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(The FASEB Journal. 2000;14:1629-1640.)
© 2000 FASEB

HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity

LIVIO TRUSOLINO, SILVIA CAVASSA, PAOLA ANGELINI, MARGHERITA ANDÒ, ANDREA BERTOTTI, PAOLO M. COMOGLIO and CARLA BOCCACCIO1

Institute for Cancer Research and Treatment, University of Torino Medical School, 10060 Candiolo-Torino, Italy

1Correspondence: Division of Molecular Oncology, Institute for Cancer Research, Str. Provinciale 142, 10060 Candiolo (Torino), Italy. E-mail: cboccaccio{at}ircc.unito.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte growth factor/scatter factor (HGF/SF) controls a genetic program known as ‘invasive growth’, which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth factor (EGF). Neither factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including ß1, ß3, ß4, and ß5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These data suggest that HGF/SF promotes cell adhesion and invasiveness by increasing the avidity of integrins for their specific ligands.—Trusolino, L., Cavassa, S., Angelini, P., Andò, M., Bertotti, A., Comoglio, P. M., Boccaccio, C. HGF/scatter factor selectively promotes cell invasion by increasing integrin avidity.


Key Words: growth factors • tyrosine kinase receptors • MET • adhesion • integrin activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR (HGF/SF) is the prototype of the plasminogen-related growth factors (PRGFs), a family of cytokines including macrophage-stimulating protein (MSP). PRGFs are endowed with the distinctive property of inducing epithelial and other cell types to execute a genetic program defined as ‘invasive growth’ (1) . This includes proliferation, disruption of intercellular junctions and migration (‘scatter’) through the extracellular matrix (ECM), protection from apoptosis, and eventually leads to expression of differentiated phenotypes. In epithelia, dissociation of compact cell sheets induced by HGF/SF is transient and followed by reorganization in tubules (branching morphogenesis) that compose the parenchymal architecture of several organs (2) . The invasive growth signal is specifically transduced by the tyrosine kinase receptors encoded by the oncogenes MET (HGF/SF receptor), RON (MSP receptor), and sea (3 4 5) .

Induction of physiological invasive growth by PRGFs takes place during embryonic as well as postnatal development and regeneration of many epithelial and nonepithelial tissues (6) . Deregulation of the HGF/SF intracellular signals after MET oncogenic activation leads to the malignant counterpart of the invasive growth program. This enables cancer cells to invade the surrounding matrix and survive through foreign environments to form metastases in distant organs. Point mutations in the kinase domain of MET have been found in hereditary and sporadic cases of papillary renal carcinoma and in sporadic hepatocellular carcinoma (7 , 8) . In vitro, these mutations cause constitutive activation of a transforming and invasive signal (9 , 10) . MET overexpression in the absence of mutations is found in aggressive carcinomas. In colorectal carcinoma, MET gene amplification confers a selective advantage that supports the ability to metastasize to the liver (11 , 12) . In osteo- and rabdomyosarcomas derived from mesenchymal cells that physiologically express HGF/SF, MET ectopic (over)expression causes autocrine and paracrine loops to form, which foster cell invasiveness and tumor malignancy (13 , 14) . Correlation between MET activation and increased metastatic potential observed in human tumors is confirmed by in vitro and in vivo experiments. These show that constitutive activation of the tyrosine-kinase encoded by the MET oncogene, induced by MET mutations and/or by coexpression of HGF/SF and MET, confers invasive properties to cells in vitro and metastatic ability after implant in mice (15 16 17) .

The HGF/SF signaling pathways responsible for invasive growth have been mostly elucidated. A conserved sequence encompassing two tyrosines (Y1349VHVXXXY1356VNV in Met) in the carboxyl-terminal intracellular tail is a distinctive feature of PRGF receptors. On phosphorylation, this sequence provides a multifunctional docking site indispensable and sufficient to recruit and activate several signal transducers. These include the adaptor protein Grb-2 that activates Ras, the p85 docking subunit for phosphatidylinositol 3-kinase (PI3-K), the signal transducer and activator of transcription (Stat) -3, and the multifunctional adaptor Gab-1 (18 19 20) .

Concomitant activation of multiple pathways accounts for the complexity of the resulting biological response to PRGFs. However, the many aspects of invasive growth can be separated from each other and ascribed at least in part to specific signaling effectors (21 , 22) . The Ras pathway is required to elicit either proliferation and transformation or cell scatter (21 , 23 , 24) . PI3-K is necessary and sufficient to trigger cell motility (25 26 27) . Tubule induction by HGF/SF is a multistep phenomenon that requires, besides Ras and PI3-K activation, the presence of the specific HGF/SF receptor substrate Gab1 (20) and the integrity of the Stat pathway (19) .

Although respective functions of signal transducers have been extensively analyzed, there is still much to learn about the effectors that mediate the invasive growth response to HGF/SF. Three essential events make the cells’ journey across the matrix possible. The first is disruption of intercellular junctions, which allows cell dissociation and reshaping into a motile phenotype. The second is ECM digestion, which facilitates cell movement and stromal infiltration. Finally, invading cells must perform constant and dynamic remodeling of adhesive contacts with ECM, which, by engaging surface integrin receptors, provides a support for cell migration and a consensus that protects from apoptosis. The ability of HGF/SF to deconstruct cell–cell contacts, as well as HGF/SF-dependent induction and/or activation of ECM proteases, has been documented (26 , 28 29 30) . In contrast, little is known about the effect of HGF/SF on cell–matrix interactions during the invasive process.

Accordingly, the aim of this study was to investigate whether HGF/SF can selectively affect integrin expression, function, and topographical distribution when promoting cell invasiveness. Within this context, a critical issue is to use a cell model in which cell motility and invasion are elicited uniquely because of the stimulation of integrin activity.

To this end, we chose MDA-MB-231, a cell line that responds to HGF/SF with a markedly increased invasiveness of basement membranes and stromal matrices. These cells express barely detectable amounts of E-cadherin; therefore, they do not form adherens junctions and display a constitutive fibroblastoid phenotype. Here we show that MDA-MB-231 cells synthesize high basal levels of matrix metalloprotease-9 (MMP-9) and urokinase-type plasminogen activator (uPA), which are not further increased by HGF/SF. Thus, in this cell model, HGF/SF stimulation does not affect either cell–cell junction architecture or matrix digestion. In contrast, HGF/SF, unlike conventional growth factors such as epidermal growth factor (EGF) and insulin, can selectively increase the adhesive properties of a multiple repertoire of integrins to their specific ligands. This distinctive feature is necessary and sufficient to induce efficient invasion of basement membranes and ECM components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, reagents, and antibodies
MDA-MB-231 (ATCC, Rockville, Md.) is a human mammary adenocarcinoma cell line. Cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gibco BRL, Grand Island, N.Y.) and starved in DMEM containing 0.1% bovine serum albumin (BSA) for 24 h. Cell culture and stimulation were performed at 37°C in a 5% CO2 water-saturated atmosphere.

HGF/SF was a generous gift from Genentech (San Francisco, Calif.); transforming growth factor ß1 (TGF-ß1), EGF, and insulin were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Matrigel (Collaborative Research, Bedford, Mass.) is a solubilized basement membrane preparation, extracted from mouse sarcoma, containing laminin, collagen type IV, proteoglycans, and growth factors. Laminin-1, laminin-5, fibronectin, vitronectin, LY294002, and wortmannin were from Sigma (St. Louis, Mo.).

Anti-Met monoclonal antibody (mAb) DO24 was obtained as described (31) . Anti-phosphotyrosine polyclonal antibody was purchased from UBI (Lake Placid, N.Y.; anti-E-cadherin mAb was from Transduction Laboratories (Lexington, Ky.). mAbs against integrins were as follows: MAR4, against ß1, was a gift from Sylvie Ménard (Istituto Nazionale Tumori, Milano, Italy) and Pier Carlo Marchisio (DIBIT-San Raffaele Scientific Institute, Milano, Italy). 3E1 mAb (against ß4) and function-blocking mAbs 6S6 (against ß1), ASC-3 (against ß4), LM609 (against the {alpha}Vß3 complex), and P1F6 (against the {alpha}Vß5 complex) were all from Chemicon International, Inc. (El Segundo, Calif.). A rabbit polyclonal antiserum against ß1 integrins was kindly supplied by Ivan de Curtis (DIBIT-San Raffaele Scientific Institute, Milano, Italy).

Surface biotinylation, detergent extractions, immunoprecipitation, and Western blotting
For surface biotinylation, confluent monolayers were washed three times at 4°C with Hank’s balanced salt biotinylation buffer, pH 7.4 (HBB, containing 1.3 mM CaCl2, 0.4 mM MgSO4, 5 mM KCl, 138 mM NaCl, 5.6 mM D-glucose, and 25 mM HEPES, pH 7.4). Sulfosuccinimido biotin (0.5 mg/ml in HBB, Pierce, Rockford, Ill.) was applied twice to the cells for 20 min at 4°C. The reaction was stopped by incubating four times at 4°C with minimal essential medium containing Hank’s balanced salts, 0.6% BSA, 20 mM HEPES, pH 7.4.

For immunoprecipitation, cells were lysed with ice-cold RIPA buffer [50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5% deoxycholate, 5 mM EDTA, 1 mM Na3VO4] containing inhibitors of proteases (2 mM PMSF, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 5 µg/ml aprotinin) for 20 min on ice. Extracts were clarified at 12,000 g for 15 min, normalized with the BCA Protein Assay Reagent Kit (Pierce), and incubated with different mAbs for 2 h at 4°C. The immunocomplexes were collected with affinity-purified rabbit anti-mouse immunoglobulin G (Pierce) coupled to protein A-Sepharose and eluted in boiling Laemmli buffer. In some experiments aimed at obtaining an actin-enriched fraction, cells that had been either left untreated or stimulated with HGF/SF were initially extracted with a buffer containing 0.2% Triton X-100, 100 mM KCl, 200 mM sucrose, 10 mM EGTA, 2 mM MgCl2, 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 1 min (membrane-soluble fraction). The cells were rinsed several times before adding a second buffer containing 1% Tween-40, 0.5% deoxycholate, 10 mM NaCl, 2 mM MgCl2, 20 mM Tris-HCl, pH 7.5, for 10 min (actin). Total cellular proteins were extracted by solubilizing the cells in boiling Laemmli buffer. Samples were sonicated in order to decrease viscosity and adjusted to a protein concentration of 100 µg/each.

Solubilized proteins were electrophoresed on 8% SDS-polyacrylamide slab gels under reducing conditions and transferred to nylon membranes (Hybond, Amersham, Amersham, U.K.). For detection of biotinylated proteins, filters were probed with peroxidase-conjugated streptavidin. Nonbiotinylated proteins were detected by incubation of filters with specific antibodies, followed by peroxidase-conjugated secondary antibodies. Peroxidase reaction was detected by the enhanced chemiluminescence system (Amersham) and visualized on Kodak X-OMAT AR films.

Zymography
MDA-MB-231 cells were plated at 60–80% confluence in DMEM supplemented with 10% FCS. After 12 h, cells were washed twice with phosphate-buffered saline (PBS) and starved with serum-free DMEM for 24 h. Medium was replaced with fresh serum-free DMEM supplemented with HGF/SF or TGF-ß. Culture supernatants were harvested at different times and the cellular debris was removed by centrifugation at 1000 g. Cells were washed twice with PBS and lysed with 0.5% Triton X-100 in 0.1 M Tris-HCl, pH 8.1, for 10 min under constant shaking. Cell lysates were centrifuged at 10,000 g for 20 min at 4°C. Culture supernatants and cell extracts were immediately processed as follows. For MMP-9 and MMP-2 detection, cell extracts or supernatants were incubated with gelatin-Sepharose (Sigma) and equilibrated with 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM CaCl2, 0.02% Tween-20, 10 mM EDTA in an end-over-end mixer for 3 h at 4°C. Beads were eluted with Laemmli buffer and loaded on SDS 8% polyacrylamide gels containing 1 mg/ml of gelatin A (from porcine skin, Sigma). After electrophoresis, gels were washed with 2.5% Triton X-100 and with H2O at room temperature, then incubated in a buffer containing 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 20 mM CaCl2 at 37°C for 24–48 h, as described (32) . For uPA detection, cell extracts or supernatants were solubilized in Laemmli buffer and loaded on SDS 8% polyacrylamide gels containing 0.1% casein (Sigma) and 5 µg/ml plasminogen (Sigma). After electrophoresis, gels were washed with 2.5% Triton X-100 and with H2O at room temperature, then incubated in a buffer containing 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 15 mM EDTA, at 37°C for 24–48 h. Zymograms were stained with 0.5% Coomassie brilliant blue R-250 in 50% methanol, 10% acetic acid.

Cell migration and invasion assays
Assays were performed in Transwell chambers (6.5 mm, Costar, Cambridge, Mass.). To evaluate cell invasion, the upper sides of the porous polycarbonate filters (8.0 µm pore size) were coated with a concentration range (15–150 µg/cm2) of reconstituted Matrigel basement membrane. Matrigel solution (50 µl) was added to each filter and dried under a hood according to the manufacturer’s instructions and previously described methods (33) . The lower sides of the filters were coated with a fixed Matrigel concentration (5 µg/cm2). Invasion through isolated ECM components was performed with filters coated on the lower side with laminin-1, laminin-5, fibronectin, or vitronectin (10 µg/ml). 5 x 104 serum-starved MDA-MB-231 cells were seeded on the upper side of the filters and incubated in DMEM, 0.1% BSA with 0–100 ng/ml HGF/SF. After 6–24 h, cells on the upper side of the filters were mechanically removed. Cells that migrated onto the lower side of the filters were fixed with 11% glutaraldehyde in PBS and stained with 0.1% crystal violet in 20% methanol. Cells that migrated onto the lower side of Transwell filters were counted in four grids using a phase-contrast light microscope fitted with a 32 grid eyepiece at a total magnification of 100x. Data presented are the means ± SD of triplicate wells from two experiments.

Cell proliferation assay
24-well plates (Costar) were coated with 10 µg/ml fibronectin or polylysine in PBS, pH 7.4. Proteins were allowed to bind overnight at 4°C before the wells were rinsed and blocked for 2 h at 37°C with 2% heat-denatured BSA (RIA grade; Sigma) in PBS, pH 7.4. 2 x 104 cells/well were plated in DMEM 10% FCS, grown for 24 h, then starved for 24 h in serum-free DMEM, 1% BSA. HGF/SF (0–200 ng/ml), EGF (0–200 ng/ml), or FCS (2.5–15%) together with 3H-thymidine (Amersham) to a final concentration of 5 µCi/ml were added to the culture medium. After incubation for 20 h, monolayers were fixed with 10% TCA and solubilized with 10% SDS. The incorporated radioactivity was quantitated in a liquid scintillation spectrometer (1600 TR Liquid Scintillation Analyzer, Packard Instruments, Downers Grove, Ill.). The data presented are the means ± SD of quadruplicate wells from two experiments.

Cell adhesion assay
Assays were performed according to ref 34 with minor modifications. In brief, 96-well microtiter plates (Nunc, Naperville, Ill.) were coated with a concentration range (2.5–20 µg/ml) of laminin-1, laminin-5, fibronectin, or vitronectin in PBS, pH 7.4. Proteins were allowed to bind overnight at 4°C before the wells were rinsed and blocked for 2 h at 37°C with 2% heat-denatured BSA in PBS, pH 7.4. Starved cells were harvested, washed twice with serum-free medium and added to the wells at a concentration of 104 cells/0.1 ml of the same medium. After incubation for 45 min at 37°C in the presence or absence of HGF/SF, EGF, or insulin, wells were gently washed in PBS. In some assays, cell plating was performed in the presence of 25 µM LY294002 or 100 nM wortmannin. In experiments to evaluate inhibition of adhesion, cells were incubated before plating in the presence of appropriate dilutions of the function-blocking mAbs for 30 min at 4°C. Adherent cells were fixed in 11% glutaraldehyde in PBS and stained with 0.1% crystal violet in 20% methanol. Cell numbers were obtained by counting all cells in the well using a phase-contrast light microscope at a total magnification of 25x. All data presented are the means ± SD of quadruplicate wells from two experiments. Nonspecific cell adhesion as measured on BSA-coated wells has been subtracted.

Indirect immunofluorescence microscopy
Serum-starved cells were plated onto 24-well plates (Costar) containing 1.4 cm2 glass coverslips, previously coated with matrix substrates as described for adhesion assays. After incubation for 45 min at 37°C in the presence or absence of HGF/SF (100 ng/ml), cells were fixed in a freshly prepared solution containing 3% formaldehyde (from paraformaldehyde) and 2% sucrose in PBS, pH 7.6, for 5 min at room temperature. Cells were permeabilized by soaking coverslips in HEPES-Triton X-100 buffer (20 mM HEPES, pH 7.4, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, and 0.5% Triton X-100) for 3 min at room temperature. Indirect immunofluorescence was performed as previously reported (35) . In brief, after saturation with PBS-2% BSA for 15 min at 37°C, the primary antibodies were layered onto cells and incubated in a moist chamber for 30 min. After rinsing in PBS-0.2% BSA, coverslips were incubated with the appropriate rhodamine-tagged secondary antibody (Dakopatts, Glostrup, Denmark) for 30 min at 37°C in the presence of 2 µg/ml of fluorescein-labeled phalloidin (Sigma). Coverslips were mounted in Mowiol 4–88 (Hoechst AG) and observed in a photomicroscope (Axiophot, Zeiss) equipped with epifluorescence lamp and planapochromatic oil immersion lenses. Fluorescence images were recorded on Kodak T-Max 400 photographic films exposed at 1000 ISO and developed in T-Max Developer for 10 min at 20°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MDA-MB-231 cells are endowed with an invasive phenotype
Human breast carcinoma MDA-MB-231 is a cell line that displays a spindle-shaped ‘scattered’ phenotype and grows in culture without forming packed islands typical of epithelia (Fig. 1A ). It is likely that MDA-MB-231 cells cannot form adherens junctions, as they express very low levels of E-cadherin compared to epithelioid cell lines such as GTL-16 and A549. This is shown in Western blot of total cell extracts with anti-E-cadherin antibodies (Fig. 1B ). Zymography experiments demonstrate that MDA-MB-231 cells express high basal levels of active extracellular matrix proteases, such as uPA and MMP-9 (Fig. 1C , D , E , F ). uPA was detected in both extracts (Fig. 1C ) and culture supernatants (Fig. 1D ) of unstimulated cells run in casein/plasminogen gels, where it was present as a single digestion band of ~55 kDa. Pro-MMP-9 (which is active toward type IV collagen) was detected in both cell extracts (Fig. 1E ) and culture supernatants (Fig. 1F ) of unstimulated cells run in gelatin gels. Here pro-MMP9 was present as a single band of 92 kDa. In time course experiments (6–48 h), HGF/SF or EGF stimulation did not increase the levels of uPA or pro-MMP-9 (Fig. 1C , D , E , F , and not shown). A weak induction of the activated form of MMP-9 was observed in cell extracts stimulated by HGF/SF as a band of ~85 kDa. This was very slight when compared to the one induced by TGF-ß, which, as expected, significantly enhanced proteolytic activity of uPA from culture supernatants and of pro-MMP9 from both supernatants and cell extracts (Fig. 1C , D , E , F ). In gelatin gels, no MMP-2 (72 kDa) was observed in either condition. The HGF/SF receptor, encoded by the MET oncogene, was present and became phosphorylated after standard HGF/SF treatment, as shown by immunoprecipitation with anti-Met antibodies and Western blot with anti-phosphotyrosine and anti-Met (Fig. 1G, H ). EGF receptor has been previously reported to be expressed but not constitutively activated in MDA-MB-231 cells (36) .



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  		Figure 1. MDA-MB-231 cells are endowed with an invasive phenotype. A) Micrograph (bar: 5 µm) of a fixed and stained MDA-MB-231 culture showing the constitutive spindle-shaped and scattered cell phenotype. B) Western blot of total extracts of MDA-MB-231 and epithelial cell lines GTL16 and A549, probed with antibodies to E-cadherin (arrow). C—F) Zymograms of cell extracts (C, E) and supernatants (D, F) from MDA-MB-231 cells either unstimulated (Unst.) or treated with HGF/SF or TGF-ß for 12 h. uPA is detected in plasminogen-casein gels as a single band of ~55 kDa. Pro-MMP-9 is detected in gelatin gels as a single band of ~92 kDa. MMP-9 is present only in cell extracts as a band of ~85 kDa. G, H) Western blots of MDA-MB-231 extracts immunoprecipitated with anti-Met antibodies after a 10 min stimulation in the absence (Unst.) or presence of HGF/SF (100 ng/ml). Filters were probed with antibodies either to phosphotyrosine (G) or to the Met ß chain (H).

Taken together, these data suggest that MDA-MB-231 cells display a spontaneous motile phenotype due to lack of adherens junctions and to a high basal production of ECM proteases. Both features seem not to be affected by treatment with exogenous growth factors.

HGF/SF promotes MDA-MB-231 cell invasion through reconstituted basement membranes
We thus tested the invasive properties of MDA-MB-231 cells across reconstituted basement membranes. Invasion assays were performed in two-chamber Transwell devices whose filters were coated on both sides with a polymerized layer of Matrigel, which contained laminin, collagen type IV, proteoglycans, and growth factors. On the upper side of the filters, a concentration range (15–150 µg/cm2) of Matrigel was tested; on the lower side, a fixed concentration (5 µg/cm2) was maintained. Serum-starved cells were plated on the upper side of the filters and incubated in the presence of a concentration range of HGF/SF (0–400 ng/ml, added to both Transwell chambers) in time course experiments (2–24 h).

In the absence of HGF/SF, MDA-MB-231 cells spontaneously invaded the basement membrane and stopped on the lower side of the filters. As Matrigel concentration increased, cell invasion was delayed and the total number of migrating cells was dramatically reduced, and virtually abolished at 45 µg/cm2. Under these tightly controlled conditions, HGF/SF induced a dose-dependent stimulation of cell invasion, which became remarkably evident in the presence of 45 µg/cm2 of Matrigel after 6 h of incubation (Fig. 2A, B ). In contrast, stimulation of cells with saturating amounts of insulin or EGF did not result in increased invasiveness (not shown). HGF/SF did not induce proliferation of MDA-MB-231 cells either alone or in the presence of 10% FCS. This was assessed by 3H-thymidine incorporation during HGF/SF stimulation up to 24 h (Fig. 2C ). Coating of culture plates with Matrigel or other extracellular components such as fibronectin did not interfere with the effect of HGF/SF and/or serum on proliferation.



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  		Figure 2. HGF/SF promotes MDA-MB-231 cell invasion through reconstituted basement membranes. A, B) Micrographs (bar: 50 µm) of Transwell filter lower sides fixed with glutaraldehyde and stained with crystal violet. MDA-MB-231 cells were seeded onto the upper sides of Matrigel-coated filters, and incubated in the absence (A) or presence (B) of HGF/SF (100 ng/ml) for 6 h. C) Histogram of 3H-thymidine incorporation (cpm) by MDA-MB-231 cells during a 24 h incubation in the absence (Unst.) or presence of HGF/SF, serum, or both.

In MDA-MB-231 cells, HGF/SF was not able to elicit the completion of the physiological invasive growth program. In fact, it did not induce formation of branching tubules in cells cultured in three-dimensional matrices made of either type I collagen, Matrigel, or a mixture of both (not shown).

HGF/SF stimulates cell adhesion to several matrix ligands
The observation that HGF/SF increases MDA-MB-231 cell invasion through artificially reconstituted matrices prompted us to investigate whether this growth factor can stimulate integrin-mediated cell adhesion. Initial assays were performed to characterize the surface adhesive repertoire of MDA-MB-231 cells. By using a battery of integrin-specific mAbs in immunoprecipitation experiments on membrane biotinylated cell monolayers, we found that MDA-MB-231 cells express the {alpha}3ß1, {alpha}5ß1, {alpha}6ß4, {alpha}vß1, {alpha}vß3, and {alpha}vß5 integrins (not shown).

Based on the above findings, adhesion assays were performed on several ECM ligands before and after exposure to HGF/SF. Subconfluent cultures were detached from culture dishes and plated in serum-free conditions onto a plastic substrate coated with a concentration range (2.5 to 20 µg/ml) of laminin-1, laminin-5, fibronectin, and vitronectin. Cells that had been either left untreated or incubated in the presence of noninhibitory anti-integrin antibodies adhered poorly to all substrates tested (not shown). When the same kind of adhesion assay was performed in the presence of increasing concentrations of HGF/SF, adhesion was enhanced on all substrates in a dose-dependent manner (Fig. 3A , B , C , D , graphs). Nonspecific adhesion processes were not found to be affected by HGF/SF since HGF/SF treatment did not exert any effect on cell adhesion to dishes coated with BSA (not shown). Moreover, the increase of adhesion was not due to a faster kinetics of attachment, as it was maintained even after 3 h of incubation. Finally, adhesion was enhanced by HGF/SF in a selective way. In fact, stimulation of cells with saturating amounts of EGF (Fig. 3A , B , C , D , graphs) or insulin (not shown) did not result in increased adhesion to any of the substrates tested.



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  		Figure 3. HGF/SF, but not EGF, stimulates cell adhesion to and migration through several matrix ligands. Histograms represent MDA-MB-231 cells attached 45 min after cell resuspension and plating onto laminin-1 (A), laminin-5 (B), fibronectin (C), and vitronectin (D) in the presence of increasing concentrations of HGF-SF (closed bars) or EGF (open bars). Basal adhesion in the absence of growth factor stimulation has been normalized to one. Micrographs are low magnification images of the lower sides of Transwell filters fixed with glutaraldehyde and stained with crystal violet. MDA-MB-231 cells were seeded onto filters coated on the lower side with 10 µg/cm2 laminin-1 (A), laminin-5 (B), fibronectin (C), or vitronectin (D) and incubated 6 h in the absence of growth factors (Unst.) or in the presence of 100 ng/ml HGF/SF or 100 ng/ml EGF. Cell numbers ± SD are indicated in brackets.

HGF/SF promotes MDA-MB-231 cell invasiveness through different matrices
Based on the observation that HGF/SF increases the adhesive capability of integrins, we decided to test whether HGF/SF stimulation specifically promotes cell migration toward defined ECM components. As commercial preparations of reconstituted matrices such as Matrigel usually contain serum factors and proteases that do not allow selective evaluation of integrin-dependent cell migration, we used a two-chamber Transwell system using purified samples of isolated matrix ligands.

To this aim, cells were serum-starved, detached, and plated onto Transwell filters coated on the lower side with laminin-1, laminin-5, fibronectin, or vitronectin. Treatment with HGF/SF increased cell migration toward all the substrates with a variable degree of efficiency (Fig. 3A , B , C , D , micrographs). In contrast, MDA-MB-231 migration was not affected by EGF (Fig. 3A , B , C , D , micrographs). These experiments indicate that the selective ability of HGF/SF to modulate integrin adhesive function not only results in increased static adhesion of cells onto 2-dimensional layers, but also induces a migratory phenotype across 3-dimensional matrices.

HGF/SF promotes adhesion via ß1, ß3, ß4, and ß5 integrins
We next investigated whether the ability of HGF/SF to affect cell–matrix interactions was mediated by one or more integrins. Under basal conditions, MDA-MB-231 cell adhesion to both laminin-1 and -5 was cooperatively controlled by ß1 and ß4 integrins. Function-blocking mAbs against either integrin could partially impair adhesion to laminin-1 when added individually and almost totally when added together (Fig. 4A ). As expected, mAbs against ß3 and ß5 heterodimers did not influence cell adhesion to laminins (not shown). In the presence of HGF/SF, cell adhesion was enhanced compared to untreated cells when either ß1 or ß4 integrins where blocked by single-antibody treatment. Stimulation with HGF/SF was almost totally ineffective only when both ß1 and ß4 integrins were blocked by their respective inhibitory mAbs (Fig. 4A ). Superimposable results were obtained when laminin-5 was used as an adhesive substrate (not shown). Taken together, these results demonstrate that HGF/SF enhances adhesion efficiency of MDA-MB-231 cells to laminins through {alpha}3ß1 and {alpha}6ß4 integrins.



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  		Figure 4. HGF/SF activates ß1, ß3, ß4, and ß5 integrins. Histograms represent MDA-MB-231 cells attached onto dishes coated with 10 µg laminin-1 (A) or vitronectin (B) after 45 min in the absence (-) or presence (+) of HGF/SF (100 ng/ml). Before plating, cells were resuspended and incubated in the absence (-) or presence of single or multiple blocking mAbs (Anti-ß) against integrin ß subunits 1 and 4 (which mediate attachment to laminins) or 1, 3 and 5 (which mediate attachment to fibronectin and vitronectin). Basal adhesion levels in the absence of HGF-SF or inhibitory mAbs have been normalized to one.

Data obtained from adhesion assays onto fibronectin and vitronectin indicated multiple cooperativity in the recognition of both matrix ligands between ß1, ß3, and ß5 integrins. Cell adhesion to vitronectin was partially inhibited by treating cells with single mAbs against ß1, ß3, or ß5 integrins (Fig. 4B ). Adhesion was more efficiently blocked, but not totally abolished, by adding a combined mixture of inhibitory mAbs against two integrin subunits (Fig. 4B ). Only when mAbs against ß1, ß3, and ß5 integrins were added collectively was adhesion completely inhibited (Fig. 4B ). Control function-blocking mAbs against the ß4 subunit were not able to impair adhesion (not shown). Similar to what was observed for laminins, treatment with HGF/SF was completely ineffective only in the presence of mAbs against all integrins involved. Even when two integrins of three were blocked, the presence of a residual active heterodimer was able to increase adhesion in response to HGF/SF stimulation (Fig. 4B ). Data obtained with cells plated on fibronectin were almost identical to those described for vitronectin (not shown). Therefore, HGF/SF potentiates cell adhesion to fibronectin and vitronectin by affecting ß1, ß3, and ß5 integrins.

In conclusion, HGF/SF is able to promote adhesion to a vast number of epithelial and stromal matrix ligands by influencing a multiple repertoire of integrins including ß1, ß3, ß4, and ß5 heterodimers.

HGF/SF modifies integrin subcellular distribution without affecting expression
The ability of HGF/SF to enhance integrin-mediated adhesion could be due to either quantitative changes of the integrin surface levels—i.e., accelerated conversion of precursor heterodimers to mature forms and increased membrane delivery—or functional activation of already exposed integrins. To verify both hypotheses, MDA-MB-231 cells were treated with a suboptimal concentration of HGF/SF (100 ng/ml) for various times corresponding to the standard timing of adhesion assays. Cells were then surface-biotinylated and extracted with an SDS-containing lysis buffer that should recover both diffusible and cytoskeleton-associated integrins. Immunoprecipitation experiments on biotinylated cell extracts using excess ß1, ß3, ß4, or ß5-specific mAbs showed no significant modifications of the integrin membrane expression for all the subunits tested (Fig. 5 ). Since no changes in integrin expression levels could be observed on HGF/SF treatment, we interpret HGF/SF-promoted adhesion as a conversion of the integrin functional state from partially to fully active, with consequent enhancement of ligand binding capability.



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  		Figure 5. HGF/SF does not affect integrin expression. Western blots of MDA-MB-231 cells treated with HGF/SF (100 ng/ml) for the indicated times, surface-biotinylated, extracted, and immunoprecipitated with antibodies to integrin subunits ß1 (A), ß3 (B), ß4 (C), or ß5 (D). Probing with streptavidin reveals also coprecipitated {alpha} integrin subunits.

Ligand binding of integrins can be regulated through induction of either affinity (conformation) or avidity (clustering) changes. To address this question, immunofluorescence experiments were performed on MDA-MB-231 cells adhering onto glass coverslips coated with the same substrates used in adhesion assays. After attachment in the presence or absence of HGF/SF, cells were fixed and treated with permeabilization buffer (0.5% Triton X-100). This procedure extracts freely diffusing molecules yet maintains cytoskeletal connections (34 , 37) , hence preserving those integrins that are engaged in newly forming adhesive structures.

In untreated cells plated onto laminin-5 (Fig. 6A ), fibronectin (Fig. 6C ), and vitronectin (Fig. 6E ), ß1 integrin displayed a superimposable pattern of diffuse grainy immunoreactivity. In contrast, HGF/SF treatment resulted in clustering of the heterodimer at the cell periphery: specifically, ß1 integrin was highly enriched along the cell spreading front and in lamellipodia (Fig. 6B, D, F ). This indicates that HGF/SF is able to promote ß1 integrin aggregation and recruitment at nascent adhesive contacts and motility structures. ß3, ß4, and ß5 integrins also underwent such a topographical redistribution on HGF/SF stimulation (not shown).



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  		Figure 6. HGF/SF modifies integrin subcellular distribution. Immunofluorescence micrographs (bar: 5 µm) showing ß1 integrin subunit distribution in MDA-MB-231 cells plated onto laminin-5 (A, B), fibronectin (C, D), or vitronectin (E, F) in the absence (Unst.) or presence of HGF/SF (100 ng/ml). Arrowheads indicate integrin clusters.

Our observation that integrins are mobilized to actin-rich cell protrusions in response to HGF/SF stimulation prompted us to examine their association with microfilaments in more detail using an in situ extraction scheme that solubilizes proteins to an extent that correlates with their cytoskeletal associations (38 39 40) . Specifically, membrane and actin fractions were obtained using sequentially a Triton X-100 buffer for soluble membrane proteins and a two-detergent buffer (1% Tween-40/0.5% deoxycholate) that removes the bulk of the actin cytoskeleton. The relative amount of ß1 integrin present in both fractions was detected by immunoprecipitation and subsequent immunoblotting with ß1-specific antibodies (Fig. 7 ). Notably, HGF/SF stimulation resulted in a substantial reduction in the amount of ß1 heterodimers in the soluble fraction and an increase in the amount of integrin associated with the actin cytoskeleton. These findings provide evidence that the redistribution of ß1 integrin to lamellipodia and membrane ruffles that we detected by immunofluorescence microscopy is indeed due to an increase in its association with F-actin.



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  		Figure 7. HGF/SF increases the association of ß1 integrins with the actin cytoskeleton. MDA-MB231 cells were either left untreated or stimulated with HGF/SF (100 ng/ml) for 20 min. The cells were then sequentially extracted to obtain membrane (Sol.) and actin (Act.) fractions. After solubilization, ß1 integrins were immunoprecipitated from each fraction using an anti-ß1 mAb, resolved by SDS-PAGE, and detected by immunoblotting using a ß1-specific polyclonal antibody.

HGF/SF affects integrin adhesion and avidity through a PI3-K-dependent mechanism
To begin investigating which signaling pathway(s) is involved in HGF/SF-driven enhancement of integrin activity, we tested the contribution of PI3-K, a well-known regulator of migratory responses (25 26 27) , to both adhesion and clustering. MDA-MB-231 cells were plated onto laminin-1, laminin-5, fibronectin, and vitronectin in the presence of two pharmacologically distinct PI3-K inhibitors, LY294002 and wortmannin, under basal conditions or after HGF/SF stimulation. Addition of LY294002 (Fig. 8A ) or wortmannin (not shown) negated the effects of HGF/SF on cell adhesion onto all matrix ligands. Unstimulated adhesion was also somewhat reduced, indicating that a basal level of PI3-K activity is necessary for cell attachment.



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  		Figure 8. PI3-K inhibitors block HGF/SF-mediated adhesion and integrin clustering. A) Adhesion assays were carried out on laminin-1 (Lam-1), laminin-5 (Lam-5), fibronectin (Fn), and vitronectin (Vn) in the presence of 25 µM LY294002 or control DMSO with no stimulation or after treatment with HGF/SF (100 ng/ml). Similar effects were observed when analysis was performed in the presence of 100 nm wortmannin (our unpublished data). B—D) Immunofluorescence micrographs (bar: 5 µm) showing ß1 integrin subunit distribution in MDA-MB-231 cells plated onto fibronectin in the absence of HGF/SF (B), in the presence of 100 ng/ml HGF/SF (C), and on concomitant treatment with HGF/SF and 25 µm LY294002 (D). Arrowheads indicate integrin clusters.

To further define whether PI3-K is involved in HGF/SF-dependent regulation of integrin avidity, we performed immunofluorescence experiments on cells adhering onto the promiscuous substrate fibronectin in the presence of HGF/SF alone or in combination with LY294002. As expected, treatment with HGF/SF resulted in aggregation of ß1 integrins along the cell periphery (Fig. 8C ). In contrast, HGF/SF stimulation in the presence of the inhibitor did not induce integrin clustering so that integrin topographical distribution was superimposable to that of untreated cells (compare Figs. 8B, D ).

Taken together, these results suggest that HGF/SF-triggered pathways require functional PI3-K for optimal adhesion and for up-regulation of integrin avidity. Furthermore, the observation that LY294002 strongly inhibits HGF/SF-promoted attachment onto all substrates tested indicates that this enzyme commonly controls the ability of HGF/SF to affect integrin activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An essential process during tissue morphogenesis, wound repair, and tumor progression is the ability of some cells to detach from the primary site of growth, trespass tissue boundaries, and move through inappropriate compartments. These events are largely regulated by extracellular signals, including growth factors belonging to the PRGF family, together with cell–cell and cell–matrix interactions. Numerous studies have demonstrated that the first step along this migratory sequence is loss of function of adhesion molecules involved in the maintenance of tissue compaction, namely, the cadherin–catenin complex (41 , 42) . What has been less clear, however, is an understanding of the mechanisms whereby cells invade through basement membranes and stroma. Indeed, little information is available about how cells, once dissociated from the tissue of origin, can survive, proliferate, and migrate through foreign districts. A likely explanation is that they acquire the ability to recognize a modified extracellular environment by displaying a versatile set of adhesive receptors, namely, by either neo-expression or functional activation of integrins. The optimal interaction between migrating elements and the ECM provides a mechanical guide for attachment and movement (43) , keeps cells responsive to growth factors (44) , and conveys a survival message protecting from apoptosis (45) .

In this paper we demonstrate that in the MDA-MB-231 cell system, HGF/SF governs efficient execution of the invasive program by enhancing the adhesive capabilities of a multiple repertoire of integrins. This function is peculiar for HGF/SF, not being exerted by conventional growth factors such as EGF, and accounts per se for the generation and maintenance of the migratory phenotype. Moreover, in the case of our cellular model, invasiveness stimulated by HGF/SF is an exclusive consequence of integrin activation. In fact, HGF/SF markedly increases MDA-MB-231 invasiveness without affecting either cell–cell junction architecture or matrix digestion. Accordingly, these cells represent the ideal model to dissect the specific role of cell–matrix interactions during HGF/SF-driven invasive phenomena in the absence of other potentially confounding events.

HGF/SF-dependent triggering of efficient substrate binding capability is not accompanied by quantitative changes in integrin expression levels; rather, it relies on qualitative modulation of integrin recognition of matrix ligands, a process known as activation or inside-out signaling (46) . Activation of integrins is a complex phenomenon resulting from the acute integration of biochemical and structural events. These include conformational changes within their extracellular domains, reorganization of their cytoplasmic connections, and redistribution along the plane of the plasma membrane. The outcome is a transient stabilization of cell–substratum interactions (47 48 49 50) . The events that trigger integrin activation are poorly understood. Integrins can be artificially activated by divalent cations such as Mg2+, Ca2+, and Mn2+ and by treating cells with specific monoclonal anti-integrin antibodies (51 52 53) . At the physiological level, this phenomenon has been investigated in platelets and leukocytes, but little information is available for epithelial and other cells that are part of compact tissues and adhere to basement membranes. Our study provides the first evidence of a growth factor-dependent, simultaneous activation of multiple integrins in epithelial cells. In addition, we show that this activation is instrumental in promoting cell invasiveness across basement membranes and through stromal matrices.

HGF/SF treatment results in integrin clustering at adhesive sites and motility structures, thus increasing local concentrations of integrin receptors. It is likely that aggregated integrins would form a high-density, high-valency complex endowed with enhanced avidity for matrix substrates because of the proximity of ligand binding sites. This is in turn responsible for potentiation of adhesion and migration efficiency, even if affinity changes cannot be ruled out.

A vast number of earlier studies have shown that the dynamics of integrin recruitment to the actin cytoskeleton can be controlled at various levels, with different tyrosine kinases, tyrosine phosphatases, and PI3-K being intimately involved in this process (54 55 56) . Which signaling pathways are used by HGF/SF to manipulate integrin local concentration in MDA-MB-231 cells? As a part of the current study, we specifically addressed the question as to whether or not HGF/SF governs integrin adhesive activity through the PI3-K signaling pathways. Our data clearly indicate that this is the case. First, we found that two pharmacological inhibitors of PI3-K, LY29004 and wortmannin, completely abolished the proadhesive effect of HGF/SF on all substrates tested. Second, we observed that inhibition of PI3-K activity was accompanied by HGF/SF inability to recruit integrins at the newly forming adhesive apparatus. This finding is consistent with the current knowledge on the role of PI3-K in cell movement and migration: phosphatidylinositides generated by PI3-K are able to activate a GTP-GDP exchanger for the small GTPase Rac, homologous to Ras (57) . Activated Rac, in turn, controls organization of the actin cytoskeleton, inducing formation of membrane ruffles and lamellipodia (24) , where integrin clusters are found on HGF/SF stimulation.

An important issue deserving further consideration is that HGF/SF can protect epithelial cells from anoikis, a form of programmed cell death occurring when adherent cells are detached from their physiological substrates (45 , 58 , 59) . It is tempting to speculate that the survival message conveyed by HGF/SF resides at least partially in its ability to activate the function of a multiple set of integrins, thus supplying versatile adhesive information that may confer resistance to anoikis. From this point of view, the ability of HGF/SF to activate integrins results in a double selective advantage: it provides functional receptors for invasive growth and protects cells from massive apoptosis.

In summary, our study provides the first evidence that a single growth factor can activate multiple integrins in epithelial cells through coordinated regulation of avidity changes. Because HGF/SF affects the activity of integrins involved in the recognition of both basal lamina and stromal components, it can render epithelial cells competent to recognize previously unknown ECM components. This feature provides a mechanistic explanation for the peculiar function of HGF/SF in promoting cell invasion.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Maria Prat for the DO24 mAb. The skillful technical assistance of Mrs. Raffaella Albano, Mrs. Laura Palmas, and Mrs. Giovanna Petruccelli is gratefully acknowledged. We thank Mrs. Antonella Cignetto for secretarial help and Mrs. Elaine Wright for editing the manuscript. This work has been supported by Associazione Italiana per la Ricerca sul Cancro, The Giovanni Armenise-Harvard Foundation for Advanced Scientific Research, and European Commission grant no. BMH4-CT98–3852 (to P.M.C.).

Received for publication September 17, 1999. Revision received February 3, 2000.
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 RESULTS
 DISCUSSION
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