The 67 Kda Laminin Receptor Increases Tumor Aggressiveness by Remodeling Laminin-1

Endocrine-Related Cancer (2005) 12 393–406

The 67 kDa laminin receptor increases tumor aggressiveness by remodeling laminin-1

V Berno, D Porrini, F Castiglioni, M Campiglio, P Casalini, S M Pupa, A Balsari1,SMe´nard and E Tagliabue

Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1 20133 Milan, Italy 1Institute of Pathology, University of Milan, 20133 Milan, Italy (Requests for offprints should be addressed to S Me´nard; Email: [email protected])

Abstract The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the a6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure. Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which — including those encoding a3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors — were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion through extracellular matrix degradation. Endocrine-Related Cancer (2005) 12 393–406

Endocrine-Related Cancer (2005) 12 393–406 DOI:10.1677/erc.1.00870 1351-0088/05/012–393 g 2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/25/2021 09:21:22PM via free access Berno et al.: Tumor invasion and 67LR-modiﬁed laminin-1

Introduction Materials and methods Cell lines and culture conditions The recent view of tumors as functional tissue dynami- cally interconnected with the microenvironment sug- Human breast carcinoma cell line MDAMB231 gests that the remodeling of the extracellular matrix (American Type Culture Collection Manassas, VA, (ECM) around the tumor is relevant in tumor invasion USA) was maintained in RPMI 1640 (Sigma) supple- of surrounding tissues and dissemination to distant mented with 10% fetal calf serum (FCS), 1% sites (Pupa et al. 2002). The key molecules in this L-glutamine, and antibiotics (100 mg/ml) at 37 xCina process are adhesion molecules and their receptors, as humidified 5% CO2 atmosphere. Experiments to de- well as proteolytic enzymes that degrade basement termine the effect of laminin-1 or peptide G-treated membrane components (Liotta & Kohn 2001). The laminin-1 were carried out in serum-free medium. high-affinity 67 kDa laminin receptor (67LR) expressed Separation of MDAMB231 cells according to 67LR by tumor cells is known to be critically involved in the expression levels was performed by incubating cells with metastatic process (Castronovo et al. 1990, 1992, Cioce anti-67LR MLuC5 monoclonal antibody (MAb) fol- et al. 1991, Martignone et al. 1993, Sobel 1993, Basolo lowed by anti-mouse immunoglobulin (Ig)-conjugated et al. 1996, Sanjuań et al. 1996, Van den Bruˆle et al. magnetic beads (Miltenyi Biotec GmbH, Bergisch 1996, Fontanini et al. 1997, Waltregny et al. 1998, Gladbach, Germany) according to the manufacture’s Molino et al. 2003), although its exact mechanism procedure. remains unclear. In vitro data indicate that the 67LR promotes adhesion, but this activity cannot account Reagents and antibodies for its role in invasion since stabilization of the tumor Peptide G (IPCNNKGAHSVGLMWWMLAR), cor- cells at the primary site is implied. responding to amino acids 161–180 of the 37 kDa Furthermore, after the initial description of 67LR, precursor protein of 67LR and scrambled peptide X it became clear that integrins are the primary mediators (PMLRWGCHIAMVNKLSWGNA) were synthesized of cell adhesion and signal transduction to the nucleus. by Neosystem (Strasbourg, France). Murine laminin-1 The 67LR is an accessory molecule of the a6 integrin and human purified TIMP-2 were obtained from Sigma subunit (Ardini et al. 1997), and confocal microscope and Chemicon International, Inc. (Temecula, CA, studies (Starkey et al. 1999) confirmed the presence of USA) respectively. MAbs directed against a6-chain 67LR in the vicinity of focal adhesion plaques associated integrin (MAR6) (Bottini et al. 1993), 67LR (MLuC5) witha6b1integrin. However, this molecule isalso shedin (Martignone et al. 1992), talin (8D4 clone) (Sigma) and soluble form by tumor cells in amounts proportional to laminin (LAM-89 clone) (Sigma) were used. Treatment its overexpression (Karpatovaét al. 1996). The correla- of laminin-1 with peptide G or X was performed by tion between the magnitude of the shedding and the incubating adhesion molecules with distinct peptides at enhanced invasiveness in ‘in vitro’ experiments (Starkey a 1 : 1 (w/w) ratio for 1 h at 37 xC. et al. 1999) is consistent with the notion that shed 67LR promotes aggressive malignant behavior in solid tumors. Soluble 67LR, as well as a 20-amino-acid Indirect immunofluorescence and confocal peptide (peptide G) corresponding to the 67LR laminin microscopy analyses binding site, change the conformation of laminin upon Glass coverslips were left uncoated or were coated with interaction with this adhesion molecule (Magnifico et al. laminin-1 or laminin-1 pre-incubated with peptide G 1996). These 67LR-induced conformational changes or peptide X overnight at 37 xC. MDAMB231 cells result in increased proteolytic cleavage of laminin and serum-starved for 24 h were plated, allowed to grow for consequent generation of proteolytic fragments that 30 min, fixed with 3.7% formaldehyde in the medium, promote tumor cell migration (Ardini et al. 2002). saturated with 5% BSA and permeabilized with 0.1% Because receptor recognition domains of laminin are Triton-X 100 in PBS. For phalloidin staining, cells were conformation dependent (Mercurio 1995), 67LR- incubated with fluorescein isothiocyanate (FITC)- induced remodeling of laminin and unmasking of cryptic conjugated phalloidin (Molecular Probes Inc., Eugene, sites might modulate the interaction between tumor cells OR, USA) and observed with an inverted microscope and the ECM, with consequences for metastatic poten- connected to a Nikon DXM camera. Captured images tial. To test this hypothesis we investigated the effect of were processed using the Image PC software package. peptide G-modified laminin-1 on cellular processes For cell surface labeling, cells on coverslips were washed involved in metastasis such as adhesion, migration, with PBS, incubated with MLuC5 (1 : 100), MAR6 invasion and activation of proteolytic enzymes. (1 : 50) and 8D4 (1 : 50) primary antibody for 1 h at

394 Downloadedwww.endocrinology-journals.org from Bioscientifica.com at 09/25/2021 09:21:22PM via free access Endocrine-Related Cancer (2005) 12 393–406 room temperature and incubated with specific second- chamber separated by an 8 mm pore membrane. Cell ary FITC (Alexa 488)- or TRITC (Alexa 546)- or Texas- suspension containing 4r104 cells in RPMI medium red (Alexa 633)-conjugated goat anti-mouse antibody without growth factors was transferred to the upper for 45 min (Molecular Probes Inc.). After three washes wells after addition of untreated (LN-1), peptide G in PBS, coverslips were mounted on glass slides using (pG-LN-1)- or peptide X-treated (pX-LN-1) laminin-1, Mowiol (Calbiochem, San Diego, CA, USA) and ob- peptide G (pG) or peptide X (pX) alone to the lower served with a confocal microscope (Microradiance chamber as attractants. Control chambers contained 2000, BioRad) equipped with Ar (488 nm) and HeNe no added chemoattractants. After incubation at 37 xC (543 nm) lasers. Images were taken using a r60 oil im- for 4 h, migratory cells were dissociated from the bottom mersion lens (1024r1024 pixels). Images were analyzed of the membrane by incubation with cell detachment using Lasersharp 2000 software. Control cells were ex- buffer. Regarding high or low 67LR-expressing posed to relevant secondary antibodies alone and MDAMB231 cells, they were allowed to migrate for showed no significant degree of labeling. 24 h using native laminin-1 as chemoattractant. Detached cells were lysed and stained for 15 min at Cell adhesion assay room temperature with CyQuant GR dye (Chemicon International, Inc), which exhibits strong fluorescence Equal amounts (10 mg/ml) of intact or peptide G-treated enhancement when bound to cellular nucleic acids. laminin-1 were adsorbed on 96-well plates (Greiner Fluorescence was measured using TECAN plate reader Labortechnik, Frickenhausen, Germany) for 1 h at with a 480/520 nm filter set. 37 xC. MDAMB231 cells (1r104 cells per well) in serum-free culture medium were added and allowed Cell invasion assay to adhere at 37 xC. After 1 h, plates were filled with PBS, inverted and shaken in a tank of PBS for Invasion assays were performed in a Matrigel invasion 15 min. Regarding high or low 67LR-expressing cells, chamber (Becton Dickinson Labware, Bedford MA). MDAMB231 cells separated as reported were plated For chemotaxis assay, 750 ml of serum-free culture on native laminin-1 for 6 h at 37 xC. Afterwards they medium and equal amounts (10 mg/ml) of native (LN- were filled with PBS, inverted and shaken in a tank of 1), peptide G (pG-LN-1)- or peptide X-treated PBS for 15 min. Adherent cells were fixed in ice-cold (pX-LN-1) laminin-1, peptide G (pG) or peptide X 10% trichloroacetic acid, labeled with sulphorhod- (pX) alone were added to the lower wells, whereas amine B and evaluated based on optical density at 500 ml MDAMB231 cells were transferred to the upper 4 550 nm wavelength. wells (4r10 cells per well). For experiments with MDAMB231 pre-incubated with treated or untreated ELISA assay laminin-1, lower chambers were filled with culture medium and cells previously seeded for 30 min on native Equal amounts (20 mg/ml) of native, peptide G- or or modified laminin-1 (10 mg/ml) at 37 xC were trans- peptide X-treated laminin-1 were adsorbed in 96-well ferred into the upper chambers as described for the plates (Greiner Labortechnik) for 3 h at 37 xC. Anti- chemotaxis analysis. After 6 h, cells on the upper side laminin MAb diluted 1 : 200 was incubated for 1 h at of the filters were mechanically removed. High or low 37 xC. After several washings with PBS, anti-mouse Ig 67LR-expressing MDAMB231 cells were allowed to horseradish peroxidase (HPR)-conjugated antibody migrate for 24 h using native laminin-1 as chemoat- was incubated for 1 h at 37 xC. After additional wash- tractant. Filters were fixed and stained with Diff-Quick ing, the immune complexes were detected by adding solution (Dade Behring, Duedingen, Switzerland). The 3,3k,5k,5 tetramethylbenzidine (TMB) (Sigma) as sub- number of migrated cells underneath the membranes strate for 30 min at room temperature in the dark. The was counted in four high-power fields. enzymatic reaction was arrested by adding 1 M H2SO4. Plates were read at 450 nm using an automated ELISA Superarray reader. The GEArray gene expression array system consisting of 97 cDNA fragments, printed in tetra-spot format, Cell migration assay from genes associated with metastasis and with control The effects of peptide G-treated laminin-1 or laminin-1 sequences (PUC18 as negative control, b-actin and on tumor cell migration were determined by a modified GAPDH for loading) was obtained from SuperArray Boyden chamber assay using a 96-well cell migration (Bethesda, MD, USA). Total cellular RNA was iso- kit (Chemicon International, Inc.), with each well lated with Trizol reagent (Invitrogen) and RNA

www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/25/2021395 09:21:22PM via free access Berno et al.: Tumor invasion and 67LR-modified laminin-1 integrity was assessed by visualization of ethidium Proteases bromide-stained gels. Microarray analysis was carried Matrix metalloproteinases: ADAMTS1 (Meth 1), out according to the manufacturer’s instructions. Briefly ADAMTS8 (Meth 2), MMP1 (collagenase-1), MMP2 cDNA was prepared from total RNA obtained from (gelatinase A), MMP3 (stromelysin-1), MMP7 (matrily- cells plated on native or peptide G-modified laminin-1 sin), MMP8(neutrophilcollagenase),MMP9(gelatinase for 30 min, by reverse transcription with MMLV B), MMP10 (stromelysin-2), MMP11 (stromelysin-3), reverse trascriptase, radiolabeled using [a-32P]dCTP MMP12 (macrophage elastase), MMP13 (collagenase- (10 mC/ml, Amersham) and hybridized under specified 3), MMP14 (MT1-MMP), MMP15, MMP16, MMP17, conditions to a positively charged nylon membrane MMP20 (enamelysin), MMP24, MMP26. Serine protei- containing the array DNA. After washing, arrays were nases: CTSG (cathepsin G), PLAT (tPA), PLAU (uPA), visualized by autoradiography and tetra-spot images PLAUR (uPAR), TMPRSS4. were converted to numerical data using the free ScanAlyze software. GEArray Analyzer software was Cysteine proteinases used to match raw data with the gene list for metastasis CASP8, CASP9, CST3 (cystatin C), CTSB (cathepsin GEArray. Raw signal intensities were corrected for B), CTSL (cathepsin L). Other related genes: CTSD background by subtracting signal intensity of a nega- (cathepsin D), HPSE (heparanase), MGEA5 (mening- tive control or blank, and normalized to signal in- ioma associated hyaluronidase). Protease inhibitors: tensity of a housekeeping gene using image-analysis SERPINB2 (PAI-2), SERPINB5 (maspin), SER- software (AtlasImage version 2.0). For each gene, PINE1 (PAI-1), TIMP-1, TIMP-2, TIMP-3. values corresponding to two different treatments were compared and a gene expression difference higher than RT-PCR that observed for housekeeping genes (1.6-fold) was Reverse transcription was carried out at 42 xC for considered indicative of change due to cell treatment. 60 min using 2 mg total RNA, 75 pmol oligo(dT) The array includes the genes described below. primer, 0.5 mM of each dNTP and 50 U of MMLV reverse transcriptase (Invitrogen). RT product was Cell adhesion molecules amplified with primer pairs specific for the genes to be Integrins: ITGA1 (integrin a1), ITGA2 (integrin a2/ studied. Conditions and primer sequences are avalaible LFA1b), ITGA2B (integrin a2b), ITGA3 (integrin a3), on request. GAPDH cDNA was amplified in parallel ITGA4 (integrin a4/VLA-4), ITGA5 (integrin a5), to evaluate the efficiency of RNA extraction and ITGA6 (integrin a6), ITGA7 (integrin a7), ITGA8 cDNA synthesis. GAPDH primers were 5k-TCCAC- (integrin a8), ITGA9 (integrin a9), ITGA10 (integrin CACCCTGTTGCTGTA-3k and primer 5k-ACCACA- a10), ITGA11 (integrin a11), ITGAL (integrin aL/ GTCCATGCCATCAC-3k, with an expected product of LFA1a/CD11A), ITGAM (integrin aM), ITGAV 452 bp. Each RT-PCR product was electrophoresed on (integrin aV), ITGAX (integrin aX), ITGB1 (integrin a 1% agarose gel, visualized by ethidium bromide b1), ITGB2 (integrin b2), ITGB3 (integrin b3/CD61), staining and the optical density (OD) of the bands was ITGB4 (integrin b4), ITGB5 (integrin b5), ITGB6 measured with Image Quant 5.2 Software (Molecular (integrin b6), ITGB7 (integrin b7), ITGB8 (integrin b8). Diagnostics, San Francisco, CA, USA). The intensity Immunoglobulin (Ig) Superfamily: CEACAM5 (CEA), of the bands was normalized for every sample, relative DCC, ICAM1, MICA (MUC-18), NCAM1, NRCAM, to the intensity of the respective GAPDH bands, accor- PECAM1, VCAM1. Cadherin and catenins: CDH1 ding to the following formula: OD gene/OD GAPDH. (E-cadherin), CTNNA1 (catenin a), CTNNAL1 (cate- For each gene, results are then expressed as the ratio nin a like 1), CTNNB1 (catenin b), CTNND1 (catenin pG-LN-1/LN-1. d1), CTNND2 (catenin d2). Selectins: SELE (ELAM- 1/E-selectin), SELL (L-selectin), SELP (P-selectin). Other related genes: CD44, CNTN1. Detection of metalloproteinase activity Cells were grown for 30 min on plates precoated with Extracellular matrix proteins laminin-1 or peptide G-treated laminin-1. Aliquots of CAV1 (caveolin-1), COL18A1 (LOC51695/endostatin), cell supernatants were electrophoresed through a 10% COL1A1, COL4A2, ECM1 (extracellular matrix 1), SDS–PAGE gel polymerized with 0.1% gelatin (Sig- FGB (fibrinogen b), FN1 (fibronectin-1), LAMB1 ma). Gels were washed three times for 20 min each in (laminin B1), LAMC1 (laminin B2), SPARC, SPP1 2.5% Triton X-100 to remove SDS, and incubated (OPN, osteopontin), THBS1 (TSP-1), THBS2 (TSP-2), overnight at 37 xC in collagenase buffer containing THBS3 (TSP-3), VTN (vitronectin). 50 mM Tris–HCl (pH 7.5), 200 mM NaCl and 10 mM

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CaCl2. Gelatinolytic activity was visualized as clear cellular extensions colocalized in adhesion plaques as blue bands by staining with 0.5% Coomassie Blue and identified by talin labeling (Fig. 2A), whereas in cells destaining with 10% methanol and 5% acetic acid. seeded on peptide G-treated laminin-1, 67LR was Previous data were confirmed using the MMP gelati- distinct from the a6 subunit in filopodia, especially at nase activity assay kit (Chemicon International, Inc.) the root of such protrusions, in addition to colocalizing which is based on cleavage of biotinylated gelatinase with a6 integrin subunit in focal adhesion plaques substrate by active MMP-2 enzyme. The remaining (Fig. 2B and C). These results point to the different biotinylated fragments were added to biotin-binding behavior of cells interacting with 67LR-modified 96-well plates and detected with streptavidin enzyme laminin-1 as compared with cells interacting with the complex. Addition of enzyme substrate results in a unmodified molecule. colored product, detectable based on optical density at To determine whether 67LR-modified laminin-1 af- 450 nm. fects activities related to tumor aggressiveness, in vitro cell adhesion and migration in the presence of peptide Statistics G-treated or untreated laminin-1 were analyzed. MDAMB231 cells seeded in wells containing native Data are expressed as the means S.D. of three t or peptide G-treated laminin-1 showed a significant independent experiments. Statistical analysis was increase in adhesion compared with cells seeded on performed using an unpaired Student’s t-test. P values uncoated matrix; a small but statistically significant of 0.05 were considered statistically significant. < increase in adhesion was observed on the modified vs native laminin-1 (P=0.006). Cells incubated in the Results presence of peptide X-treated laminin-1 showed adhesion comparable with that of native laminin-1 while Effect of 67LR-treated laminin-1 on tumor cells plated in the presence of peptide X or G displayed cell morphology and aggressiveness an adhesion superimposable on that observed for MDAMB231 human breast carcinoma cells plated on control (Fig. 3A). By ELISA analysis with anti- laminin-1 untreated or treated with the 67LR mimic laminin-1 antibodies, results for binding of peptide peptide G were analyzed for morphology after fixation G-treated laminin-1 to the plastic substrate were similar and staining with FITC-phalloidin. Cells plated on to those of untreated or peptide X-treated laminin-1 native laminin-1 presented the actin bundles organized (Fig. 3B). When MDAMB231 cells separated accord- as multiple cables parallel to margins (Fig. 1B), whereas ing to 67LR expression were seeded for 6 h on native cells plated on peptide G-modified laminin-1 exhibited laminin-1 a significant increase in adhesion (P< a polygonal array of filament bundles (Fig. 1C). Cells 0.0001) was found in high 67LR-expressing cells plated on peptide X-treated laminin-1 or uncoated sub- compared with cells presenting a low level of the strate presented an elongated-shaped and round mor- laminin-1 receptor. Chemotaxis assay to quantitate the phology respectively (Fig. 1D and A). To investigate effect of 67LR-induced conformational change on whether 67LR shed from tumor cells had effects on the laminin-1-stimulated cell motility revealed a 2.5-fold actin infrastructure similar to those induced by peptide or 3-fold increase in MDAMB231 cell motility when G-modified laminin-1, MDAMB231cells — which are native or peptide G-treated laminin-1, respectively, was heterogeneous for the expression of monomeric laminin used as chemoattractant (Fig. 3C). The difference in receptor — were separated according to 67LR expres- motility between unmodified and peptide G-modified sion. By cytofluorimetric analysis, two cell subpopu- laminin-1 was quite small but still statistically signi- lations were found to display a different expression of ficant (P=0.02). Cells in the presence of both peptides the 67LR with a mean fluorescence intensity of 328t X and G alone or peptide X-treated laminin-1 showed 166 (high 67LR) and 67t177 (low 67LR). After incu- migration comparable with that of cells without chemo- bation on native laminin-1 for 6 h, high 67LR-ex- attractant or with native laminin-1. MDAMB231 cells pressing cells presented the actin bundles organized as separated according to 67LR expression and allowed cells plated on peptide G-modified laminin-1 (Fig. 1E). to migrate for 24 h using native laminin-1 as chemo- On the other hand, cells presenting a low level of 67LR attractant, revealed that only those presenting high showed an elongated morphology similar to that of levels of 67LR increased their migration compared cells plated on native laminin-1 (Fig. 1F). Confocal with cells expressing low levels, although this difference microscopic analysis of cells plated on native laminin-1 did not reach statistical significance (Fig. 3C). revealed a6 integrin subunit, the canonical laminin-1 Because the invasive phenotype consists of coordi- receptor, and 67LR concentrated at the tips of the nated attachment, detachment, migration and matrix

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Figure 1 Morphology of breast carcinoma cells seeded on different laminin forms. MDAMB231 cells plated for 1 h on glass coverslips left uncoated (A) or coated with LN-1 (B), pG-LN-1 (C) or pX-LN-1 (D). MDAMB231 cells expressing high (E) or low (F) levels of 67LR seeded on LN-1 for 6 h. After fixation, cells were stained with FITC-phalloidin, and viewed using a fluorescence microscope. Photographs are representative of different actin stainings obtained in three separate experiments. The percentages of cells presenting the different morphologies were: 76t1.4% (B), 67t4.3% (C), 71t3% (D), 65t2% (E) and 77t0.5% (F). Scale bars, 10 mm. degradation, we analyzed the effect of 67LR-induced native laminin-1 was comparable with that in the ab- conformational change in laminin-1 on the ability of sence of chemoattractant (Fig. 4A). However, extend- the tumor cells to penetrate the basement membrane. ing incubation time with native laminin-1 was found to MDAMB231 cells seeded in invasion chambers con- induce basement membrane penetration related to taining a membrane coated with a uniform layer of expression of 67LR (Fig. 4A) (P=0.04, high 67LR vs basement membrane (Matrigel) migrated within 6 h low 67LR). Furthermore, cells pretreated with mod- when the peptide G-modified laminin-1 was present in ified laminin-1 for 30 min before invasion assay in the the lower chamber (P=0.035), whereas migration of absence of chemoattractant revealed a 6-fold increase these cells through the membrane in the presence of in invasion as compared with cells pretreated with

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Figure 2 Distribution of 67LR and a6 in breast carcinoma cells. Indirect immunofluorescence was used to examine localization of 67LR (blue), a6 integrin (green) and talin (red) in breast carcinoma cells grown on glass coverslips coated with LN-1 (A) or pG-LN-1 (B) for 1 h. Panel C shows higher magnified image of filopodia (red boxed area in panel B). Overlay of individual fluorescence images was carried out using LaserSharp 2000 Software. Photographs are representative of different 67LR and a6 integrin staining in three separate experiments. The percentages of cells presenting the different morphologies were 75t1.9% (A) and 68t3.7% (B). Scale bars, 5 mm. unmodified laminin-1 (P=0.005) (Fig. 4B) suggesting fibronectin and laminin B1; and MMPs such as that laminin-1 modified by interaction with the 67LR stromelysin-3, MMP-8, MMP-7 and MT1-MMP, as provides a message that promotes invasion. well as the cysteine proteases cathepsin L and cystatin C; and the serine protease tPA were dramatically overexpressed in modified laminin-1-seeded cells. Effect of 67LR-treated laminin-1 on gene Expression of molecules involved in regulation of expression in breast cancer cells proteolytic enzymes such as PAI-1, TIMP-1 and -2 was To examine the signal derived from 67LR-modified also augmented. laminin-1, MDAMB231 cells plated on native or Microarray results were verified for some selected peptide G-modified laminin-1 for 30 min were analyzed genes by RT-PCR analysis (Fig. 5A) with similar for gene expression by cDNA superarray, including results even if the fold-change in mRNA levels genes for cell adhesion molecules, ECM proteins, that resulted was lower compared with array analysis proteases and their inhibitors. Arrays were hybridized (Fig. 5B). TIMP-2, cystatin C, ECM1, stromelysin-3, with 32P-labeled cDNA probes generated from RNA a1 and a3 integrin subunits were more transcribed in preparations of MDAMB231 cells seeded on untreated cells plated on peptide G-modified laminin compared or peptide G-treated laminin-1. Spot intensities were with cells plated on native laminin-1, whereas vitro- normalized relative to expression levels of four house- nectin had higher expression levels in cells plated on keeping genes. For each gene, values corresponding to unmodified vs modified laminin-1. The MMP-2, whose two different treatments were examined. Comparative expression was not found to change in microarray analysis of values corresponding to each gene revealed analyses, was equally transcribed in cells exposed to differential expression for 19 genes (Table 1) presenting 67LR-modified or native laminin-1. higher variation than that observed for housekeeping Because MT1-MMP and TIMP-2, the two molecules genes (1.6-fold). Seventeen of these genes were up- involved in MMP-2 activation, were both up-modu- modulated in MDAMB231 cells seeded on peptide lated in cells seeded on peptide G-treated laminin-1, G-modified laminin-1 compared with cells treated with the activity of this metalloproteinase was further unmodified laminin-1, which presented up-modulation investigated by zymographic analysis of the culture of only two ECM proteins: vitronectin and osteopontin. supernatant from these cells; the clear band corre- Cells plated on peptide G-treated laminin-1 showed sponding to activated MMP-2 was more notable overexpression of 12% (7/58) of ECM proteins and compared with cells seeded on native laminin-1, adhesion molecules vs 26% (10/38) of proteases and whereas levels of pro-enzyme did not change in any their regulators. In particular, all a-subunits of inte- condition (Fig. 6A). Accordingly, using the MMP grins (1, 3 and 5) as well as catenin d1, ECM1, gelatinase activity assay kit, MDAMB231 cells showed

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A 1.00 Adhesion a significant (P=0.01) increase in MMP-2 gelatinolytic activity when exposed to peptide G-modified vs native * **laminin-1 (Fig. 6B). To determine the relevance of 0.75 increased MMP-2 activity in the invasiveness of cells pretreated with modified laminin-1, invasion of MDAMB231 cells was assayed in the presence or ab- 0.50 sence of the MMP inhibitor TIMP-2. Addition of such an inhibitor reduced the migration of these cells through

O.D. (550 nm) the membrane by 33%, while the same treatment 0.25 reduced the invasiveness of cells exposed to native laminin-1 by only 7% (P=0.0003). Moreover, TIMP- 2-mediated inhibition of invasiveness was stronger in 0 cells expressing high levels (23%) rather than low levels pG pX (13%) of 67LR (P=0.014) (Fig. 6C). LN-1 control pG-LN-1 pX-LN-1 high 67LRlow 67LR

B 0.30 Elisa Discussion The present study demonstrates that the interaction of 67LR-modiﬁed laminin-1 with tumor cells enhances 0.20 their ability to invade and increases expression and activation of proteolytic enzymes and their regulators. As indicated by morphological analysis, MDAMB231 breast carcinoma cells line grown on peptide G-treated

O.D. (450 nm) 0.10 laminin-1 exhibited actin-containing structures different from those formed in the presence of native laminin- 1. Speciﬁcally, the former cells developed ﬁlopodia, extensions by which cells establish dynamic adhesion 0 and motility on the substratum where they are shaped pG pX LN-1 (O’Connor et al. 1990, Mitchison & Cramer 1996, control pG-LN-1 pX-LN-1 Adams 2001). The functional involvement of 67LR in C 30000 Chemotaxis the dynamics of these actin-containing motility structures is also supported by the localization of this

20000 adsorbed on a 96-well plate. After 6 h plates were filled with PBS, inverted and shaken in a tank of PBS for 15 min. Control represents adhesion of cells plated on uncoated substrate. (B) Adhesion of LN-1, pG-LN-1 or pX-LN-1 to culture substrates detected using anti-laminin MAb followed 10000 by anti-mouse Ig HPR-conjugated antibody together with TMB Fluorescence (480 nm) as substrate and H2SO4 for arresting the enzymatic reaction. The immune complexes were evaluated spectrophotometrically at 450 nm using an automated ELISA 0 reader. Control represents uncoated wells. (C) Migration of MDAMB231 cells on LN-1, pG-LN-1, pX-LN-1, pG or pX. pG pX LN-1 After 4 h migrating cells were evaluated using a CyQuant GR control pG-LN-1 pX-LN-1 high 67LRlow 67LR dye solution together with a lysis buffer. High or low 67LR- expressing MDAMB231cells were allowed to migrate for 24 h Figure 3 Effect of peptide G-modified laminin-1 on tumor cell using LN-1 as chemoattractant. Control represents migration adhesion and migration. (A) Adhesion of MDAMB231 cells on of cells in absence of any chemoattractant. All experiments LN-1, pG-LN-1, pX-LN-1, pG or pX adsorbed on a 96-well were repeated three times; error bars represent standard plate overnight at 37 xC. After 1 h plates were filled with PBS, deviations of the means calculated in separate experiments; inverted and shaken in a tank of PBS for 15 min. High or low *, statistically significant (P<0.05) as determined using 67LR-expressing MDAMB231 cells were seeded on LN-1 Student’s t-test.

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A 80 differences between unmodified and 67LR-modified laminin-1 in two steps of cell motility, i.e. adhesion and * migration, do not explain the mechanism by which 67LR is associated with tumor invasiveness. By con- 60 trast, the enhanced ability, compared with the native adhesion molecule, of tumor cells induced by modified laminin-1 to traverse a Matrigel basement suggests that 40 * the invasive potential upon interaction with laminin-1 conformationally modified by 67LR underlies the high Migrated cells metastatic behaviour of tumors overexpressing the 20 monomeric laminin-1 receptor. The highly increased production of proteolytic enzymes by tumor cells treated with 67LR-modified 0 laminin-1 compared with native laminin supports this pG pX hypothesis. Indeed, of the 24 integrin subunits and 19 LN-1 control pX-LN-1 non-integrin cell adhesion molecules investigated in pG-LN-1 high 67LRlow 67LR microarray analysis, only 3 (12%) and 1 (5%), respec- B 400 tively, showed a change. By contrast, 10 of 38 (26%) proteases and their regulators were up-modulated by ** cell binding to 67LR-treated laminin-1. Specifically, 300 expression was enhanced in 26% of MMPs, 20% of serine proteases, 40% of cysteine proteases and 50% of molecules involved in their regulation — such as 200 TIMPs and PAI-1 — with consequent modulation of enzyme activation. Consistent with these findings,

Migrated cells Givant-Horwitz et al. (2004) recently reported the down-modulation of MMP-2 collagenolytic activity 100 in A375SM melanoma cells transfected with a monomeric laminin receptor precursor antisense construct. The a3 integrin subunit was among the receptor 0 molecules up-modulated in cells seeded on 67LR-

LN-1 treated laminin-1. This integrin usually does not par- control pG-LN-1pX-LN-1 ticipate significantly in laminin-1 binding to the cell surface, but does act in laminin-5 binding (Carter et al. Figure 4 Invasion of MDAMB231 cells. Cell migration in vitro 1990). Thus, up-modulation of the a3 subunit implies was measured using Matrigel as an ECM barrier in modified Boyden chambers with LN-1, pG-LN-1, pX-LN-1, pG or pX as an increased probability of interaction with laminin-5, chemoattractants (A) or as pretreatment agents (B). After 6 h, known to be implicated in tumor cell invasion (Shang filters were fixed and stained with crystal violet and viewed et al. 2001, Wang et al. 2004). The similar degree of up- under light microscope with r20 magnification. Cells modulation for both a5 and its ligand fibronectin migrating to the underside were counted in four microscopic (Robinson et al. 2004) points to the ability of tumor fields. High or low 67LR-expressing MDAMB231 cells were cells interacting with 67LR-treated laminin-1 to allowed to migrate for 24 h using LN-1 as chemoattractant. Migrating cells were fixed, stained and counted as described modify the composition of the ECM as well as the above. Controls represent migration of cells in the absence of ability to interact with it. A similar speculation can be any chemoattractant or following any pretreatment. All made for the a1 and laminin B1 chain, both of which experiments were repeated three times; error bars represent presented 4- to 5-fold expression up-modulation. More- standard deviations of the means calculated in separate over, a1 is involved in protrusive cell-matrix contacts, experiments; *, statistically significant (P<0.05) as determined using Student’s t-test. such as filopodia, which are usually associated with rapid membrane remodelling, transient matrix adhesions and exploratory cell movements (Gardner et al. receptor in filopodia distinct from that of the canonical 1996, Hynes 2002). The 67LR-induced membrane laminin-1 receptor a6 integrin subunit with which the protrusions with a matrix degradation activity suggest monomeric laminin-1 receptor reportedly colocalizes in that expression of genes with cytoskeletal function focal adhesion plaques (Starkey et al. 1999). The small might also be modulated by peptide G-modified

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Table 1 Peptide G-modiﬁed laminin-1-dependent genes differentially expressed by MBAMB231 cells

pG-LN-1/ Genebank Description Gene name LN-1

Cell adhesion molecules M59911 Integrin, a3 (antigen CD49C, a3 subunit of VLA-3 receptor) Integrin a3 14.11 X68742 Integrin, a1 Integrin a1 5.60 AF062343 Catenin (cadherin-associated protein), d1 Catenin d1 3.25 X06256 Integrin, a5 (ﬁbronectin receptor, a polypeptide) Integrin a5 3.19 Extracellular matrix proteins NM_004425 Homo sapiens extracellular matrix protein 1 (ECM1), ECM1 10.96 transcript variant 1 M61916 Laminin B1 chain Laminin B1 4.06 X02761 Fibronectin 1 Fibronectin-1 2.03 X03168 Vitronectin (serum spreading factor, somatomedin B, Vitronectin 0.25 complement S-protein) M83248 Homo sapiens secreted phosphoprotein 1 (osteopontin, Osteopontin 0.14 bone sialoprotein I, early T-lymphocyte activation 1) (SPP1), Proteases X57766 Human stromelysin-3 Stromelysin-3 5.50 NM_002424 Matrix metalloproteinase 8 (neutrophil collagenase) Neutrophil collagenase 3.27 X07819 Matrix metalloproteinase 7 (matrilysin, uterine) Matrilysin, uterine 2.70 D26512 Homo sapiens mRNA for membrane-type matrix metalloproteinase 1 MT1-MMP 2.15 NM_000930 Homo sapiens plasminogen activator, tissue (PLATa) tPA 2.10 NM_000099 Homo sapiens cystatin C (amyloid angiopathy and cerebral Cystatin C 1.85 hemorrhage) (CST3) X12451 Cathepsin L Cathepsin L 1.79 Inhibitors M16006 Plasminogen activator inhibitor, type I (PAI-1) PAI-1 4.79 NM_003255 Tissue inhibitor of metalloproteinase 2 TIMP-2 3.14 NM_003254 Tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, TIMP-1 2.27 collagenase inhibitor)

laminin-1 even though RT-PCR analyses did not (Gasparini et al. 1995, Gebarowska et al. 2002), we reveal differences in tropomyosin expression in cells found that laminin modified by interaction with the stimulated with treated vs untreated laminin-1 (data monomeric receptor strongly increases the expression not shown). of ECM1, a secreted glycoprotein implicated in cell The supposition of induction by 67LR of a ‘dif- proliferation, angiogenesis and differentiation. In pri- ferent’ matrix is strengthened by the reduction of mary breast carcinomas, ECM1 expression has been vitronectin and osteopontin, two cell adhesion mole- correlated with tumor metastatic properties (Wang cules whose overexpression has been linked to differ- et al. 2003). The most direct evidence for the role entiation and tumor progression respectively (Senger of ECM1 in angiogenesis is the finding that purified et al. 1983, Panda et al. 1997, Aaboe et al. 2003). recombinant ECM1 stimulates the proliferation of However, even cell–cell interaction was affected by cultured endothelial cells and promotes blood vessel 67LR-modified laminin-1, as indicated by the signifi- formation in the chorioallantoic membrane of chicken cantly increased expression of catenin d1, a component embryos (Han et al. 2001). Because growth and of the adherens junction and with a role in cell motility. metastasis are critically dependent on the formation Indeed, ectopic expression of this molecule alters cell of new vessels, and because 67LR is also overexpressed morphology, inducing the elaboration of lamellipodia in endothelial cells during neoangiogenesis (Stitt et al. and increasing the scattering response to hepatocyte 1998, Tanaka et al. 2000, McKenna et al. 2001), growth factor treatment (Lu et al. 1999). laminin-1 modification induced by the monomeric Consistent with the reported association between laminin receptor might also support tumor progression 67LR expression and tumor high neovascularization through the induction of angiogenesis.

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A B

LN-1 pG-LN-1 Integrin 499 bp alpha 3

Integrin 240 bp alpha 1

775 bp ECM 1

256 bp Vitronectin

342 bp Stromelysin-3

380 bp Cystatin-C

859 bp TIMP-2

503 bp MMP-2

Microarray 452 bp GAPDH RT-PCR

0.1 1 10 100 Ratio of gene expression (pG-LN-1/LN-1)

Figure 5 (A) Validation of microarray results for selected genes by RT-PCR. Comparison of expression analyses in cells seeded on LN-1 or pG-LN-1 of transcripts for a3 and a1 integrin subunits, ECM1, vitronectin, stromelysin 3, cystatin C, TIMP-2, MMP-2. GAPDH cDNA was ampliﬁed in parallel to evaluate the efﬁciency of RNA extraction and cDNA synthesis. (B) Comparative expression levels of a panel of eight genes assayed by RT-PCR and microarray.

Most of the proteolytic enzymes up-regulated by stimulated by modified laminin-1. The changes in modified laminin-1 interaction such as cystatin-C, expression of such MMP regulators explain the cathepsin L and tPA might participate in ECM significant increase of MMP-2 activation upon treat- degradation (Birkedal-Hansen 1995, Fata et al. 2004), ment with 67LR-modified laminin-1 without mRNA but the metalloproteinases appear to play the major modification and suggest that 67LR-enhanced inva- role (Egeblad & Werb 2002). Enzymes such as MT1- siveness is strongly dependent on MMP-2 activation. MMP, MMP-7 and -8 which were considerably up- The role of this protease is proved by TIMP-2 inhi- modulated have been widely described to cleave bition experiments. The synthesis and activation of collagen, laminin and fibronectin respectively, and to different proteases induce physical alterations of ECM be highly involved in tumor invasion mechanisms. The proteins, which expose cryptic domains (Ardini et al. need for a delicate balance between proteases and 2002, Khan et al. 2002, Palmieri et al. 2003), and their inhibitors in each phase of the migration process facilitate the activation of different signal transduction might provide a functional explanation for the up- molecules that regulate cytoskeleton arrangement and regulation of TIMPs and PAI-1 in the same cells cellular response in pathological conditions. The

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superarray data were confirmed by RT-PCR. The A lower ratio of PCR assay compared with array results control LN-1 pG-LN-1 can be explained by different saturation levels of the two methodologies. 72 kDa Together, our results on the interaction of tumor 64 kDa pro MMP-2 active MMp-2 cells with 67LR-modified laminin-1 to enhance tumor cell invasion, point to a mechanism by which the role of 67LR in tumor cell dissemination stems from its ability to modify laminin-1. This conclusion is stren- B 0.4 gthened by data obtained investigating the effects * induced by native laminin-1 in tumor cells expressing high or low levels of 67LR. Indeed, alterations in actin organization as well as adhesion, migration and espe- 0.3 cially invasion were found in 67LR highly positive cells but not in low-positive cells when these cells were seeded for extensive periods on unmodified laminin-1. 0.2 As the 67LR shedding is proportional to its expression level (Karpatovaét al. 1996, Starkey et al. 1999), these O.D. (450 mm)

Gelatinase activity ﬁndings support a role for 67LR in changing laminin-1 0.1 structure and consequently in promoting the metastatic phenotype. Therefore, this laminin-1 receptor appears to play a prominent role in the continually evolving interactions 0 between neoplastic cells and the ECM that are essential LN-1 for tumor invasion and metastasis. control pG-LN-1

C 40 * * Acknowledgements We thank Mrs Piera Aiello and Mrs Cristina Ghirelli 30 for technical assistance and Mrs Laura Mameli for manuscript preparation.

20 Funding This work was supported by grants from 10 Associazone Italiana per la Ricerca sol Canco (AIRC). The authors declare that there is no conﬂict % Inhibition of invasion by TIMP-2 by % Inhibition of invasion of interest that would prejudice the impartiality of 0 this scientiﬁc work.

LN-1 pG-LN-1 high 67LRlow 67LR

Figure 6 Analysis of MMP-2 activity and its involvement in cell invasion. (A) Conditioned medium of MDAMB231 cells results in a colored product, detected based on optical density seeded on plates left uncoated (control), or coated with LN-1 at 450 nm. Control represents supernatant of cells plated or pG-LN-1 for 30 min, was assessed for gelatinase activity by on uncoated substrate. Error bars represent standard zymography. (B) MMP-2 activity was also tested with a deviations of the means calculated in separate experiments; gelatinase activity assay kit that utilizes a biotinylated *, statistically signiﬁcant (P<0.05) as determined using gelatinase substrate, which is cleaved by active MMP-2 Student’s t-test. (C) Inhibition of invasion by TIMP-2 in enzyme. The remaining biotinylated fragments are then MDAMB231 cells pretreated with LN-1 or pG-LN-1 and added to biotin-binding 96-well plates and detected with in MDAMB231 cells selected for high or low level of 67LR streptavidin-enzyme complex. Addition of enzyme substrate expression.

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