-stimulated signaling with ERK and AKT activation reduced to approximately 28 and 6%, respectively, of the activation observed in control cells (Fig. 6BGo). Interestingly, inhibition of {alpha}v-integrins also led to substantial reductions in both ERK and AKT activation levels, to approximately 32 and 49%, respectively, of that observed by control cells (Fig. 6BGo). This finding is of particular significance, given that MCF-10A cells lack the {alpha}vβ3 integrin, which has been the main focus of ECM/integrin interactions in modulating IGF-I signaling. These data provide the first evidence for the important interaction between VN-binding integrins and the IGF-IR in regulating IGF-I-IGFBP-VN complex stimulated cell signaling in breast cells.

IGF-I-IGFBP-VN complex-stimulated migration is mediated via the PI3-K/AKT pathway
To determine the relative contributions of the two signaling pathways in IGF-I-IGFBP-VN complex-stimulated migration, cells were preincubated with specific pharmacological inhibitors of the ERK/MAPK and PI3-K/AKT pathways, U0126 and LY294002, respectively (Fig. 6AGo). Doses of 20 µM LY294002 and 10 µM U0126 were shown to be specific for inhibition of AKT and ERK 1/2, respectively, with no significant nonspecific or cross-pathway inhibition by either inhibitor observed in both MCF-7 and MCF-10A cell lines (Fig. 7Go, A and B). Inhibition of ERK 1/2 by U0126 decreased MCF-7 migration induced by IGF-I-IGFBP-3-VN and IGF-I-IGFBP-5-VN complexes to 86.0 ± 3.4 and 78 ± 3.6% of that observed for vehicle controls, respectively (Fig. 7CGo). A greater decrease in migration was observed in cells treated with LY294002, with responses of 43.2 ± 2.3 and 35.9 ± 2.8% of vehicle controls observed for IGF-I-IGFBP-3-VN and IGF-I-IGFBP-5-VN complexes, respectively (P < 0.05) (Fig. 7CGo). Furthermore, incubation of the cells with a combination of both inhibitors led to a further inhibition of IGF-I-IGFBP-VN-stimulated migration with responses of 11.9 ± 0.75 and 11.9 ± 0.36% of vehicle controls wells, respectively, for IGFBP-3- and IGFBP-5-containing complexes (P < 0.05) (Fig. 7CGo).


Figure 7
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  		FIG. 7. Involvement of ERK/MAPK and PI3-K/AKT pathways in IGF-I-IGFBP-VN-stimulated migration. MCF-7 and MCF-10A cells were pretreated with the MAPK inhibitor (U0126, 10 µM) and/or the PI3-K/AKT inhibitor (LY294002, 20 µM) for 1 h before seeding into wells precoated with IGF-I-IGFBP-VN complexes for 10 min (A and B). Cells were then lysed for immunoblot analysis of specific inhibition of ERK 1/2 and AKT activation by pharmacological inhibitors. Representative blots of three replicates are shown on the top of histograms. Histograms represent results from densitometric analysis of immunoblots, with the ratio of ERK (A) and AKT (B) activation in the presence of pharmacological inhibitors expressed relative to activation of vehicle control-treated cells. Pretreated MCF-7 (C) and MCF-10A cells (D) were seeded into Transwell inserts that had the lower well and membrane surface precoated with IGF-I (30 ng/ml), IGFBP-3 or -5 (90 ng/ml), and VN (1 µg/ml) and allowed to migrate for 5 h. Data are expressed as a percentage of the response observed in vehicle control wells containing an equivalent concentration of dimethylsulfoxide. The asterisks indicate treatments in which migration was significantly inhibited (P < 0.05). The data presented are pooled from three experiments with treatments tested in at least triplicate wells. Cell viability of serum-starved MCF-7 (E) and MCF-10A cells (F) were determined after pretreatment with LY294002 (LY 20 µM) and U0126 (U 10 µM). Cells were pretreated as described above and then seeded into 96-well plates that had been precoated with IGF-I-IGFBP-5-VN complexes and incubated for 5 h at 37 C with 5% CO2. Cell viability was then assessed using MTS reagent as described in Materials and Methods. The asterisk indicates treatments that significantly inhibited cell viability, compared with vehicle control-treated cells (P < 0.05). In all cases, error bars indicate SEM.

 
Incubation of MCF-10A cells with 10 µM U0126 had no significant effect on either IGF-I-IGFBP-3-VN or IGF-I-IGFBP-5-VN complex-stimulated migration, with responses of 96.1 ± 3.6 and 96.8 ± 4.2% of vehicle controls, respectively (Fig. 7DGo). However, incubation of MCF-10A cells with 20 µM LY294002 significantly inhibited migration in response to both IGF-I-IGFBP-3-VN (46.9 ± 2.2%) and IGF-I-IGFBP-5-VN (44.8 ± 2.8%) complexes (Fig. 7DGo). Incubation of MCF-10A cells with both U0126 and LY294002 led to a greater inhibition of migration than found with either inhibitor alone, in response to IGF-I-IGFBP-3-VN complexes (Fig. 7DGo). Importantly, the inhibition of cell migration in response to LY294002 was not due to effects on cell viability because LY294002 was determined to have no significant effect on cell viability in either MCF-7 or MCF-10A cell lines (Fig. 7Go, E and F). Taken together, these data suggest a central role for PI3-K/AKT activation in the enhanced migration observed in response to IGF-I-IGFBP-VN complexes in both MCF-7 and MCF-10A cell types.

Overexpression of wild-type and activated AKT enhances IGF-I-IGFBP-VN-stimulated migration
To further determine the importance of AKT in IGF-I-IGFBP-VN-stimulated migration, expression constructs containing Myc-His tagged mouse wild-type AKT-1 (WT-AKT) and N-terminal myristoylated AKT-1 (MYR-AKT), which produces an activated form of AKT, were transiently transfected into MCF-10A cells. Transient transfections produced an approximate 200% increase in AKT levels, with equivalent levels of both WT-AKT and MYR-AKT expressed (Fig. 8AGo). Whereas IGF-I-IGFBP-VN complexes are already potent stimulators of MCF-10A cell migration, the expression of WT-AKT and MYR-AKT led to further significant increases in migration of 127.8 ± 8.5% (WT-AKT) and 151.7 ± 8.3% (MYR-AKT) of empty vector control cells (pUSEamp), respectively (P < 0.05) (Fig. 8BGo). Expression of activated AKT in MCF-10A cells was also observed to increase migration above that of wild-type AKT, with this effect bordering on statistical significance (P = 0.053). The effect of AKT overexpression was independent of an overall increase in basal levels of migration because subsequent assays revealed there was no difference in the levels of migration in response to VN alone for cells expressing either WT-AKT or MYR-AKT, compared with control cells (Fig. 8CGo). Moreover, the increase in migration observed with overexpression of AKT still requires activation of the IGF-IR because [L24][A31]IGF-I significantly reduced cellular migration in both control and MYR-AKT-expressing cells, compared with the migration induced by IGF-I-IGFBP-5-VN complexes containing native IGF-I (P < 0.05) (Fig. 8DGo). Similarly, optimal migration of both control and MYR-AKT expressing cells also required the involvement of VN-binding integrins, with antibody mediated inhibition of {alpha}v-integrins significantly decreasing cell migration in response to IGF-I-IGFBP-5-VN complexes (Fig. 8EGo). These results provide further evidence for an important interaction between the IGF-IR and VN-binding integrins in IGF-I-IGFBP-VN-stimulated migration, which is enhanced by PI3-K/AKT pathway activation.


Figure 8
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  		FIG. 8. Overexpression of wild-type (WT) and activated (MYR) AKT enhances IGF-I-IGFBP-VN-stimulated migration. A, Western immunodetection of WT- and MYR-AKT expression levels in MCF-10A cells after transfection with pUSEamp expression constructs. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Transiently transfected MCF-10A cells expressing WT-AKT, MYR-AKT, and empty vector (pUSEamp) were seeded into Transwell inserts that had the lower well and membrane surface precoated with IGF-I-IGFBP-5-VN (B) or VN alone (C) and allowed to migrate over 5 h. Data are expressed as a percentage of the response stimulated in empty vector control cells. Expression of both wild-type and activated AKT significantly increased migration in response to the IGF-I-IGFBP-5-VN complex, compared with empty vector control cells (*, P < 0.05). Data are pooled from three experiments (n = 15). D, MCF-10A cells expressing MYR-AKT or empty vector (pUSEamp) were seeded into Transwell inserts precoated as above, with either native IGF-I or [L24][A31]IGF-I in the presence of IGFBP-5 and allowed to migrate for 5 h. Data are expressed as a percentage of the response stimulated by native IGF-I containing complexes in empty vector control cells. E, MCF-10A cells expressing MYR-AKT or empty vector (pUSEamp) were preincubated with monoclonal blocking antibodies against the {alpha}v-integrin subunit (1:10) or control mouse IgG (25 µg/ml) for 30 min at room temperature, before seeding into Transwell inserts precoated with IGF-I-IGFBP-5-VN complexes. Cells were allowed to migrate for 5 h, after which the migration of empty vector control cells in the presence of control IgG antibodies was taken as 100%. In all cases (B–E), the asterisk indicates treatments that significantly increased migration above empty vector control cells (P < 0.05). A cross indicates significant inhibition of migration in comparison with native IGF-I (D) or control IgG (E) (P < 0.05). In D and E, results are from two duplicate experiments with each treatment tested in six wells per experiment (n = 12). In all cases where appropriate, the lower well and membrane surface of Transwell inserts were precoated with native IGF-I or [L24][A31]IGF-I (30 ng/ml), IGFBP-3/-5 (90 ng/ml), and VN (1 µg/ml). Error bars indicate SEM.

 
Substrate-bound complexes containing IGF-I and IGFBPs can stimulate equivalent functional responses to those added in the solution phase
Traditionally, in vitro approaches to studying the effects of growth factors on cells have involved adding growth factors in solution, whereby they are free to rapidly diffuse throughout the culture medium. Because cells in vivo are imbedded in a complex ECM, it is likely that soluble growth factors would interact with components of the ECM before binding to their respective cell surface receptors to modulate cell function. As such, we wanted to compare the functional effects of both solution-phase and substrate-bound IGF-I-IGFBP complexes. Thus, serum-starved MCF-10A cells were seeded into Transwell inserts in which the lower chamber and membrane surface had either been precoated with IGF-I and IGFBPs, with unbound factors removed by washing (substrate-bound) or had IGF-I and IGFBPs added into the media of the lower chamber immediately before seeding of cells (solution-phase). The concentration of IGF-I and IGFBPs used in the solution-phase treatments was identical with the concentration added to substrate-bound treatments before the prebinding and wash steps. As expected, the addition of IGF-I in solution produced a significant increase in MCF-10A cell migration, compared with substrate-bound IGF-I, with responses of 118.9 ± 6.3 and 158.6 ± 5.5% of the control wells observed, respectively (P < 0.05) (Fig. 9AGo). This is not surprising because in the absence of IGFBPs, minimal IGF-I would remain bound to VN in the substrate-bound approach. However, there were no significant differences in the response of MCF-10A cells to IGF-I in the presence of either IGFBP-3 or IGFBP-5 with both treatment strategies (Fig. 9AGo). Analysis of AKT activation also revealed similar patterns of AKT phosphorylation in both MCF-10A (Fig. 9BGo) and MCF-7 (Fig. 9CGo) cell lines with both treatment strategies. Indeed, the only major difference in stimulation of AKT activation by IGF-I between the substrate-bound and solution-phase strategies was in the absence of either IGFBP-3 or IGFBP-5 (Fig. 9Go, B and C). Thus, similar levels of AKT activation were induced by IGF-I in the presence of IGFBP-3 and IGFBP-5 by substrate-bound or solution-phase factors in both the MCF-10A and MCF-7 cell lines.


Figure 9
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  		FIG. 9. VN-bound IGF-I-IGFBP complexes stimulate functional responses equivalent to solution-phase complexes. A, Stimulation of MCF-10A migration in response to both substrate-bound and solution-phase IGF-I-IGFBP complexes. Transwells were precoated with substrate-bound complexes as described in Materials and Methods. For solution-phase treatments, wells were treated as above except IGF-I and IGFBPs were not added to VN-coated lower wells until immediately before seeding of cells in the upper chamber. Cells were then allowed to migrate for 5 h and quantified as already described. Asterisk indicates a significant increase in migration between substrate-bound and solution-phase strategies for each treatment (P < 0.05). B and C, Comparison of AKT activation by substrate-bound and solution-phase complexes. Six-well plates were precoated with substrate-bound complexes as described in Materials and Methods. Solution-phase IGF-I and IGFBPs were added to VN-coated wells immediately before the addition of cells. After incubation of cells for the indicated times, cells were lysed and levels of phosphorylated AKT determined by immunoblot analysis. Membranes were subsequently stripped and reprobed to determine total levels of AKT. Representative results of multiple experiments are shown. SFM, Serum-free media.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well accepted that cell migration is one of the key events leading to tumor dissemination. Altered cellular interactions with the extracellular microenvironment and exposure to elevated levels of mitogenic hormones and growth factors, such as the IGFs, are considered to be two factors that are pivotal in breast cancer metastasis. Our laboratory has shown previously that IGFBPs, namely IGFBP-2, -3, -4, and -5, enhance IGF-I binding to VN by forming a heterotrimeric complex comprised of IGF-I-IGFBP-VN (15). The studies reported here demonstrate that substrate-bound IGF-I-IGFBP-VN complexes are potent stimulators of both normal and cancerous breast cell migration. Furthermore, this response is mediated via a PI3-K/AKT-dependent mechanism involving interactions between the IGF-IR and VN-binding integrins. This is therefore the first significant investigation into the effects of substrate-bound IGF-I-IGFBP-VN complexes on breast cell migration and the mechanisms underpinning these responses.

IGFs are recognized as having important roles in normal mammary gland development in which they have been demonstrated to regulate ductal morphogenesis, glandular development, and prevention of apoptosis during involution (25). Thus, IGFs can modulate processes important in both normal mammary gland development and the progression of breast cancer, including proliferation, invasion, and resistance to apoptosis. Indeed, we report that substrate-bound complexes containing IGF-I are potent stimulators of cellular migration and PI3-K/AKT pathway activation in both malignant and normal breast cells. This provides additional evidence in support of previous studies demonstrating IGF-I stimulates migration of breast cancer cells (36, 37).

It is clear that the IGF system is complex and the biological effects of the IGFs are determined by diverse interactions between many different molecules. However, this complex regulatory system is disrupted in breast cancer, which ultimately leads to excess IGF-IR signaling and the development of the invasive phenotype. Moreover, IGF-IR activation stimulates the invasion of breast cancer cells through collagen IV (38) and Matrigel (39), with inhibition of the IGF-IR blocking the metastasis of breast cancer cells (39, 40). Our studies using both the [L24][A31]IGF-I analog and antibody inhibition of the IGF-IR demonstrate an important involvement of the IGF-IR in the increased migratory response induced by substrate-bound IGF-I-IGFBP-VN complexes.

The IGF system is widely acknowledged as a highly relevant growth regulatory system in breast cancer. Similarly, VN has been associated with breast tumors in vivo, displaying a distinct distribution pattern, compared with normal tissue (41). With increased expression of VN and VN-binding integrins reported at the leading edge of migrating tumor cells (18, 19, 20), the interactions among the IGF-IR, VN, and VN-binding integrins would appear to have a particularly relevant role to breast cancer metastasis. However, to date the mechanisms behind the enhanced migration in response to IGFs associated with VN have remained unclear. Using IGF-I analogs that bind IGFBPs poorly, we demonstrate a critical functional involvement of IGFBP-3 and IGFBP-5 in heterotrimeric complex-stimulated cell migration. Thus, IGF-I, through the involvement of IGFBPs, can be captured by VN, providing a local reservoir of IGF-I in the pericellular ECM, which can interact with the IGF-IR. It has previously been shown that cellular responses to IGF-I can be mediated by IGF-I association with the ECM via IGFBP-5, thereby stabilizing IGF-I matrix concentrations and promoting receptor interactions (42). As such, we believe that the prebinding approach adopted in this study may more accurately reflect the in vivo situation, therefore making these results especially biologically significant.

Numerous growth factor receptors interact with ECM components to modulate cellular functions, suggesting an important interaction between growth factor receptors and the ECM (43, 44, 45). There is accumulating evidence for direct cooperation between the IGF-IR and {alpha}v-integrins as the signaling pathways between these receptors are clearly interconnected (34, 46, 47). Indeed, Clemmons and Maile (47) have demonstrated a requirement for {alpha}vβ3 ligand occupancy for vascular cells to respond optimally to IGF-I and showed that blocking VN binding to {alpha}vβ3 with disintegrin antagonists results in the abolition of IGF-I-stimulated responses such as cell migration, IGF-IR autophosphorylation, and activation of PI3-K. It seems, however, that this regulation is not unidirectional because IGF-IR activation by IGF-I can modulate integrin signaling (48). Furthermore, cooperation between the IGF-IR and {alpha}vβ5 integrin has already been demonstrated to promote tumor cell metastasis in vivo independent of tumor cell growth (49). Our study also provides additional evidence for an important interaction between the IGF-IR and VN-binding integrins because blockade of these receptors was demonstrated to abolish IGF-I-IGFBP-VN-stimulated cellular migration. Again, these results corroborate previous findings in which inhibition of the IGF-IR and {alpha}vβ5 integrin reduced IGF-I-stimulated migration of MCF-7 cells on VN (36).

Antibody inhibition of the IGF-IR substantially reduced the migration of both MCF-10A and MCF-7 cells in response to substrate-bound IGF-I-IGFBP-VN complexes (Fig. 3Go). This effect was observed to be most prominent in MCF-10A cells, compared with MCF-7 cells, which may reflect the lower level of IGF-IR expression in MCF-10A cells. Studies were also undertaken to determine the role of integrins in IGF-I-IGFBP-VN-stimulated cell migration because integrin/matrix associations are required for optimal growth factor signaling (45). Overall, antibody inhibition of the {alpha}v-integrin subunit led to the greatest reduction in cell migration responses to IGF-I-IGFBP-VN complexes in both cell types (Fig. 3Go). More specifically, the {alpha}vβ5 integrin was determined to be the major VN-binding integrin involved in mediating IGF-I-IGFBP-VN-stimulated cell migration in both cell types (Fig. 3Go). In contrast, antibody inhibition of the β1 integrin subunit was observed to have little effect on migration in either cell type, which was unexpected, given previously reported interactions between the β1 integrin and the IGF-IR (50, 51, 52). However, similar results have been reported using MCF-7 cells, whereby inhibition of the {alpha}vβ5 integrin, but not the β1 integrin subunit, inhibited IGF-I-stimulated migration on VN (36). Interestingly, in this same study, the role for these integrins was reversed when type IV collagen was used in place of VN (36). Thus, whereas both the IGF-IR- and VN-binding integrins are involved in IGF-I-IGFBP-VN-stimulated cell migration, the relative contribution of these receptors to this response is likely to be due to not only the cell type-specific expression levels of these receptors but also the specific ECM protein that the cells encounter. Nevertheless, these results demonstrate an important role for both the IGF-IR- and VN-binding integrins, most prominently {alpha}vβ5, in the MCF-7 and MCF-10A cell migration responses observed. Furthermore, the data support a critical involvement of integrin receptors in IGF-stimulated cell migration.

One mechanism by which growth factor receptors and integrins can collaborate is via synergistic responses in their respective downstream signaling pathways (45, 53, 54). Substrate-bound IGF-I-IGFBP-VN complexes were demonstrated herein to induce synergistic increases in intracellular signal transduction, in particular, an increased and sustained activation of the PI3-K/AKT pathway. Furthermore, in both MCF-7 and MCF-10A cell lines, synergistic increases in AKT activation occurred when all components of the complex were present. Our results also indicate a pivotal role for AKT in promoting breast cell migration as pharmacological inhibition of the PI3-K/AKT pathway significantly attenuated IGF-I-IGFBP-VN complex-stimulated cell migration (Fig. 6Go). Moreover, the overexpression of wild-type and activated AKT 1 further increased cell migration in response to IGF-I-IGFBP-VN complexes, supporting previous studies demonstrating the importance of the PI3-K/AKT pathway in IGF-I-dependent motility of cancer cells (37, 55).

In addition to AKT’s well established role in cell survival, proliferation, and metabolism, AKT has important pathological functions in tumorigenesis and metastasis, whereby AKT is hyperactivated in many human cancers (56). There is accumulating evidence indicating that AKT promotes cell motility in fibroblasts and tumor cells (57, 58, 59) in which AKT, through interactions with proteins such as Rac, Cdc42, and Girdin, can modulate actin organization and microtubule stabilization at the leading edge of migrating cells (57, 60). Moreover, a number of clinical studies correlate increased AKT expression and activation with more invasive and metastatic disease, which in turn, are associated with poor prognosis (56, 61, 62, 63). Conversely, there have been conflicting reports indicating that AKT 1 can block breast cancer cell motility (64), with isoform-specific roles of AKT 1 and AKT 2 demonstrated in IGF-IR-overexpressing MCF-10A cells (65). Nevertheless, our results support a role for AKT 1 in promoting cellular migration in response to IGF-I-IGFBP-VN complexes. However, future studies using RNA interference or dominant-negative AKT approaches will be important to determine whether activation of AKT is critical to the enhanced cell migration stimulated by IGF-I-IGFBP-VN complexes.

IGF-I-IGFBP-VN complexes were not as potent stimulators of cell migration in the highly metastatic MDA-MB-231 cell line because these cells were highly motile on VN alone. We hypothesize that this response is due to these cells expressing the {alpha}vβ3 integrin, which on ligation by VN can modulate signaling pathways downstream of the IGF-IR (66), hence adopting a highly migratory phenotype in the presence of VN alone. Previous studies found similar results with MDA-MB-231 cells in response to IGF-II-VN complexes (14). This was hypothesized to be due to the relatively low level of IGF-IR expression by these cells (~7 x 103 IGF-IR/cell) or the presence of the {alpha}vβ3 integrin, which is not present in MCF-7 cells (14). Supporting the hypothesized role of the {alpha}vβ3 integrin in this response, we demonstrate here that MCF-7-β3 cells, which express the normally deficient {alpha}vβ3 integrin, showed similar responses to those found with MDA-MB-231 cells, illustrating the importance of the {alpha}vβ3 integrin and its ligand VN in highly invasive breast cancer cells.

Because traditional in vitro approaches to studying the effects of growth factors on cells have involved adding these soluble growth factors in solution, we wanted to compare the functional effects of both solution-phase and substrate-bound IGF-I-IGFBP complexes. It was observed that substrate-bound IGF-I-IGFBP complexes induce comparable functional effects to those found with IGF-I-IGFBP treatments added in solution phase (Fig. 9Go). Two possible mechanisms may account for these equivalent functional responses. First, the majority of IGF-I added into wells during the prebinding step, in the presence of IGFBP-3 and IGFBP-5, is incorporated into the substrate-bound VN complexes. This would therefore provide equivalent amounts of IGF-I to that present in the solution-phase treatments. However, recent preliminary findings in our laboratory indicate that, depending on the molar ratio of VN, IGFBPs, and IGF-I used in the prebinding steps, between 20 and 75% of the IGF-I added into the solution phase remains in substrate-bound complexes (data not shown). This suggests that less IGF-I would be present in the substrate-bound complexes in the study reported here than was present in the solution-phase treatments. Yet despite this, similar responses were observed. We hypothesize that the capture of IGF-I within the pericellular environment may provide a local reservoir of IGF-I to promote increased interactions with cell surface receptors such as the IGF-IR- and VN-binding integrins. Lower amounts of IGF-I would therefore be required to induce comparable functional effects, at least in these short-term assays. However, future, more detailed studies to determine the quantities of substrate-bound growth factors and indeed specific receptor interactions will be critical in fully elucidating these mechanisms.

In summary, our results indicate that IGF-I when bound to VN via IGFBP-3 or -5 to form a heterotrimeric substrate-bound complex is a potent stimulator of MCF-7 and MCF-10A cell migration. These novel complexes appear to facilitate cooperation between the IGF-IR- and VN-binding integrins, resulting in enhanced and sustained PI3-K/AKT pathway activation, which is pivotal to the increased migratory response observed. Because both IGF-I and VN are implicated in tumor biology, we propose the IGF-I-IGFBP-VN interaction is an important mechanism promoting breast cancer metastasis. Understanding the processes that lead to the establishment of secondary tumor bodies and strategies to halt the spread of cancer beyond the primary site are therefore highly valuable, yet few interventions have targeted this aspect of breast disease. As such, future studies investigating the dysregulation, or overexpression, of these receptors and their ligands in contributing to the dissemination of tumors will be of particular importance. Furthermore, because cell migration is a critical process in wound healing and tissue remodeling, these interactions may also be of significant value in the emerging field of tissue engineering for application in a range of tissue repair therapies.


   Acknowledgments
 
We thank Mr. Andrew Ramsay and members of the Tissue Repair and Regeneration Program (Institute of Health and Biomedical Innovation, Queensland University of Technology) for providing technical advice. We also thank Dr. Stephen Myers, Dr. Janet Martin, Dr. Robert Pauley, Dr. Chris Shmidt, and Dr. John Price for providing cell lines used in this study.


   Footnotes
 
This work was supported by a grant from the National Health and Medical Research Grant 290511 (Australia).

Disclosure statement: J.A.K. and Z.U. are inventors on Growth Factor Complex Australia (WO 02/24219 A1). Z.U. consults for Tissue Therapies Ltd. B.G.H., J.A.K., D.I.L., and Z.U. have equity interests in Tissue Therapies Ltd. D.R.V.L. has nothing to declare.

First Published Online December 13, 2007

Abbreviations: ECM, Extracellular matrix; IGFBP, IGF-binding protein; IGF-IR, type I IGF receptor; LR3, long R3; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MYR-AKT, myristoylated AKT-1; PI3-K, phosphatidylinositide 3-kinase; VN, vitronectin; WT-AKT, wild-type AKT-1.

Received June 5, 2007.

Accepted for publication November 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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