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Endocrinology Vol. 144, No. 6 2417-2424
Copyright © 2003 by The Endocrine Society

Insulin-Like Growth Factor-II Bound to Vitronectin Enhances MCF-7 Breast Cancer Cell Migration

Anthony Noble, Chris Towne, Lisa Chopin, David Leavesley and Zee Upton

Tissue BioRegeneration and Integration Research Program, Center for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4000, Australia

Address all correspondence and requests for reprints to: Anthony M. Noble, Tissue BioRegeneration and Integration Program, Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. E-mail: a.noble{at}qut.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that IGF-II binds the extracellular matrix protein vitronectin (VN) with an affinity similar to that for the type-1 IGF receptor (IGF-1R). In view of this finding, and given the cited role of VN in cell motility and adhesion, we aimed to elucidate the functional consequences of this interaction on cellular processes relevant to breast carcinoma. We demonstrate that this complex slightly inhibits cell attachment and has little effect on protein synthesis in MCF-7 breast cancer cells. However, prebinding IGF-II to immobilized VN was found to significantly enhance breast cancer cell migration through Transwells. Interestingly, IGF-II bound to VN, and not IGF-II in solution in the presence of VN, seems to be responsible for the effects on cell migration. Furthermore, studies using analogs of IGF-II with reduced affinity for the IGF-1R or IGF binding proteins indicate that this response involves the IGF-1R but is independent of IGF binding proteins. This is the first study demonstrating that IGF-II:VN complexes enhance migration of cells. This may prove to be especially relevant, given that overexpression of IGF-II and VN are features of many tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I and IGF-II ARE mitogenic peptide growth factors involved in a broad range of cellular processes, including hyperplasia, DNA synthesis, differentiation, cell cycle progression, and inhibition of apoptosis (1, 2, 3). These effects are mediated through binding to their tyrosine-kinase-linked cell surface receptor, the type 1 IGF receptor (IGF-1R). The IGFs are also tightly regulated by a family of specific binding proteins, termed IGF-binding proteins (IGFBPs), whose primary role is to bind free IGFs and thereby moderate their half-life, specificity, and activity (4).

The IGFs have previously been linked with processes related to breast cancer progression and metastasis, such as cell cycle control, inhibition of apoptosis, and cell migration (3). In addition, loss of control of the IGF system is believed to be highly relevant to cancer progression. For example, high levels of the IGF-I and IGF-1R and low IGFBP levels are associated with poor long-term prognosis in breast cancer (5, 6).

Altered contact between cells and their associated extracellular matrix (ECM) is a prerequisite for breast cancer cell migration and, thus, metastasis (7). Cell migration requires interactions with adhesive molecules, such as vitronectin (VN). Indeed, interaction of cells with VN has been reported to be essential for IGF-stimulated migration (8, 9, 10, 11). Recently, VN has been shown to bind directly to IGF-II (12), whereas IGF-I can bind to VN in the presence of select IGFBPs (13, 14). The finding that VN, an ECM organization and adhesion molecule, binds IGF-II with an affinity that is similar to that of IGF-II for IGF-1R (12), its biologically relevant receptor, reveals a specific physical link between IGF action and VN in the ECM. In addition, recent studies in our laboratory have demonstrated that IGF-II bound to VN can stimulate synergistic functional responses in human keratinocytes in vitro (13). Taken together, these findings suggest that the formation of IGF-II:VN complexes may play an important role in modulating the effect of IGF-II in VN-rich ECM.

VN is a glycoprotein that is highly abundant in the blood and in the ECM. Primarily synthesized in the liver, but expressed by many other cell types, VN circulates in the blood in a closed conformation and is deposited in the ECM in an open, or extended, conformation (15). Both conformations are believed to bind IGF-II (12, 13, 16) and also bind multiple other ligands, including collagen (17), glycosaminoglycans (18), many other ECM proteins, and a wide variety of integrins, particularly the {alpha}v-integrins. Indeed, the primary role of VN is as an ECM organization molecule that provides adhesive links to these cell-surface integrin receptors via an RGD-binding motif. The VN receptors ({alpha}v-integrins) have been shown to regulate the actin cytoskeleton rearrangement required for growth and invasion; hence, VN binding coordinates cell adhesion and movement (19, 20).

VN has been implicated in a number of cellular processes associated with cancer. These range from control of metastasis (21) to reattachment at remote sites (15) and angiogenesis (22). Moreover, metastatic cancer cells have increased expression of {alpha}v-integrins, and an increased expression of VN correlates with metastatic potential in gliomas and colorectal adenocarcinomas (23, 24). Hence, loss of controlled adhesion, by up-regulation of VN and its receptors, may be a key determinant of metastatic ability (25, 26).

IGF-II has been demonstrated to stimulate increased migration in breast carcinoma cells (9). One mechanism through which this has been hypothesized to occur is by increased {alpha}vß3-expression, thus facilitating an increased interaction with VN in the ECM. Studies examining functional interactions between VN and the IGF system demonstrate that VN and IGF are tightly regulated and closely associated. This is illustrated by demonstration that occupancy of both the {alpha}vß3-VN receptor and the IGF-1R are prerequisites for breast cancer cell migration (9, 10, 27) and by the finding that the insulin receptor substrate 1, a downstream target of IGF-1R, associates with the VN receptors, the {alpha}v-integrins (28). Furthermore, breast tissues are known to be highly responsive to IGFs and are exposed to extensive hormone-stimulated matrix remodeling in their normal life cycle. Changes in these systems are also relevant in the cancer phenotype. The high level of interaction between VN and the IGF system in breast cancer, along with the demonstrated synergy of IGFs complexed to VN in stimulating keratinocyte protein synthesis (13), suggests that the IGF-II:VN complex will be biologically relevant in breast cancer cells, perhaps modulating tumor development and progression. This study aimed to examine the effect of the IGF-II:VN complex on processes important in breast cancer development and progression. Thus, the attachment, protein synthesis, and migration of poorly metastatic MCF-7 and highly metastatic MDA-MB-231 breast cancer cells seeded on IGF-II bound to an immobilized VN substratum in vitro were examined and are reported here.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The MCF-7 (ATCC, Manassas, VA; no. HTB-22) cell line was obtained from Mr. Steven Myers (Centre for Molecular Biotechnology) and was grown in DMEM/Ham’s F12 (DMEM/F12) media (1:1) (Life Technologies, Inc., Mulgrave, Victoria, Australia) containing 10% FCS. Media was changed daily and cells passaged at 80% confluence using 0.25% trypsin/0.5 mM EDTA solution (Oxoid, Hampshire, UK).

The MDA-MB-231 (ATCC no. HTB-26) human breast carcinoma cell line was obtained from Associate Professor Erik Thompson (St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia). The MDA-MB-231 cell line was grown in DMEM/F12 media (1:1) (Life Technologies, Inc.) containing 10% FCS. Media was changed daily and cells passaged at 80% confluence using 0.25% trypsin/0.5 mM EDTA solution (Oxoid).

Prebinding of IGF-II to VN
Most in vitro assays examining cell function add exogenous factors in solution; hence, the cells are bathed in the solution containing the substance throughout the assay. This is not the environment that cells encounter in vivo. Rather, cells in tissues are supported and surrounded by an ECM synthesized by cells, in which hormones and other factors are localized. In this study, which specifically addresses the binding of a growth factor to an ECM molecule, a strategy of prebinding VN and IGF-II to tissue culture plastic in 24-well plates and to the lower chamber and membrane surface of 12.0-µm-pore Transwells (Costar, New York, NY) was employed in an attempt to more accurately reflect the in vivo environment.

Preliminary studies in our laboratory have shown that maximal cell attachment in multiwell plates can be achieved by prebinding 300 ng VN to 24-well plates and 1 µg VN to 12-well plate wells; greater amounts of VN did not increase this effect. Thus, three hundred microliters of DMEM/F12 containing 300 ng VN (Promega Corp., Annandale, New South Wales, Australia) was added to 24-well tissue culture dishes or 500 µl of DMEM/F12 containing 1 µg VN was added to the lower chamber of a Transwell and incubated at 37 C for 2 h. Media containing unbound VN was removed, and the wells were washed with 1 ml HEPES binding buffer (HBB) containing 0.5% BSA (RIA-grade) (Sigma-Aldrich Corp., St. Louis, MO). Three hundred microliters of HBB containing 1.0% BSA was then added to wells and incubated at 37 C for 30 min to block nonspecific binding sites in the tissue culture dishes. The wells were then washed again with 1 ml HBB containing 0.5% BSA. Three hundred microliters of HBB containing 0.5% BSA and IGF-II (GroPep Pty. Ltd., Adelaide, South Australia, Australia) was then added, and the plates were incubated again for 2 h. The solution containing unbound IGF-II was removed, and the wells were washed with HBB and air-dried in laminar flow hoods.

Protein synthesis assays
Protein synthesis assays were performed as described in Francis et al. (1986) (29). Two hundred thousand cells that had been serum starved by incubation in serum-free media (SFM) for 4 h were seeded into each well in 1 ml SFM and incubated at 37 C/5% CO2 for 40 h. After washing and trichloracetic-acid precipitation of protein in the monolayer, incorporation of [3H]-leucine (Amersham International, Buckinghamshire, England) into de novo synthesized protein was determined by subsampling solubilized protein precipitate for ß-scintillation counting.

Attachment assays
Attachment assays were performed essentially as described by Leavesley et al. (1999) (30). Two hundred thousand cells that had been serum starved by incubation overnight in SFM containing 2 µCi [3H] leucine/ml were seeded into each well in 1 ml SFM and incubated at 37 C/5% CO2 for 4 h. The cells were serum starved for a longer period, compared with the other assays reported here, to better prime cells for the uptake of radiolabeled amino acid and to synchronize the cell-cycle state of cells before the attachment assays. After washing and trichloracetic-acid precipitation of protein in the monolayer, the amount of labeled protein (thus, number of attached cells) was determined by subsampling solubilized protein precipitate for ß-scintillation counting.

Migration assays
Migration assays were performed essentially as described by Leavesley et al. (1993) (31). Fifty thousand cells that had been serum starved by incubation in SFM for 4 h were seeded into the upper chamber of a 12.0-µm-pore Costar Transwell (12-well plate format). Cells that had migrated to the lower surface of the porous membrane, after 5 h of incubation at 37 C in 5% CO2, were fixed, then stained with Crystal Violet, in 0.1 mM borate buffer (pH 9). The number of cells attached was estimated by extracting Crystal Violet in 10% acetic acid and determining the absorbance of these extracts via spectrophotometry.

Statistical analysis
Data were analyzed by first expressing all data as a percentage of the negative control (-VN, -IGF-II). Responses were then tested for significance vs. VN-only controls and IGF-II-only controls using a two-tailed homoscedastic Student’s t test. P values < 0.05 indicate responses that were significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Attachment
VN is an adhesive molecule found throughout the ECM, and it mediates cell attachment by binding to cellular integrins through an RGD integrin-recognition sequence within the VN molecule (15). Assays testing attachment of MCF-7 cells to IGF-II prebound to VN immobilized on tissue culture plastic were performed to determine the effect of complex formation on cell attachment (Fig. 1Go). As anticipated, prebinding 300 ng VN to tissue culture plastic increased cell attachment. This resulted in increases of more than 2-fold in total cells attached (P < 0.01). Interestingly, when increasing amounts of IGF-II were prebound to the same amount of VN, this increase was reduced to approximately 150% of control wells (-VN, -IGF-II); thus, IGF-II bound to VN has an inhibitory effect on cell attachment, compared with wells containing VN alone. Statistical analysis in this situation revealed that attachment of cells to VN prebound with all amounts of IGF-II tested was significantly (P < 0.01) inhibited, compared with the effect of VN alone.



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  		Figure 1. Attachment of MCF-7 cells to tissue culture plastic coated with IGF-II prebound to VN (gray bars) or IGF-II bound to the dishes in the absence of VN (white bars). Each bar represents the average attachment obtained from three replicate experiments in which treatments were analyzed in triplicate wells. Data points where the effect of the complex is significantly different from that of VN alone are indicated by an asterisk.

 
Protein synthesis
We have previously established that the effects of IGFs in stimulating protein synthesis closely correlate with cell proliferation, as measured by increase in total cell number. Hence, the response of cells to VN:IGF-II complexes was examined through assays measuring incorporation of radioactive leucine into de novo synthesized total cell protein (Fig. 2Go). De novo protein synthesis in cells seeded onto tissue culture plastic prebound with 300 ng VN was significantly increased (~200%), compared with control wells that did not contain IGF-II or VN (P < 0.01). Increases in protein synthesis of 170–190%, compared with control wells (without IGF-II or VN), were also obtained when 10–1000 ng IGF-II were prebound to this same amount of VN. However, statistical analysis of these results revealed that IGF-II bound to VN does not elicit significant (P > 0.05) changes in protein synthesis in MCF-7 cells, compared with the effects obtained with VN alone.



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  		Figure 2. De novo protein synthesis of MCF-7 cells, in response to tissue culture plastic coated with IGF-II prebound to VN (gray bars) or IGF-II bound to the dishes in the absence of VN (white bars). Each bar represents the average response obtained from three replicate experiments in which treatments were analyzed in triplicate wells.

 
Migration
Cell migration is a key process in cancer development, and both VN and IGF-II have established roles in the mediation of cell migration. To dissect the ability of IGF-II bound to VN to alter MCF-7 cell migration from the effects of IGF-II in solution, the migration of cells through Transwells was measured using two approaches. The first involved the removal of IGF-II that remained in solution after the prebinding step described in Materials and Methods, followed by the addition of SFM in the lower chamber of the Transwells and seeding of cells into the upper chamber. The parallel assay left the media containing unbound IGF-II in the lower chamber.

When VN was prebound to the lower well of 12.0-µm-pore Transwells, a 4-fold increase in migration to the lower chamber, compared with negative control wells, was observed (P < 0.01). Prebinding 1–100 ng IGF-II to the wells, in the absence of VN, stimulated no significant increase in migration (P > 0.05). However, when 1–100 ng IGF-II was prebound to 1 µg VN in the lower chamber, 7- to 9-fold increases in cell migration were observed (Fig. 3Go). Further statistical comparison with positive controls further showed that these responses were significantly higher (P < 0.01) than the effects of IGF-II alone and VN alone. Interestingly, the parallel assays, in which IGF-II that had not bound to VN was also retained in solution, revealed similar responses to those obtained when unbound IGF-II was removed (P values > 0.05) (Fig. 4Go).



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  		Figure 3. Migration of MCF-7 cells seeded into the upper chamber of 12-µm-pore Transwells to the lower surface, in response to the lower chamber being coated with IGF-II prebound to VN (gray bars), or IGF-II bound to the dishes in the absence of VN (white bars). Each bar represents the average number of cells on the lower membrane after 5 h of incubation. Results were obtained from three replicate experiments in which treatments were analyzed in triplicate wells. Data points where the effect of the complex is significantly different from that of VN alone are indicated by an asterisk.

 


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  		Figure 4. Migration of MCF-7 cells seeded into the upper chamber of 12-µm-pore Transwells to the lower surface, in response to the lower chamber being coated with IGF-II bound to VN with unbound IGF-II removed and replaced with SFM (gray bars), and IGF-II prebound to VN with unbound IGF-II in solution retained in the lower chamber (black bars). Each data point is paired with a VN-free control (white bars) containing the same amount of IGF-II in the absence of VN. Each bar represents the average number of cells on the lower membrane after 5 h of incubation. Results were obtained from two replicate experiments in which treatments were analyzed in triplicate wells.

 
[L27]-IGF-II is an IGF-II analog that does not bind to the IGF-1R (GroPep Pty. Ltd.; Ref. 32); the receptor through which IGF-mediated migration is believed to be signaled. Hence, assays examining the ability of [L27]-IGF-II prebound to VN to stimulate migration through Transwells were conducted. These assays revealed that VN-[L27]-IGF-II complexes did not enhance MCF-7 migration beyond the level obtained with VN alone and that the level of migration was significantly less (P < 0.01) than that observed in response to native IGF-II bound to VN (Fig. 5Go). Responses to VN-[L27]-IGF-II were significantly greater (P < 0.01) than those obtained in the negative control (-VN, -IGF-II). However, this response could be attributed to the effect of VN in the complex and is confirmed by findings that there is no significant difference between the VN-[L27]-IGF-II and VN-only controls (P > 0.05). These results indicate that the enhanced migration arising from IGF-II bound to VN involves interaction of IGF-II with the IGF-1R.



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  		Figure 5. Migration of MCF-7 cells seeded into the upper chamber of 12-µm-pore Transwells to the lower chamber that had been coated with VN, native IGF-II bound to VN (gray bars), or L27-IGF-II bound to VN (black bars). Each data point is paired with a VN-free control (white bars) containing the same amount of IGF-II in the absence of VN. Each bar represents the average number of cells on the lower membrane after 5 h of incubation. Results were obtained from two replicate experiments in which treatments were analyzed in triplicate wells.

 
IGFBPs are key regulators of IGF exposure to cells. To determine whether IGFBPs are involved in the migratory responses to VN:IGF-II complexes observed here, migration assays using an IGF-II analog that binds poorly to IGFBPs, yet retains affinity for the IGF-1R, were conducted. Assays using this IGF-II analog, des(1–6)-IGF-II (GroPep Pty. Ltd.), revealed no differences in the migratory responses, compared with native IGF-II (P > 0.05), suggesting that IGF-II:VN complexes act independently of IGFBPs to enhance cell migration (Fig. 6Go).



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  		Figure 6. Migration of MCF-7 cells seeded into the upper chamber of 12-µm-pore Transwells to the lower chamber that had been prebound with VN only, native IGF-II bound to VN (gray bars), or Des(1–6) IGF-II bound to VN (black bars). Each data point is paired with a VN-free control (white bars) containing the same amount of IGF-II in the absence of VN. Each bar represents the average number of cells on the lower membrane after 5 h of incubation. Results were obtained from two replicate experiments in which treatments were analyzed in triplicate wells.

 
The MCF-7 cell line exhibits poor migration in the absence of serum or VN and, as such, is an ideal cell line to examine the effect of candidate factors that are thought to be potential stimulators of cancer metastasis. To determine whether the enhanced migration observed with this cell line would occur in a highly metastatic cell line, migration assays using the MDA-MB-231 cell line were undertaken. As expected, MDA-MB-231 cells showed a high level of migration in negative control. Migration in Transwells containing IGF-II only was no greater than that observed in negative control wells (-VN, –IGF-II) (P > 0.05). Moreover, the degree of migration in response to VN:IGF-II complexes was no greater than the effects obtained with VN only (P > 0.05) (Fig. 7Go).



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  		Figure 7. Migration of MDA-MB-231 cells seeded into the upper chamber of 12-µm-pore Transwells to the lower surface, coated with IGF-II prebound to VN (gray bars) or IGF-II bound to the dishes in the absence of VN (white bars). Each bar represents the average number of cells on the lower membrane after 5 h of incubation. Results were obtained from two replicate experiments in which treatments were analyzed in triplicate wells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
VN has previously been reported to have a critical role in the regulation of cell attachment in the ECM; hence, the ability of VN:IGF-II complexes to modulate the attachment of MCF-7 human breast cancer cells, known to express VN receptors ({alpha}v-integrins) (25), was examined. MCF-7 cells demonstrated little attachment in the absence of VN. However, an increase in attachment of more than a 2-fold was observed when 300 ng VN was prebound to tissue culture plastic. When the two highest amounts of IGF-II were prebound to VN, attachment was significantly inhibited, compared with cells exposed to VN alone. We hypothesize that the decrease in the attachment of MCF-7 cells observed in this situation may arise from IGF-II bound to VN sterically blocking the RGD integrin recognition site on VN, hence blocking the interaction of this motif with cell surface integrins (33).

IGF-II is an established promoter of cell proliferation (3). To examine whether IGF-II prebound to VN still results in proliferative effects, assays examining de novo protein synthesis in MCF-7 cells were performed. The MCF-7 cells exhibited a low basal level of protein synthesis in the control wells (-VN, -IGF-II) and a 2-fold increase above the control in response to growth in wells coated with 300 ng VN. This may reflect differences in the number of cells attached; that is, few cells did so in the –VN wells. When IGF-II was prebound to VN, small, but not significant, increases in cell protein synthesis were detected, similar to those obtained in VN-only controls; thus, the complexes do not enhance protein synthesis in this cell line. This contrasts with the situation observed in the HaCAT human keratinocyte cell line, in which IGF-II:VN complexes stimulate significantly enhanced protein synthesis (13), perhaps reflecting differences in the control of proliferation between cancerous and noncancerous cell lines.

In studies examining MCF-7 cell migration, minimal responses were observed in the absence of VN. The MCF-7 cells did, however, exhibit a greater-than-4-fold increase in migration in response to VN alone. An even-greater increase in cell migration was observed when 10 and 100 ng IGF-II was pre-bound to VN, indicating that the IGF-II:VN complexes may indeed have critical roles in modulating this cell function. Interestingly, regardless of whether the solution containing unbound IGF-II was replaced with SFM or retained, the MCF-7 cells showed similar levels of migration (Fig. 4Go).

Previously, Doerr and Jones (1997) (10) established that 10 ng/ml was the optimum concentration of IGF-I for stimulation of migration of MCF-7 cells on VN. We have demonstrated here that, when IGF-II is prebound to VN, large increases in migration are elicited in response to both 10 and 100 ng IGF-II and that similar enhancement of migration is observed when unbound IGF-II is retained in solution or removed from Transwells. These results suggest that the IGF-II:VN complex can stimulate migration of MCF-7 cells and that this would seem to be primarily attributable to IGF-II captured by VN. The ability of bound IGF-II to exert these significant effects on cell migration may, in part, be attributable to reduced interactions with the cation-independent mannose-6-phohosphate receptor (CIMPR), a receptor believed to regulate cellular exposure to IGF-II. The interaction between the CIMPR and IGF-II is known to be crucial for fetal development (9) and has also been suggested to be involved in tumor suppression (34). IGF-II bound to VN may be less able to interact with the CIMPR, compared with IGF-II in solution; thus, whereas IGF-II in solution may be removed from the cell surface, captured IGF-II is not. Hence, binding of IGF-II to VN may represent a novel means of regulating cellular exposure to IGF-II.

Further studies using IGF-II analogs with reduced affinity for the IGF-1R or IGFBPs were undertaken to dissect the underlying mechanisms through which MCF-7 cell migration was enhanced. Migration assays using [L27]-IGF-II, an IGF-II analog with little affinity for IGF-1R (32), were performed in parallel with assays using native IGF-II. These assays revealed that [L27]-IGF-II prebound to VN resulted in effects no different from those observed in response to VN alone; that is, the enhanced migration was abrogated. This would indicate that the enhanced migration observed in response to IGF-II:VN complexes is mediated by IGF-II associated with the complex, binding and stimulating the IGF-1R. Whereas it is well established that binding of IGFs to this receptor leads to a number of downstream signals stimulating both proliferation and migration, the apparently specific enhancement of migration found here in response to the VN:IGF-II complex is hypothesized to be as a result of integrin–VN interactions sensitizing the internal cellular environment to activation of the migration pathway. Previous studies by Jones et al. (1996) (9) demonstrated that activation of the IGF-1R is crucial to elicit IGF-stimulated migration; however, activation of both IGF-1R and integrin is required for maximal effect. The finding that an integrin-binding protein (VN) and an IGF-1R-binding protein (IGF-II) form a complex and that this complex is a potent stimulator of cell migration is therefore of great interest.

IGFBPs are extensively involved with IGF regulation; hence, investigation of possible interactions between IGF-II:VN complexes and IGFBPs was undertaken by performing migration assays with des(1–6)IGF-II. The results of these studies showed that des-(1–6)IGF-II had a similar ability to enhance MCF-7 migration as the native IGF-II. This indicates that IGFBPs are not involved in the enhancement of migration reported here. However, we would expect that the MCF-7 cells would have synthesized only small amounts of endogenous IGFBPs during the 5-h time course of the assays. Moreover, trypsinization of the cells immediately before seeding makes it unlikely that cell-surface-associated IGFBPs were present in the assays. Future studies will examine the effect of IGFBPs on IGF:VN complexes, because IGF-I has been recently demonstrated to bind VN in the presence of IGFBPs (13), and these trimeric complexes may also play a role in cell migration. This hypothesis is supported by recent findings by Nam et al. (2002) (14), who showed enhanced migration in response to wounding of smooth muscle cells is observed after exposure to IGF-I in the presence of both VN and IGFBP-5.

Assays examining the migration of the highly metastatic MDA-MB-231 cell line revealed no increase in migration in response to complexes above that observed in wells containing just VN. Lee et al. (35) showed that both MCF-7 and MDA-MB-231 cells express IGF-II in vitro. In MCF-7 cells, stimulation by estrogen is required to elicit this expression. Yet, in MDA-MB-231, IGF-II expression is constitutive. These expression profiles are analogous to the widely established estrogen receptor (ER) identities of these cells (i.e. MCF-7 cells are ER-positive and MDA-MB-231 cells are ER-negative). The baseline expression of IGF-II by the MDA-MB-231 cell line may account for the lack of response to IGF-II observed in assays reported here using this cell line. Furthermore, the very high rate of cell division and very fast growth of this cell line suggest that the cell cycle processes are under poor control. Additionally, specific loss of IGF-1R-mediated cell cycle control in this cell line has recently been demonstrated by Bartucci et al. (2001) (36). Other studies have demonstrated that the MDA-MB-231 cell line expresses approximately 7 x 103 IGF-1R/cell; this is 10-fold less than MCF-7 cells (~6 x 104/cell) and, crucially, is below the theoretical threshold level required for IGF-1R activation of more than1.5 x 104 IGF-1R/cell (37). MCF-7 and MDA-MB-231 also express different VN-binding cell surface integrins. MCF-7 expresses {alpha}vß5- and {alpha}vß1-integrins, whereas MDA-MB-231 expresses {alpha}vß3-integrin in addition to those integrins present on MCF-7 (25). The {alpha}vß3-integrin has been implicated in the independent stimulation of intracellular signaling pathways normally associated with the activation of growth factor receptors (38). This property may mean that those pathways stimulated by IGFs are already activated, and thus that the cells do not increase their rate of migration in response to IGF-II when {alpha}vß3 is activated.

The results of this study reveal, for the first time, that IGF-II:VN complexes significantly stimulate migration of MCF-7 cells. Interestingly, the effect of the complex on cell migration in MCF-7 cells seems to primarily arise from IGF-II bound to VN. Taken together, these data indicate that the VN:IGF-II complex is functionally relevant and may indeed be a significant factor in breast cancer development and progression. Thus, the complex may inhibit cell attachment, yet, at the same time, stimulate migration in the ECM. If indeed, VN:IGF-II complexes do promote migration (and thus, metastasis of breast cancer cells), drugs directed at inhibiting IGF-II:VN complex formation may prove to be highly effective therapeutics.


   Acknowledgments
 
We would like to acknowledge the expert technical assistance and advice given by Ms. Carolyn Hyde.


   Footnotes
 
The authors would like to thank the Queensland Cancer Fund and Queensland University of Technology ATN grant schemes for providing the funding for this project.

Abbreviations: CIMPR, Cation-independent mannose-6-phohosphate receptor; DMEM/F12, DMEM/Ham’s F12; ECM, extracellular matrix; ER, estrogen receptor; HBB, HEPES binding buffer; IGFBP, IGF-binding protein; IGF-1R, type-1 IGF receptor; SFM, serum-free media; VN, vitronectin.

Received October 30, 2002.

Accepted for publication February 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Keiss W, Yang Y, Kessler U, Hoeflich A 1994 Insulin-like growth factor-II/mannose-6-phosphate receptor: the myth continues. Horm Res 41:66–73
  2. Wood TL, Yee D 2000 IGFs and IGFBPs in the normal mammary gland and in breast cancer. J Mammary Gland Biol Neoplasia 5:1–5[CrossRef][Medline]
  3. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological action. Endocr Rev 16:3–34[Abstract/Free Full Text]
  4. Clemmons DR 1998 Role of insulin-like growth factor binding proteins in controlling IGF actions. Mol Cell Endocrinol 140:19–24[CrossRef][Medline]
  5. Flint DJ, Tonner E, Allan GJ 2000 Insulin-like growth factor binding proteins: IGF-dependent and independent effects in the mammary gland. J Mammary Gland Biol Neoplasia 5:65–73[CrossRef][Medline]
  6. Surmacz E 2000 Function of the IGF-I receptor in breast cancer. J Mammary Gland Biol Neoplasia 5:95–105[CrossRef][Medline]
  7. Gui GPH, Puddefoot JR, Vinson GP, Wells CA, Carpenter R 1997 Altered cell matrix contact: a prerequisite for breast cancer metastasis. Br J Cancer 75:623–633[Medline]
  8. Stewart AJ, Johnson MD, May FEB, Westley BR 1990 Role of IGFs and the IGF-1R in the estrogen stimulated proliferation of human breast cancer cells. J Biol Chem 265:21172–21178[Abstract/Free Full Text]
  9. Jones IJ, Prevette T, Gockerman A, Clemmons DR 1996 Ligand occupancy of the {alpha}vß3 integrin is necessary for smooth muscle cells to migrate in response to insulin like growth factor I. Proc Natl Acad Sci USA 93:2482–2487[Abstract/Free Full Text]
  10. Doerr ME, Jones JI 1996 The roles of integrins and extracellular matrix proteins in the insulin-like growth factor stimulated chemotaxis of human breast cancer cells. J Biol Chem 271:2443–2447[Abstract/Free Full Text]
  11. Clemmons DR, Horvitz G, Engleman W, Nichols T, Moralez A, Nichols GA 1999 Synthetic {alpha}vß3 antagonists inhibit insulin-like growth factor-I stimulated smooth muscle cell migration and replication. Endocrinology 140:4616–4621[Abstract/Free Full Text]
  12. Upton Z, Webb H, Hale K, Yandell CA, McMurtry JP, Francis GL, Ballard FJ 1999 Identification of vitronectin as a novel insulin-like growth factor II binding protein. Endocrinology 140:2928–2931[Abstract/Free Full Text]
  13. Upton Z, Kricker J 2001 Growth factor complex. International Patent Application WO 02/24219A1
  14. Nam TJ, Busby W, Clemmons DR 1997 Insulin growth factor binding protein 5 binds to plasminogen activator inhibitor 1. Endocrinology 138:2972–2978[Abstract/Free Full Text]
  15. Schvartz I, Segar D, Shaltiel S 1999 Vitronectin. Int J Biochem Cell Biol 31:531–544[CrossRef]
  16. McMurty JP, Francis GL, Upton Z, Walton PE, Rosselot G, Caperna TJ, Brocht DM 1996 Plasma clearance and tissue distribution of labeled chicken and human IGF-I and IGF-II in the chicken. Endocrinology 150:149–160
  17. Morris CA, Underwood PA, Bean PA, Sheehan M, Charlesworth JA 1994 Relative topography of biologically active domains of human vitronectin. J Biol Chem 269:23845–23852[Abstract/Free Full Text]
  18. Francois PP, Preissner KT, Herrman M, Haugland RP, Vaudaux P, Lew DP, Krause KH 1999 Vitronectin interaction with glycosaminoglycans. J Biol Chem 274:37611–37619[Abstract/Free Full Text]
  19. DePasquale JA 1998 Cell matrix adhesions and localization of vitronectin receptor in MCF7 human mammary carcinoma cells. Histochem Cell Biol 110:485–494[CrossRef][Medline]
  20. Huang S, Stupack D, Liu A, Cheresh D, Nemerow GR 2000 Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of {alpha}v integrins. Oncogene 19:1915–1923[CrossRef][Medline]
  21. Felding-Haberman B, O’Toole TE, Smith JW, Fransvea E, Ruggeri ZM, Ginsberg MH, Hughes PE, Pampori N, Shattil SJ, Meuller BM 2001 Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci USA 98:1853–1858[Abstract/Free Full Text]
  22. Varner JA, Brooks PC, Cherish D 1995 The integrin {alpha}vß3: angiogenesis and apoptosis. Cell Adhes Commun 3:367–374[Medline]
  23. Tomasini-Johansson BR, Sundberg C, Lindmark G, Gailiat JO, Rubin K 1994 Vitronectin in colorectal adenocarcinomas—synthesis by stromal cells in culture. Exp Cell Res 214:303–312[CrossRef][Medline]
  24. Uhm JH, Dooley NP, Kyritsis AP, Rao JS, Gladson CL 1999 Vitronectin, a glioma derived extracellular matrix protein, protects tumour cells from apoptotic death. Clin Cancer Res 5:1587–1594[Abstract/Free Full Text]
  25. Meyer T, Marshall JF, Hart IR 1998 Expression of {alpha}v integrins and vitronectin identity in breast cancer cells. Br J Cancer 77:530–536[Medline]
  26. Gladson CL, Dennis C, Rotolo TC, Kelly DR, Grammer JR 1997 Vitronectin expression in differentiating neuroblastic tumours. Am J Pathol 150:1631–1646[Abstract]
  27. Zeng B, Clemmons DR 1998 Blocking ligand occupancy of the {alpha}vß3 integrin inhibits insulin like growth factor I signaling in vascular smooth muscle cells. Proc Natl Acad Sci USA 95:11217–11222[Abstract/Free Full Text]
  28. Vuori K, Roushlahti E 1994 Association of IRS-1 with integrins. Science 266:1576–1578[Abstract/Free Full Text]
  29. Francis GL, Read LC, Ballard FJ, Bagley CJ, Upton FM, Gravestock PM, Wallace JC 1986 Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrums. J Biochem 233:207–213
  30. Leavesley DI, Stanley JM, Faull RJ 1999 Epidermal growth factor modifies the expression and function of extracellular matrix adhesion receptors expressed by peritoneal mesothelial cells from patients on CAPD. Nephrol Dial Transplant 14:1208–1216[Abstract/Free Full Text]
  31. Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA 1993 Integrin ß 1- and ß 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol 121:163–170[Abstract/Free Full Text]
  32. Roth BV, Burgisser DM, Luthi C, Humbel RE 1991 Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or deletion of residues 62–67. Biochem Biophys Res Commun 181:907–914[CrossRef][Medline]
  33. Morris CA, Underwood PA, Bean PA, Sheehan M, Charlesworth JA 1994 Relative topography of biologically active domains of human Vitronectin. J Biol Chem 269:23845–23852
  34. DaCosta SA, Schumaker LM, Ellis MJ 2000 Mannose-6-phoshate/IGF-II receptor; a bone fide tumour suppressor gene or promising candidate? J Mammary Gland Biol Neoplasia 5:85–94[CrossRef][Medline]
  35. Lee AV, Darbe P, King RJ 1994 Processing of insulin-like growth factor-II by human breast cancer cells. Mol Cell Endocrinol 99:211–220[CrossRef][Medline]
  36. Bartucci M, Morelli C, Mauro L, Ando S, Surmacz E 2000 Differential insulin-like growth factor I receptor signaling and function in the estrogen receptor positive MCF-7 and ER negative MDA-MB-231 breast cancer cells. Cancer Res 61:6747–6754
  37. Rubini M, Hongo A, D’Ambrosio C, Baserga R 1997 The IGF-1R in mitogenesis and transformation of mouse embryo fibroblasts: the role of receptor number. Exp Cell Res 230:284–292[CrossRef][Medline]
  38. Giancotti FG, Roushlati E 1999 Integrin signalling. Science 285:1028–1032[Abstract/Free Full Text]



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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.
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