This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mira, E. Articles by Martínez-A, C. Search for Related Content PubMed PubMed Citation Articles by Mira, E. Articles by Martínez-A, C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-PHENYLALANINE THIOPHENE Medline Plus Health Information Breast Cancer

Insulin-Like Growth Factor I-Triggered Cell Migration and Invasion Are Mediated by Matrix Metalloproteinase-9¹

Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Emilia Mira, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Spanish Research Council CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail: emira{at}cnb.uam.es

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the _vß₅ integrin, a vitronectin receptor. The levels of _v- and ß₅-subunits expressed in MCF-7 cells depend on the IGF-I concentration, which triggers an increase in both of these subunits. Based on these results, we suggest that IGF-I-induced MCF-7 cell migration is mediated by the MMP-9 activity on the cell surface and by _vß₅ integrin.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
TUMOR CELL migration is one of the most important events contributing to tumor dissemination, and its prevention may arrest malignant evolution (1). Insulin-like growth factor I (IGF-I) has been implicated in the migration of several cell lines in vitro and in vivo, including the estrogen receptor-positive MCF-7 breast carcinoma cell line (2, 3, 4). IGF-I is also a mitogen for human breast cancer cells (5), and the combination of the chemoattractant and mitogenic activities of IGF-I probably contributes to tumor survival and progression.

Tumor dissemination has been correlated with matrix metalloproteinases (MMP) due to their proteolytic activity on extracellular matrix proteins (6). More recently, MMP have been implicated in the release of the soluble form of several growth factors (7), and interestingly, several IGF-binding proteins have been described as MMP substrates (8, 9, 10). Together, these data enlarge the relevance of this proteinase family on sustaining an invasive phenotype. Among its members, MMP-9 exhibits a widespread expression in a variety of human cancers, suggesting its involvement in human tumor dissemination (11). Direct evidence of its role in tumor progression is derived from transfection experiments in which the MMP-9 gene in nonmetastatic cells endowed them with the ability to metastasize (12).

Besides the requirement of proteolysis, adhesion to the extracellular matrix is necessary for tumor invasion (13). Integrin molecules belong to the largest family of extracellular matrix (ECM) adhesion receptors and have been implicated in several processes, including cell migration, invasion, and metastasis (14, 15). The link between integrins and MMP comes from experimental evidence such as the colocalization of the integrin _vß₃ and MMP-2 (16) and the positive regulation of MMP-1 expression by the integrin ₂ß₁ (17). Furthermore, integrin function is a requirement for IGF-I-mediated chemotaxis; in MCF-7 cells, migration through vitronectin and type IV collagen is dependent on _vß₅ and ₂ß₁, respectively, and in smooth muscle cells, ligand occupancy of _vß₃ modulates the migration process (2, 3). The dissemination of several malignant tumor cells is also dependent on the cooperation between _vß₅ and IGF-I (4).

Although IGF-I-triggered migration of MCF-7 has been previously reported (3), the mechanism mediating the MCF-7 migratory response to IGF-I through the ECM component vitronectin has not yet been elucidated. Here we have identified MMP-9 and its proteolytic activity on the MCF-7 cell surface; we describe the IGF-I-induced changes in MMP-9 and the vitronectin receptor _vß₅ and their correlation with the migration response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture and materials
MCF-7 human breast carcinoma cells from the American Type Culture Collection (Manassas, VA) were maintained in DMEM with 10% FCS, penicillin, streptomycin, L-glutamine, and sodium pyruvate. The HT-1080 human fibrosarcoma cell line (American Type Culture Collection) was cultured in the same medium, which was replaced by serum-free medium with 0.1% BSA and the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 ng/ml) for 1 day to obtain conditioned medium. Recombinant human IGF-I was provided by Pharmacia & Upjohn, Inc. (Stockholm, Sweden). Vitronectin was purchased from Collaborative Biomedical (Bedford, MA). Transwell chambers (pore size, 8 µm) were obtained from Corning Costar (Cambridge, MA). Batimastat (BB-94) (18) was provided by Dr. F. Colotta (Pharmacia & Upjohn, Inc., Milan, Italy); aprotinin and PMA were obtained from Sigma Chemical Co. (St. Louis, MO). The monoclonal antibody (mAb) anti-IGF-I BB9E10 has been previously described (19). Anti-IGF type 1 receptor (IGF-1R; -IR3), anti-MMP-9 (Ab-1), and anti-MMP-2 (Ab-3) mAb were purchased from Oncogene (Cambridge, MA). The polyclonal anti-MMP-9 antibody (Ab), obtained by immunization with a synthetic peptide (amino acids 422–434), was purchased from Research Genetics, Inc. (Huntsville, AL). Anti-_v (K267) is a polyclonal Ab obtained by immunization with a synthetic peptide (amino acids 372–385), and anti-_v (6D1) mAb was obtained by immunizing with purified human placenta integrin _vß₅; both were produced in our laboratory. The anti-ß₅ polyclonal Ab and anti-_vß₅ (p1F6) mAb were purchased from Boehringer Ingelheim (Heidelberg, Germany), the anti-ß1 (DF5) mAb was obtained from Chemicon (Temecula, CA), and the anti-2 mAb (p1E6) was obtained from Life Technologies (Paisley, Scotland, UK).

Migration assays
MCF-7 cells (10⁵) were trypsinized and resuspended in serum-free DMEM with 0.01% BSA, then seeded in the upper chamber of vitronectin-coated transwells (5 µg/ml in PBS, 18 h at room temperature). The lower chambers were loaded with DMEM-BSA, with or without IGF-I. In some assays, cells were preincubated with Abs or inhibitors for 30 min at room temperature before seeding in the transwell. Cells were incubated for 18 h at 37 C in 5% CO₂. The chamber was disassembled, and cells on the upper surface were removed. Filters were stained, and cell numbers were calculated as previously described (3).

MMP activity measurement
Cell surface MMP activity was measured using a modification of previously described methods (20, 21). Briefly, 5 x 10⁴ cells resuspended in complete medium were seeded in 96-well microtiter plates (Nunc, Roskilde, Denmark). Cells were washed three times with HBSS (Life Technologies), and the fluorogenic peptide Dnp-Pro-ß-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH₂ (Bachem, Bubendorf, Switzerland) was added in HBSS containing 0.01% NaN₃. Plates were incubated at 37 C, and at the indicated time the increment in fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm with a Cytofluor microplate reader (Millipore Corp., Bedford, MA). When IGF-I was assayed, cells were starved for 6 h in serum-free medium and incubated with or without IGF-I for the time indicated. Medium was then removed, and after washing with HBSS, proteolytic activity was measured. When inhibitors were tested, they were preincubated for 30 min before the addition of the fluorogenic substrate. Fluorescence was normalized by cell staining with crystal violet (Sigma Chemical Co.). Zymograms were performed as previously described (20) in 10% SDS-polyacrylamide gels containing gelatin (1 mg/ml).

Fluorescence-activated cell sorting (FACS) analysis
MCF-7 cells were detached, washed with ice-cold PBS, and resuspended at 10⁶ cells/ml in PBS with 0.5% BSA and 0.01% NaN₃. Anti-MMP-9, anti-MMP-2, or anti-_vß₅ mAb were added, followed by a fluorescein isothiocyanate-labeled goat antimouse IgG (Southern Biotechnology Associates, Birmingham, AL). Cell-associated fluorescence was visualized in an EPICS XL flow cytometer (Beckman Coulter, Inc., Hialeah, FL).

PCR analysis
First strand complementary DNA (cDNA) was prepared from MCF-7-isolated messenger RNA and HT1080 total RNA. Oligonucleotide primers for PCR amplification of gelatinase B were synthesized as previously described (22), and each cycle consisted of denaturation at 94 C, annealing at 55 C, and elongation at 72 C for 30 sec in each step after 1 min at 94 C in the first cycle. The primer sequences for PCR amplification of GAPDH were as follows: sense primer, 5'-TCCTGCACCACCAAC TGCTTA-3'; and antisense primer, 5'-ACCACCCTGTTGCTGTAGCC-3'. Each cycle consisted of denaturation at 94 C and annealing at 57 C for 30 sec in each step and elongation at 72 C for 1 min after 1 min at 94 C in the first cycle.

Western blot analysis
Conditioned medium samples were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions, electroblotted, and blocked with 5% (wt/vol) nonfat milk in PBS-Tween-20 (0.1%, vol/vol). The filter was incubated with an IgG fraction of the rabbit anti-MMP-9 serum, followed by a peroxidase-conjugated goat antirabbit Ig (DAKO Corp. A/S, Glostrup, Denmark). Reactions were developed using the ECL system (Amersham, Aylesbury, UK). Cell lysates were obtained by incubating monolayer cells in lysis buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5), and 300 mM NaCl at 4 C for 15 min. After detaching cells by scraping, insoluble material was removed by centrifugation. To detect integrin subunits, samples were electrophoresed on 8% SDS-polyacrylamide gels under nonreducing conditions, then processed as described above.

Immunoprecipitation of biotin-labeled integrin
Cells were grown to 80% confluence and labeled with Sulfo-NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL) following the manufacturer’s instructions. Cell lysates were obtained as previously described (23) and were immunoprecipitated with mAb anti-_v or anti-_vß₅ (4 C, overnight), followed by agarose-conjugated goat antimouse IgG. The immunoprecipitated proteins were separated on 10% SDS-polyacrylamide gels under reducing conditions, and blots were developed using peroxidase-labeled streptavidin (Amersham) followed by enhanced chemiluminescence.

Adhesion assay
Vitronectin-coated 96-well microtiter plates (1 µg/ml in PBS, overnight at 4 C) were blocked with 5% BSA in PBS for 3 h at 37 C. MCF-7 cells (3 x 10⁵) in DMEM-BSA were preincubated with anti-_vß₅ and anti-₂ mAb for 30 min and seeded. After a 2-h incubation, plates were washed three times with DMEM-BSA, and adherent cells were stained with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.) (24).

Statistical analysis
All experiments were repeated two or three times. The mean and SD of triplicate points from one or two experiments are shown. Zymographic data and the corresponding densitometry quantitation are presented from a representative experiment. The significant differences between group values were analyzed using the Kruskal-Wallis test, and the Mann-Whitney test was used to compare paired values.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MCF-7 cell migration in response to IGF-I is inhibited by the MMP inhibitor BB-94
When seeded on vitronectin-coated Transwell chambers, MCF-7 cells migrate in response to IGF-I (3). Migration observed at 18-h incubation at an IGF-I concentration of 1 ng/ml was considered maximal; migration decreases at higher IGF-I concentrations (up to 1 µg/ml; Fig. 1A). IGF-I-mediated MCF-7 cell migration is inhibited by anti-IGF-I mAb (data not shown) as well as by anti-IGF-1R mAb, as previously described (3). This migration response to IGF-I is independent of de novo protein synthesis, as cycloheximide has no effect on this assay (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 1. MCF-7 cell migration in response to IGF-I: the effect of proteinase inhibitors. A, MCF-7 cells (105) were seeded on vitronectin-coated Transwell chambers, and the indicated amount of IGF-I was added to the lower chamber. After overnight incubation, cells on the upper surface were removed, and the filter was stained. The number of cells represents the mean ± SD counted per filter. B, Cells were preincubated for 30 min at room temperature in the absence (-) or presence of BB-94 or aprotinin (10 µg/ml) before seeding into Transwell chambers. The number of migrating cells in the absence of inhibitors is assigned a relative value of 100%. The asterisk indicates a significant difference (P < 0.05) compared with untreated cells (by Kruskal-Wallis test).

MMP-9 activity is anchored to the MCF-7 cell surface
MMP activity is usually analyzed in cell culture-conditioned medium, as most MMP family members are secreted proteases (6). To characterize the MMP involved in MCF-7 migration, we therefore analyzed MCF-7 cell-conditioned medium by gelatin zymography, using HT-1080-conditioned medium as a control (25). MCF-7-conditioned medium shows gelatinolytic activity (Fig. 2A, upper panel, lane 3) coincident with the MMP-9 induced in the HT-1080 medium after PMA treatment (Fig. 2A, upper panel, lanes 1–2). Although the MMP-9 detected is the 92-kDa latent enzyme form (25), it is activated by the denaturation-renaturation process in the gel, allowing analysis of its activity (20). To confirm the identity of this gelatinolytic activity as MMP-9, Western blot analysis was performed with a polyclonal anti-MMP-9 Ab. A specific band was detected in MCF-7-conditioned medium that coincides with the mol wt found in PMA-induced HT-1080 cell-conditioned medium and absent in the medium from untreated HT-1080 cells (Fig. 2A, lower panel).

View larger version (26K): [in this window] [in a new window]   Figure 2. MCF-7 cell surface-associated proteolytic activity identified as MMP-9. A, Conditioned medium from HT-1080 cells, untreated or treated with PMA (lanes 1 and 2, respectively), and that from MCF-7 cells (lane 3) were analyzed by gelatin zymography (upper panel) and by Western blot using an anti-MMP-9 polyclonal Ab (lower panel). B, MCF-7 cells (5 x 104) seeded in 96-well microtiter plates were washed three times and serum starved for 14 h. Fluorogenic peptide was then added, and fluorescence was measured at the time indicated. Values are the mean ± SD of two independent experiments performed in triplicate. C, Cells were preincubated alone (), with aprotinin (10 µg/ml; ), or with BB94 (10 µM; •) for 30 min before substrate addition. The fluorescence observed for cells in the absence of proteinase inhibitors was considered 100% for each time point indicated. The mean ± SD of triplicate wells are represented. The asterisks indicate a significant difference (P < 0.05) between untreated and inhibitor-treated cells for each time point (by Mann-Whitney test). D, MCF-7 cells were gently detached and incubated with the Abs indicated followed by an FITC-labeled antimouse IgG Ab, and the fluorescence intensity shift was analyzed by FACS. E, cDNA from HT-1080 treated with PMA (lane 1) and MCF-7 treated with 1 ng/ml IGF-I (lane 3) were employed in PCR amplification for 32 cycles with MMP-9- and glyceraldehyde-3-phosphate dehydrogenase-specific oligonucleotides. As a control, cDNA was replaced by H2O (lane 2).

To further identify this cell-bound MMP activity as that of MMP-9, FACS analysis was performed using MMP-9-specific mAb and MMP-2 as a negative control. Incubation of MCF-7 with the anti-MMP-9 Ab promoted a cell-associated shift in fluorescence intensity compared with that observed using anti-MMP-2 or the second Ab alone (Fig. 2D). Finally, using MMP-9-specific primers, we detected messenger RNA encoding MMP-9 in MCF-7 cells (Fig. 2E, lane 3), with PMA-treated HT-1080 cells as a control (Fig. 2E, lane 1). We conclude that MMP-9 is expressed by MCF-7 cells, is located on the cell surface, and is found in the conditioned medium, and that its activity is at least in part responsible for MCF-7 cell-associated protease activity.

IGF-I triggers changes in MMP-9 activity in MCF-7 cells
MMP-9 is present in MCF-7 cell-conditioned medium, and its activity is associated with the cell surface in the absence of IGF-I treatment. As MCF-7 cells migrate through vitronectin only in the presence of IGF-I, we analyzed the possible effect of IGF-I treatment on MMP-9 activity, measuring both cell surface-associated proteolytic activity and MMP-9 in the conditioned medium. A dose-dependent decrease (r² = 0.71; P < 0.05) in cell surface-associated MMP-9 activity was observed when starved cells were treated with IGF-I for 18 h (Fig. 3A), whereas a 2.5-fold increase in the latent soluble form of MMP-9 was detected in the conditioned medium (Fig. 3B). This treatment does not activate the latent MMP-9 form, as we observed no lower mol wt bands corresponding to activated forms (29). MMP-9 enhancement in the conditioned medium is vitronectin independent, as also found for the MMP-9 membrane-associated activity (data not shown). The MMP-9 in the conditioned medium appears to be independent of MCF-7 migration, as this zymogen form is not activated by IGF-I treatment.

View larger version (30K): [in this window] [in a new window]   Figure 3. IGF-I-triggered changes in MMP-9 levels. A, The indicated amount of IGF-I was added in serum-free medium to starved MCF-7 cells and incubated overnight. The surface proteolytic activity of untreated cells, measured as the increase in fluorescence, was assigned a relative value of 100%. The mean ± SD are shown for two independent experiments performed in triplicate. B, MCF-7 cells were assayed in migration experiments with the amount of IGF-I indicated, and the conditioned medium from the upper part of Transwell chamber was analyzed by gelatin zymography. HT-1080-conditioned medium from PMA-treated or untreated cells was used as a control. The intensity of the gelatinolytic band was obtained after scanning densitometry, assigning a relative value of 100% to the band from the conditioned medium of untreated MCF-7 cells.

View larger version (14K): [in this window] [in a new window]   Figure 4. Time course of proteolytic activity in MCF-7 cells. Serum-starved MCF-7 cells were incubated for the time indicated in the absence or presence of IGF-I (1 ng/ml), then washed, and proteolytic activity measured as described in Materials and Methods. The mean fluorescence increment ± SD of triplicate wells are represented. The asterisks indicate a significant difference (P < 0.05) between untreated and IGF-I-treated cells (by Mann-Whitney test).

IGF-I treatment increases both subunits of the _vß₅ integrin

View larger version (42K): [in this window] [in a new window]   Figure 5. Effect of IGF-I treatment on vß5 levels. A, Subconfluent MCF-7 monolayers were exposed to IGF-I at the indicated concentration for 18 h. Cell lysates (4 µg total protein/sample) were electrophoresed in SDS-PAGE, transferred to nitrocellulose filters, incubated with polyclonal anti-v or anti-ß5, or with anti-ß1 mAb. After incubation with the appropriate secondary Ab, the ECL detection system was employed. B, Subconfluent cultures of MCF-7, incubated with IGF-I (1 ng/ml) for 18 h, were surface labeled with biotin, and cell lysates were immunoprecipitated with mAb anti-v (lane 1), irrelevant IgG (lane 2), and anti-vß5 (lane 3). The immunoprecipitates were resolved under reducing conditions on SDS-PAGE and transferred, and filters were incubated with streptavidin-peroxidase. Arrows indicate the integrin subunit position in the gel.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Most MMPs described to date are soluble enzymes secreted as proenzymes, processed to generate their active forms (31), but cell surface-associated MMP activity has recently been described (32, 33). Membrane-type MMP have been identified on fibroblast cells of human carcinomas (28, 33), and in addition, the soluble MMP gelatinase A exhibits cell surface association in invasive cells (16). We have described the presence of MMP-9 in the conditioned medium of MCF-7 cells, as analyzed by Western blot and gelatin zymography. We have also detected MMP-9 on the MCF-7 cell surface; using an MMP-specific substrate, we observed activity in intact MCF-7 cells, and as expected for an MMP, this activity was inhibited by BB-94, but not by aprotinin. Furthermore, an anti-MMP-9 mAb specifically stains MCF-7 cells. These results led us to conclude that MMP-9 is expressed by MCF-7 cells and is located on the cell surface as well as in the conditioned medium. Although both MMP-2 and MMP-9 gelatinases have been found on the cell surface (16, 32, 34, 35, 36), this is, to our knowledge, the first description of MMP-9 activity on the cell membrane. At least two events must take place in or near the cell surface to allow the appearance of MMP-9 activity, the binding of the newly synthesized proform, followed by its activation. High affinity binding of pro-MMP-9 to the collagen type IV 2(IV) chain has been described in MCF10A cells (34). This binding does not, however, result in zymogen activation, which, in turn, has been associated with components of the urokinase-plasmin system (35, 36). In our experimental system, we found the inactive proform in the conditioned medium and detectable MMP-9 activity only at the cell membrane level. Our data, therefore, support the idea that although inactive MMP-9 binds to ECM components, an activation step is required at the cell membrane. The mechanism of this activation in MCF-7 requires further study, although the fact that aprotinin does not block MCF-7 invasion suggests that the plasmin system is not involved.

Our data further support the hypothesis of protease focalization on the cell membrane as a possible mechanism to increase proteolytic activity, producing more efficient invasion, as previously suggested (26, 30, 37). Long term IGF-I treatment of MCF-7 cells produces a dose-dependent decrease in MMP-9 activity on the cell surface and an increment in MMP-9 in the conditioned medium. When cycloheximide was included in the invasion assay, the results were unchanged; de novo protein synthesis is therefore not required for MCF-7 cell migration in response to IGF-I. From these observations, we hypothesize that IGF-I promotes a redistribution of an MMP-9 pool between the cell surface and the medium. Only the MMP-9 proform has been detected in the MCF-7-conditioned medium; therefore, MMP-9 in the MCF-7-conditioned medium does not appear to correlate with the invasive phenotype. A cell surface-associated protease activity would thus seem to be more relevant than a soluble form in the enhancement of a migration response. The maximal migration rate was observed with 1 ng/ml IGF-I, a dose leading to a significant increase in MCF-7 cell surface-associated MMP-9 activity. The decrease detected after 18-h incubation could be explained by the autoproteolytic activity of the protease, leading to its inactivation. In the absence of exogenous IGF-I, the increase in proteolytic activity is gradual, probably due to the autocrine IGF-I produced (38), and the concomitant inactivation must be delayed.

Cell movement can be observed as a continuous interplay between adhesion and de-adhesion events (39, 40). Adhesion receptors such as integrins are needed to promote cellular invasion (41). Indeed, our results indicate that IGF-I-triggered MCF-7 chemotaxis requires cell adhesion to a specific ECM substrate through integrins, as blocking of _vß₅ integrin binding with an antagonist Ab results in the inhibition of the IGF-I-induced invasive response on vitronectin. These data are similar to those reported previously (3). Although cell attachment to the proper ECM substrate is required for migration, it is not sufficient in itself, as MCF-7 does not invade through vitronectin in the absence of this chemoattractant. It could thus be hypothesized that some cooperation should exist between integrins and IGF-I, either by modification of the integrin levels or by affecting the integrin-mediated signaling pathways.

IGF-I treatment of MCF-7 cells at the dose that induces maximum invasion does not promote any change in the _vß₅ integrin levels. These data concur with results reported previously for several cell lines in which cell migration is regulated by mechanisms independent of changes in the integrin levels (4, 42, 43). On the other hand, IGF-I at high doses produces an increase in _vß₅ integrin levels that parallels with decreased migration. An excess of cell adhesion to the underlying ECM could therefore be negative for cell migration, as described for CHO cells, in which integrin accumulation in the cell membrane diminishes cell migration speed (40).

Adhesion to the ECM and proteolysis of ECM components are necessary for tumor invasion (16, 17, 44). A functional link between adhesion and proteolysis has been shown in melanoma cells, in which MMP-2 is induced and cellular invasion increased after vitronectin ligation to the integrin _vß₃ (45). In our system, adhesion and proteolysis are effected by the integrin _vß₅ and MMP-9, respectively. Precise regulation of these two components must take place to achieve the invasive phenotype. MMP-9 must be localized and active on the cell surface, where membrane association may affect its activity, as has also been suggested for MMP-2 (45). The levels of integrin and its activated state in response to IGF-I treatment must modify cell spreading, adhesion, and detachment, which could, in turn, affect MMP activity, leading to an invasive phenotype.

In conclusion, in addition to the role of integrins in cell migration, we highlight the importance of cell surface-anchored MMP-9 activity in mediating IGF-I chemoattractant activity. This finding emphasizes cell surface proteases as suitable targets in the prevention of cell dissemination, particularly in the case of tumor metastasis.

	Acknowledgments

 
We thank Dr. F. Colotta for providing BB-94; Dr. G. del Real, F. Roncal, R. Fernández, and M. Obrero for synthesis of oligonucleotides for PCR; Dr. J. P. Albar for the anti-v Ab; I. López-Vidriera for help with flow cytometry; Dr. F. Ortego for help with statistical analyses; and C. Mark for editorial assistance.

	Footnotes

 
¹ This work was supported in part by the European Community Human Capital and Mobility Program (N CHRX-CT94–0556). The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council and Pharmacia & Upjohn, Inc.

Received May 26, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kohn EC, Liotta LA 1995 Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res 55:1856–1862[Abstract/Free Full Text]
2. Jones JI, Prevette T, Gockerman A, Clemmons DR 1996 Ligand occupancy of the _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]
3. Doerr ME, Jones JI 1996 The roles of integrins and extracellular matrix proteins in the insulin-like growth factor I-stimulated chemotaxis of human breast cancer cells. J Biol Chem 271:2443–2447[Abstract/Free Full Text]
4. Brooks PC, Klemke RL, Schön S, Lewis JM, Schwartz MA, Cheresh DA 1997 Insulin-like growth factor receptor cooperates with integrin _vß₅ to promote tumor cell dissemination in vivo. J Clin Invest 99:1390–1398[Medline]
5. McGuire WLJ, Jackson JG, Figueroa JA, Shimasaki S, Powell DR, Yee D 1992 Regulation of insulin-like growth factor-binding protein (IGFBP) expression by breast cancer cells: use of IGFBP-1 as an inhibitor of insulin-like growth factor action. J Natl Cancer Inst 84:1336–1341[Abstract/Free Full Text]
6. Matrisian LM 1992 The matrix-degrading metalloproteinases. BioEssays 14:455–463[CrossRef][Medline]
7. Birkedal-Hansen H 1995 Proteolytic remodeling of extracelular matrix. Curr Opin Cell Biol 7:728–735[CrossRef][Medline]
8. Thrailkill KM, Quarles LD, Nagase H, Suzuki K, Serra DM, Fowlkes JL 1995 Characterization of insulin-like growth factor binding protein 5 degrading proteases produced throughout serine osteoblast differentiation. Endocrinology 136:3527–3533[Abstract]
9. Fowlkes JL, Enghild JJ, Suzuki K, Nagase H 1994 Matrix metalloproteases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures. J Biol Chem 269:25742–25746[Abstract/Free Full Text]
10. Mañes S, Mira E, Barbacid MM, Ciprés A, Fernández-Resa P, Buesa JM, Mérida I, Aracil M, Márquez G, Martínez-A C 1997 Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3. J Biol Chem 272:25706–25712[Abstract/Free Full Text]
11. Himelstein BP, Canete-Soler R, Bernhard EJ, Dilks DW, Muschel RJ 1994–95 Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion Metastasis 14:246–258
12. Bernhard EJ, Gruber SB, Muschel RJ 1994 Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc Natl Acad Sci USA 91:4293–4297[Abstract/Free Full Text]
13. Zetter BR 1993 Adhesion molecules in tumor metastasis. Semin Cancer Biol 4:219–229[Medline]
14. Varner JA, Cheresh DA 1996 Integrins and cancer. Curr Opin Cell Biol 8:724–730[CrossRef][Medline]
15. Marshall JF, Hart IR 1996 The role of _v-integrins in tumour progression and metastasis. Semin Cancer Biol 7:129–138[CrossRef][Medline]
16. Brooks PC, Strömblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA 1996 Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin _vß₃. Cell 85:683–693[CrossRef][Medline]
17. Riikonen T, Westermarck J, Koivisto L, Broberg A, Kähäri V-M, Heino J 1995 Integrin ₂ß₁ is a positive regulator of collagenase (MMP-1) and collagen 1 (I) gene expression. J Biol Chem 270:13548–13552[Abstract/Free Full Text]
18. Davies B, Brown PD, East N, Crimmin MJ, Balkwill FR 1993 A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts. Cancer Res 53:2087–2091[Abstract/Free Full Text]
19. Mañes S, Kremer L, Vangbo B, López A, Gómez-Mouton C, Peiró E, Albar JP, Mendel-Hartvig IB, Llopis R, Martínez-A C 1997 Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies. J Endocrinol 154:293–302[Abstract/Free Full Text]
20. Quesada AR, Barbacid MM, Mira E, Fernández-Resa P, Márquez G, Aracil M 1997 Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases. Clin Exp Metastasis 15:26–32[CrossRef][Medline]
21. Osada H, Kono T, Miwa K, Yamada C 1996 Phorbol-ester-stimulated human lymphoid cell lines produce a plasminogen activator modulator inducing cell-bound urokinase-type plasminogen activator in malignant tumor cell lines. Int J Cancer 65:178–185[CrossRef][Medline]
22. Leppert D, Waubant E, Galardy R, Bunnett NW, Hauser SL 1995 T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol 154:4379–4389[Abstract]
23. Xue W, Mizukami I, Todd III RF, Petty HR 1997 Urokinase-type plasminogen activator receptors associate with ß₁ and ß₃ integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res 57:1682–1689[Abstract/Free Full Text]
24. Mosmann T 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63[CrossRef][Medline]
25. Zucker S, Mancuso P, DiMassimo B, Lysik RM, Conner C, Wu C-L 1994 Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays. Clin Exp Metastasis 12:13–23[CrossRef][Medline]
26. Chen W-T 1992 Membrane proteases: roles in tissue remodeling and tumour invasion. Curr Opin Cell Biol 4:802–809[CrossRef][Medline]
27. Bickett DM, Green MD, Berman J, Dezube M, Howe AS, Brown PJ, Roth JT, McGeehan GM 1993 A high troughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Anal Biochem 212:58–64[CrossRef][Medline]
28. Okada A, Bellocq J-P, Rouyer N, Chenard M-P, Rio M-C, Chambon P, Basset P 1995 Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl Acad Sci USA 92:2730–2734[Abstract/Free Full Text]
29. Shapiro SD, Fliszar CJ, Broekelmann TJ, Mecham RP, Senior RM, Welgus HG 1995 Activation of the 92-kDa gelatinase by stromelysin and 4-aminophenylmercuric acetate. J Biol Chem 270:6351–6356[Abstract/Free Full Text]
30. Basbaum CB, Werb Z 1996 Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol 8:731–738[CrossRef][Medline]
31. Nagase H 1997 Activation mechanisms of matrix metalloproteinases. J Biol Chem 378:151–160
32. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI 1995 Mechanism of cell surface activation of 72 kDa type IV collagenase. J Biol Chem 270:5331–5338[Abstract/Free Full Text]
33. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M 1994 A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370:61–65[CrossRef][Medline]
34. Olson M, Toth M, Gervasi D, Sado Y, Ninomiya Y, Fridman R 1998 High affinity binding of latent matrix metalloproteinase-9 to the a2(IV) chain of collagen IV. J Biol Chem 273:10672–10681[Abstract/Free Full Text]
35. Ginestra A, Monea S, Seghezzi G, Dolo V, Nagase H, Mignatti P, Vittorelli M 1997 Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells. J Biol Chem 272:17216–17222[Abstract/Free Full Text]
36. Mazzieri R, Masiero L, Zanetta L, Monea S, Onisto M, Garbisa S, Mignatti P 1997 Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J 16:2319–2332[CrossRef][Medline]
37. Monsky WL, Chen W-T 1993 Proteases of cell adhesion proteins in cancer. Semin Cancer Biol 4:251–258[Medline]
38. Rohlik QT, Adams D, Kull FCJ, Jacobs S 1987 An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture. Biochem Biophys Res Commun 149:276–281[CrossRef][Medline]
39. Condic ML, Letourneau PC 1997 Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth. Nature 389:852–856[CrossRef][Medline]
40. Palecek S, Huttenlocher A, Horwitz A, Lauffenburger D 1998 Physical and biochemical regulation of integrin release during rear detachment of migrating cells. J Cell Sci 111:929–940[Abstract]
41. Huttenlocher A, Sandborg RR, Horwitz AF 1995 Adhesion in cell migration. Curr Opin Cell Biol 7:697–706[CrossRef][Medline]
42. Kuijpers T, Mul E, Blom M, Kovach N, Gaeta F, Tollefson V, Elices M, Harlan J 1993 Freezing adhesion molecules in a state of high avidity binding blocks eosinophil migration. J Exp Med 178:279–284[Abstract/Free Full Text]
43. Yu D-H, Qu C-K, Henegariu O, Lu X, Feng G-S 1998 Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. J Biol Chem 273:21125–21131[Abstract/Free Full Text]
44. Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z, Damsky CH 1995 Cooperative signalling by ₅ß₁ and ₄ß₁ integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol 129:867–879[Abstract/Free Full Text]
45. Bafetti LM, Young TN, Itoh Y, Stack MS 1998 Intact vitronectin induces matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2 expression and enhanced cellular invasion by melanoma cells. J Biol Chem 273:143–149[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mira, E. Articles by Martínez-A, C. Search for Related Content PubMed PubMed Citation Articles by Mira, E. Articles by Martínez-A, C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB (L)-PHENYLALANINE THIOPHENE Medline Plus Health Information Breast Cancer