IntroductionTumor invasion and metastasis is governed by a reciprocalcross-signaling between cancer cells and host cells at theprimary tumor site and distant organs. Progression of solidtumors is associated with a remarkable disorganisation of thestromal compartments that normally maintain the mucosal andepithelial cell architecture and functions. In response to signalsproduced by cancer cells, the tumor stroma is invaded byseveral cell types. The best characterized mechanism is thehypoxia-dependent angiogenic switch in which host-cell-derived endothelial cells invade into the tumor stroma to formnew blood vessels (Carmeliet and Jain, 2000). Other host cellsthat invade the tumor are fibroblast-like cells (Roni et al., 2003;Yang et al., 2001). Solid tumor implantation in transgenic micethat express GFP under the control of the VEGF promoter leadsto induction of VEGF-promoter activity in myofibroblast-likecells. Subsequently, GFP-positive myofibroblast-like cellsinvade the tumor and can be seen throughout the tumormass (Fukumura et al., 1998). During avascular growth ofdeveloping hepatic metastases, myofibroblast-like cells arealready present, before endothelial cell recruitment (Olaso etal., 2003). Thus, myofibroblasts might provide pro-invasiveinformation at the invasion front of cancer cells (De Wever et

al., 2004). Does invasion of myofibroblasts into the epithelialcompartment precede the invasion of cancer cells into the hostcompartment? Barriers that myofibroblasts have to traverse arecollagen type I-enriched extracellular matrix (ECM) and thebasement membrane. Molecular cross-signaling betweencancer cells and myofibroblasts might stimulate migration ofboth cell types towards each other and modify the adjacentECM and basement membrane. The result is breakdown ofnormal tissue boundaries (De Wever and Mareel, 2003).

Transforming growth factor (TGF)-β1 is implicated in thesignaling from cancer cells to myofibroblasts. TGF-β is achemotactic agent for fibroblasts (Postlethwaite et al., 1987)and is secreted by colon-cancer cells as a latent complex storedin the ECM. Myofibroblasts release bioactive TGF-β fromthe latent complex through proteolytic and non-proteolyticmechanisms (Derynck et al., 2001). Bioactive TGF-β1activates an heterodimeric cell surface complex of twotransmembrane receptor serine/threonine kinases, leading toSmad and non-Smad signal transduction (Derynck and Zhang,2003). Non-Smad signal transduction comprises mitogenactivated protein kinase (MAPK) pathways. Functional TGF-β receptors are required for TGF-β-mediated activation ofMAPKs (Mulder, 2000). MAPKs are serine/threonine kinases

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Invasion of stromal host cells, such as myofibroblasts, intothe epithelial cancer compartment may precede epithelialcancer invasion into the stroma. We investigated howcolon cancer-derived myofibroblasts invade extracellularmatrices in vitro in the presence of colon cancer cells.Myofibroblast spheroids invade collagen type I in a stellatepattern to form a dendritic network of extensions uponco-culture with HCT-8/E11 colon cancer cells. Singlemyofibroblasts also invade Matrigel™ when stimulated byHCT-8/E11 colon cancer cells. The confrontation of cancercells with extracellular matrices and myofibroblasts,showed that cancer-cell-derived transforming growthfactor-β (TGF-β) is required and sufficient for invasion ofmyofibroblasts. In myofibroblasts, N-cadherin expressed atthe tips of filopodia is upregulated by TGF-β. FunctionalN-cadherin activity is implicated in TGF-β stimulated

invasion as evidenced by the neutralizing anti-N-cadherinmonoclonal antibody (GC-4 mAb), and specific N-cadherinknock-down by short interference RNA (siRNA). TGF-β1stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts.Pharmacological inhibition of JNK alleviates TGF-βstimulated invasion, N-cadherin expression and woundhealing migration. Neutralization of N-cadherin activityby the GC-4 or by a 10-mer N-cadherin peptide or bysiRNA reduces directional migration, filopodia formation,polarization and Golgi-complex reorientation duringwound healing. Taken together, our study identifies a newmechanism in which cancer cells contribute to thecoordination of invasion of stromal myofibroblasts.

Key words: Spheroid, Cross-signaling, JNK

Summary

Critical role of N-cadherin in myofibroblast invasionand migration in vitro stimulated by colon-cancer-cell-derived TGF- β or woundingOlivier De Wever 1, Wendy Westbroek 2, An Verloes 1, Nele Bloemen 1, Marc Bracke 1, Christian Gespach 3,Erik Bruyneel 1 and Marc Mareel 1,*1Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine and 2Department of Dermatology, Ghent UniversityHospital, De Pintelaan 185, 9000 Gent, Belgium3INSERM U482, Hôpital Saint-Antoine, 184 rue du Faubourg, Saint-Antoine, 75571 Paris CEDEX 12, France*Author for correspondence (e-mail: marc.mareel@ugent.be)

Accepted 20 May 2004Journal of Cell Science 117, 4691-4703 Published by The Company of Biologists 2004doi:10.1242/jcs.01322

Research Article

JCS ePress online publication date 25 August 2004

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that are activated in response to a wide variety of extracellularstimuli, including TGF-β1. Three distinct groups of MAPKshave been identified in mammalian cells: extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs) andp38 MAPK. Activated MAPKs target transcription factors andSmads. The serine/threonine kinase JNK promotes both Smadsignal transduction and phosphorylation of Jun. JNK activityis necessary for TGF-β-induced fibronectin expression inHT1080 fibrosarcoma cells (Hocevar et al., 1999) and thephosphorylation of paxillin, an essential signal for migration(Huang et al., 2003).

N-cadherin is a transmembrane glycoprotein composed ofextracellular domains that mediate homophilic interactionsbetween neighboring cells, predominantly via a peptidedomain containing the His-Ala-Val (HAV) amino acidsequence, which is located near the N-terminus. Thecytoplasmic domain of N-cadherin is anchored to theintracellular actin cytoskeleton by interacting with the α-, β-and γ-catenin complexes. N-cadherin has been considered apath-finding molecule involved in invasion, migration andneurite outgrowth (Doherty and Walsh, 1996; Williams et al.,2000; Hazan et al., 2000). Homophilic interaction of N-cadherin induces its rapid and strong anchoring to actinfilaments (Lambert et al., 2002). Furthermore, theestablishment of cadherin-mediated contacts has the potentialto translate adhesive homophilic recognition into changes inactin-mediated cell shape and surface adhesion, and, therefore,cellular motility and invasion (Ehrlich et al., 2002).

The paucity of information on the regulatory circuits thatcontrol the interplay between cancer cells and stromalmyofibroblasts can be attributed to the difficulty to mimick thetumor environment. In this study, we used combined in vitroapproaches to unravel the signal coming from cancer cellsaffecting the invasive potential of stromal myofibroblasts intocollagen and Matrigel. We further investigated how cancer-cell-derived TGF-β1 regulates the invasion of myofibroblasts.Our studies highlight the crucial role of JNK, and N-cadherinas signaling elements involved in TGF-β1- and wound-mediated migration and invasion.

Materials and MethodsCell cultureThe human colon cancer cell line HCT-8/E11 (Vermeulen et al., 1995)and the rat myofibroblast cell line DHD-Fib (Dimanche-Boitrel et al.,1994) were cultured as described. Primary myofibroblasts werederived from human colon cancer biopsies (Van Hoorde et al.,1999). Stromal cell lines were cultured in Dulbecco’s modifiedEagle’s medium (DMEM) (Life Technologies, Ghent, Belgium)supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillinand 100 µg/ml streptomycin (Life Technologies, Ghent, Belgium);they were scored immunocytochemically and considered asmyofibroblasts when >95% were positive for vimentin, prolyl 4-hydroxylase and α-smooth muscle actin and <5% were positive forsmooth muscle myosin and negative for cytokeratin; their identity wasroutinely controlled during the experiments. Myofibroblast cultureswere used until passage 10.

Chemicals and antibodiesThe 10-mer HAV-comprizing peptide [NH2-LRAHAVDING-amide;human (hu)N-CAD10] was used, which is homologous to the HAVregion in the amino-terminal part of human and rat N-cadherin. A

scrambled human N-cadherin 10-mer peptide (NH2-LHDANVGRIA-amide; huN-CAD10scr) served as a control. All decapeptides (AnsynthService, Roosendaal, The Netherlands) were separated by HPLC to atleast 95% purity; they were used at 200 µg/ml, the concentration atwhich huN-CAD10 or E-CAD10 decapeptides exhibited a maximaleffect on invasion and aggregation in our previous experiments(Willems et al., 1995; Noë et al., 1999; Nawrocki-Raby et al., 2003).

The following primary antibodies were used: anti-Golgi (6F4C5,kindly provided by P. Courtoy, ULB, Brussels, Belgium)(Chicheportiche et al., 1984), anti-N-cadherin (recognizing theextracellular domain of N-cadherin, clone GC-4), isotype-matchednon-immune IgG1 (MOPC-21) further indicated as control IgG,anti-α-catenin, anti-β-catenin, anti-vimentin, anti-cytokeratin, anti-smooth muscle myosin, anti-α-smooth muscle actin and anti-tubulin(Sigma, St Louis, MO); anti-human prolyl 4-hydroxylase (DAKO,Glostrup, Denmark); anti-phosphotyrosine, anti-ERK1/2, anti-phospho ERK1/2, (Santa Cruz Biotechnology, Santa Cruz, CA); anti-p38 MAPK and anti-phospho p38 MAPK (Biosource, Brussels,Belgium); anti-JNK (Upstate Biotechnology, Campro Scientific, TheNetherlands); anti-Jun, anti-phospho Jun (ser63), and anti-phosphoJun (ser73) (Cell Signaling, Westburg, The Netherlands); anti-N-cadherin (recognizing the cytoplasmic domain of N-cadherin;Takara Shuzo, Shiga, Japan); anti-cadherin-11 (Zymed, Sanbio, TheNetherlands); anti-TGF-β (R&D systems, Abingdon, UK). Secondaryantibodies coupled to horseradish peroxidase and antibodies coupledto fluorescein or biotin were obtained from Amersham PharmaciaBiotechnology (Little Chalfont, UK). An ELISA to determine humanTGF-β1 in biological fluids, bioactive recombinant (r)TGF-β1 andlatent-rTGF-β1 were from R&D systems. Phalloidin-FITC waspurchased from Sigma. SP600125 (used at 0.18-18 µM), a reversibleATP-competitive inhibitor of JNK with 300-fold greater selectivity forJNK than ERK and p38 MAPK (Bennett et al., 2001) was fromCalbiochem (La Jolla, CA). The MEK1 inhibitor PD98059 (used at25 µM) and the p38 MAPK inhibitor SB203580 (used at 5 µM), werefrom Alexis Corporation (San Diego, CA) and New England Biolabs(Beverly, MA), respectively. The following ECM proteins were used:collagen type I (Upstate Biotechnology, Lake Placid, NY) andMatrigel (Becton Dickinson and Company, Franklin Lakes, NJ).

ElectroporationHuman colon myofibroblasts were plated on Petri dishes and, at 70-80% confluency, were trypsinized and collected in an Nucleofector™certified cuvette (Amaxa GmBH, Cologne, Germany). A mixture of100 µl Nucleofector solution and 3 µg of short interference RNA(siRNA) was added. The cells were electroporated in the Nucleofectorelectroporator with the P22 specific Nucleofector program. siRNAstargeting N-cadherin (GenBank/EMBL/DDBJ accession numberNM_001792) were designed by Qiagen (Leusden, The Netherlands).Inhibition of N-cadherin expression was achieved by RNAinterference using a 1:1 mixture of the following double-strandedoligoribonucleotides: siN-CAD2 5′-AGUGGCAAGUGGCAGU-AAA-3 ′ and siN-CAD3 5′-GGAGUCAGCAGAAGUUGAA-3′ orsiN-CAD3 5′-GGAGUCAGCAGAAGUUGAA-3′ and siN-CAD4 5′-CCGUGUCUGUUACAGUUAU-3′. To verify the specificity of theknockdown effect, we used an oligonucleotide sequence with noknown mamalian target (con 5′-UUCUCCGAACGUGUCACGU-3′)as a control.

Preparation of colon-cancer-conditioned medium About 703106 HCT-8/E11 cells on 175-cm2 tissue flasks werewashed three times with 10 ml of serum-free DMEM and incubatedfor 48 hours at 37°C with 15 ml serum-free DMEM. The medium washarvested, centrifuged at 1250 g for 5 minutes at 4°C and passedthrough a 0.22 µm filter. Conditioned medium (CM) was stored at–20°C, which did not alter its biological activity for at least 6 months.

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To prevent the possibility that depletion of nutrients in cancer cell CMis responsible for any observed effect on myofibroblast cultures, weconcentrated the medium ten times by using centriprep tubes YM-10(Amicon, Millipore, Bedford, MA) and diluted the resulting solutionwith serum-free DMEM until the original volume (14 ml) wasreached.

Preparation of spheroid-myofibroblast aggregatesSix ml of a suspension containing 43105 myofibroblasts/ml wereincubated in a 50-ml Erlenmeyer flask on a Gyrotory shaker at 37°Cfor 2 days. The cultures were viewed under a macroscope equippedwith a calibrated ocular grid and spheroid cells with a diameterbetween 100 and 300 µm were collected by removing the supernatant(Bracke et al., 2001).

Spheroid invasion into collagen type IA collagen solution was prepared for three wells of a 6-well plate bymixing the following pre-cooled (at 4°C) components in a sterile 50-ml Erlenmeyer flask on melting ice: 2.1 ml of collagen type I (3.98mg/ml) (Upstate Biotechnology) 0.8 ml of MEM (103), 4.6 ml ofcalcium- and magnesium-free Hanks’ balanced salt solution pH 7.4,0.8 ml of NaHCO3 (0.25 M). We added 0.15 ml of NaOH (1 M) tomake the solution alkaline. The solution was mixed gently bypipetting, avoiding the introduction of air bubbles. The final solutionlooks purple, the phenol red pH indicator shows a pH >9. Of thissolution, 1.25 ml was poured in three wells to cover the bottom. Thisbottom layer prevents contact of spheroid cells with the plastic of thewell. We let the collagen set on a flat surface at 37°C in air that waswater-saturated and contained 10% CO2 for at least 2 hours. Thespheroid cells were suspended in the remaining collagen type Isolution, and 1.25 ml of this spheroid-cell-suspension was poured inthe three wells. After collagen gelation, 1 ml of the following media(serum-free CMHCT-8/E11, serum-free DMEM with testing products indesired concentrations, serum-free DMEM containing 106 HCT-8/E11 cells) was carefully added. We refreshed the medium every 2days, and scored invasion every day, up to day 7. Invasion was scoredby two independent observers (O.D.W. and A.V.).

Matrigel invasion assayTranswell chambers with polycarbonate membrane filters (6.5 mmdiameter, 8 µm pore size) were overlaid with a Matrigel gel. The filterwas placed in a 6-well plate which had as a bottom layer a collagengel containing either 106 HCT-8/E11 colon cancer cells or rTGF-β1(0.1-10 ng/ml) as a chemo-attractant. Collagen type I is used as asubstrate to mix cells in a 3-D gel instead of seeding them on 2-Dsubstrate; this was necessary, because we wanted to have more cellsin the small space of the lower compartment. Notice, that there is nocorrelation with the physiologic situation in which cancer cells reston the basement membrane and myofibroblasts reside in the collagentype I. To the upper compartment of the Transwell chamber, 43105

myofibroblasts were added. After 48 hours, the myofibroblasts thathad invaded to the membrane’s undersurface were stained with 0.4mg/ml 4′,6-diamino-2-phenylindole (DAPI) (Sigma) and werecounted on 50 fields/filter (Hall and Brooks, 2001).

Wound healing migration assayHuman colon myofibroblasts were grown in 6-well tissue culturedishes until confluent. Cultures were incubated for 3 minutes withCa2+- and Mg2+-free phosphate-buffered saline (PBS) pH 7.4. Afterremoval of PBS, confluent monolayers were wounded with a sterilizedrazor blade to remove part of the cell monolayer sheet (André et al.,1999). Wounded monolayers were washed three times with serum-free DMEM to remove dead cells. Myofibroblast migration occurred

in the presence of 1 ml DMEM with or without monoclonal antibody(mAb) or peptides or pharmacological inhibitors and was assessedafter 24 hours by counting the number of intersections at differentdistances. Starting point of migration was revealed by a small incisionin the culture dish that was made with the razor blade.

Polarization and Golgi-complex reorientationConfluent DHD-Fib myofibroblasts grown on glass coverslips werewounded with a plastic pipet tip. After 8 hours of migration, cells werefixed in 4% paraformaldehyde diluted in PBS, blocked in PBScontaining 50 mM NH4Cl, permeabilized in 0.2% Triton X-100 inPBS, and stained with a rat specific anti-Golgi mAb. The orientationof the Golgi complex was assessed as described previously (Nobesand Hall, 1999). The significance of the inhibition of the Golgi-complex reorientation was determined by the Student’s t-test(P<0.05). Samples were viewed by fluorescence microscopy (Dialux20) (Leitz, Wetzlar, Germany) and photographed with the LeitzOrthomat E camera system.

F-actin staining and numerical evaluation of filopodiaConfluent human colon myofibroblasts grown on glass coverslipswere wounded with a plastic pipet tip. After 8 hours of migration,cells were fixed in 4% paraformaldehyde with 1% glutaraldehyde inPBS (to preserve fine actin structures such as filopodia), blocked in50 mM NH4Cl in PBS, permeabilized in 0.2% Triton X-100 in PBS,and stained with phalloidin-FITC. The number of filopodia werecounted and averaged from 50 myofibroblasts in each condition.Samples were viewed by the Axiovert 200 microscope (Carl Zeiss,Göttingen, Germany).

Protein analysisTotal cell lysis, immunoprecipitation and western blot analyses wereperformed as previously described (De Wever et al., 2004). Scanningdensitometry was carried out with the Quantity One program (Bio-Rad). Quantitative determination of human TGF-β1 in CM wasperformed with ELISAs following the manufacturer’s instructions(R&D systems).

Statistical analysisAll functional assays were performed at least in triplicate unlessotherwise indicated. All values are give the means±s.d. or 95%confidence interval. Significance of Matrigel invasion was determinedwith Statview (Brainpower; Calabasas, CA) on an Apple Macintoshsystem. Comparisons were performed using an unpaired Student’s t-test. P<0.01 was considered to indicate a significant difference(indicated by *).

ResultsColon-cancer-cell-derived TGF-β is necessary andsufficient to stimulate invasion of myofibroblasts intocollagen type I and Matrigel in vitroAfter 48 hours, spheroid myofibroblasts that were suspendedin collagen type I gel, a major constituent of the stromal ECM,were still non-invasive. By contrast, when HCT-8/E11 coloncancer cells were co-cultured on top of the collagen or whentreated with CM of HCT-8/E11 (CMHCT-8/E11), spheroidmyofibroblasts invade collagen type I in a stellate patternradiating from the spheroid (Fig. 1A). Cell density at thesurface of the spheroid appeared to decrease, suggesting that aresident population of myofibroblast spheroid cells invades the

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gel, rather than only new cells generated by cell proliferation.When myofibroblasts invaded the collagen, a reorganization ofthe collagen into thick collagen fibers (straps) occurred thataligned parallel to the axes between spheroids (data notshown). This phenomenon is explained by the traction exertedby the invasive myofibroblasts that combine to realign collagenfibers into a ligament-like ‘strap’ on the axis between thespheroids (Sawhney and Howard, 2002). Invasion andformation of the strap was independent of the distance betweenthe spheroids (distances less than 5 mm). Since HCT-8/E11cells produced between 200 and 500 pg/ml TGF-β1 in the CM,we next examined the role of this cytokine in the invasion ofthe myofibroblasts. The addition of neutralizing TGF-β mAb

but not control IgG1 inhibits the invasion-stimulating effect ofHCT-8/E11 colon cancer cells and their CM. To confirmthat colon-cancer-cell-derived TGF-β1 plays a role inmyofibroblast invasion we used recombinant (r)TGF-β1 ineither its latent or its bioactive form. After 48 hours, both latentand bioactive rTGF-β1 stimulated myofibroblast spheroidinvasion, as observed for HCT-8/E11 cells and CMHCT-8/E11

(data not shown). We cultured these myofibroblasts further byexchanging the medium on day 3 and 5. After 7 days, untreatedspheroid myofibroblasts were invasive (Fig. 1B). Thisphenotype did not depend on autocrine TGF-β1 secretion bymyofibroblasts as evidenced by using TGF-β neutralizingmAb. Long-term invasion of myofibroblast spheroids wasfurther increased by both latent and bioactive rTGF-β1,resulting in a remarkable stellate pattern of invasion intocollagen type I. The larger magnification in Fig. 1B shows thatmyofibroblasts originating from two different myofibroblastspheroids establish cell-cell contacts via dendritic-likeextensions.

Next, we examined the behavior of single myofibroblastsseeded on top of Matrigel-coated filters, to mimic invasionthrough the basement membrane. The lower compartmentcontained a collagen gel mixed with 106 HCT-8/E11 cellsalone, combined with a neutralizing TGF-β mAb, or withcontrol IgG1. As shown in Fig. 1C, HCT-8/E11 colon cancercells significantly stimulate the invasion of myofibroblasts intoMatrigel after 72 hours in a TGF-β-dependent way. The roleof TGF-β in invasion was confirmed by embedding rTGF-β1 in the collagen gels of the lower compartment. Atconcentrations ranging from 0.1 to 10 ng/ml, rTGF-β1stimulated the invasion of myofibroblasts into the Matrigel ina dose-dependent manner.

N-cadherin is essential for TGF-β1 stimulated invasionof myofibroblastsWe have previously demonstrated that N-cadherin is expressedby human colon myofibroblasts at the tips of contactingfilopodia (Van Hoorde et al., 1999). In the Matrigel invasionassay, a single treatment by the N-cadherin-neutralizing GC-4mAb was tested at concentrations ranging from 20 to 160µg/ml (Fig. 2A). Invasion of the myofibroblasts was reducedby 70% in the presence of 80-160 µg/ml GC-4 mAb. Since

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Fig. 1.Colon-cancer-cell-derived TGF-β is necessary and sufficientto stimulate invasion of myofibroblasts into collagen type I andMatrigel. (A) Myofibroblast spheroids were embedded in a collagentype I gel on top of which serum-free DMEM was added without(untreated), with 106 HCT-8/E11 cells or with CMHCT-8/E11cells, inthe presence of control IgG1 or of neutralizing TGF-β mAb.(B) Myofibroblast spheroids in collagen type I were cultured for 7days with the indicated treatments. The medium was refreshed ondays 3 and 5. A representative phase-contrast micrograph is shownfor each condition, taken after 48 hours (panel A) or after 7 days(panel B). Experiments in A and B were repeated at least three timesand had similar results. Scale bars, 100 µm. (C) Single-cellmyofibroblasts at a density of 43105 were seeded upon a Matrigelcoated filter. HCT-8/E11 cells at a density of106 were seeded orrTGF-β1 was placed in the lower compartment in the presence ofcontrol IgG1 or neutralizing TGF-β mAb. Bars indicate means ofthree results ± s.d. Asterisks show statistically significant differencefrom untreated control.

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control IgG1 showed no effect, this experiment stronglysuggests a specific role for N-cadherin in rTGF-β1-stimulatedmyofibroblast invasion. With the GC-4 mAb (80 µg/ml), asimilar level of inhibition was seen with HCT-8/E11 coloncancer cells as stimulators of invasion. Fourty-eight-hourinvasion of myofibroblasts in collagen type I stimulated byCMHCT-8/E11was also inhibited by a single dose of GC-4 mAb.Inhibition was also achieved in a 7-day assay with rTGF-β1 asa stimulant, provided the GC-4 mAb was added at day 1, 3 and5. Here, also the control IgG1 had no effect (Fig. 2B) andspontaneous invasion of myofibroblasts was also sensitive toinhibition with the N-cadherin neutralizing GC-4 mAb.

To further strengthen the idea that N-cadherin expression isessential in TGF-β-stimulated invasion, we knocked down N-cadherin expression in human colon myofibroblasts usingsiRNA. For this, myofibroblasts were electroporated with two

double-stranded oligonucleotides derived from two distinctregions of N-cadherin cDNA. Myofibroblasts electroporatedeither with control oligonucleotide or without were used ascontrols. As revealed by western blot analysis 2 and 4 daysafter electroporation, two paired combinations of siN-cadherinoligonucleotides (siN-CAD2 with siN-CAD3 and siN-CAD3with siN-CAD4) efficiently reduced N-cadherin (up to 97%)but not cadherin-11 expression (Fig. 2C). Tubulin expressionwas used as a control for equal protein loading. Electroporatedspheroid myofibroblasts were subjected to the spheroid-cell-collagen-invasion-assay in the presence of rTGF-β1. As shownin Fig. 2D, inhibition of N-cadherin expression by siRNAsubstantially inhibited TGF-β-stimulated invasion. We thusconclude that, N-cadherin expression is necessary to observethe pro-invasive effect of TGF-β, at least in the spheroid-cell-invasion-assay and in the cell system analyzed.

Fig. 2.Role of N-cadherin in rTGF-β1-stimulated invasion of myofibroblasts. (A) 43105 single myofibroblasts were seeded upon a Matrigelcoated filter with rTGF-β1 or HCT-8/E11 cells in the lower compartment to stimulate invasion. N-cadherin was neutralized by the GC-4 mAbwhich was added together with the myofibroblasts. Bars indicate means of three results ± s.d. Asterisks show statistically significant inhibitionof invasion. (B) Myofibroblast spheroids cultured in collagen type I for 48 hours in the presence of HCT-8/E11 cells, and cultured in collagentype I for 7 days untreated or treated with rTGF-β1, were challenged with control IgG1 (80 µg/ml) or GC-4 mAb (80 µg/ml). In the 7-dayexperiment, medium was exchanged on day 3 and 5. A representative phase-contrast micrograph is shown for each condition. Scale bar, 100µm. (C) Western blot, showing the effect of combinations of siN-CAD oligonucleotides on N-cadherin expression and cadherin-11 expressionin myofibroblasts. Tubulin was used as loading control. (D) Spheroid myofibroblasts of cells that had been electroporated with siN-CADoligonucleotides and cultured in collagen type I for 2 or 4 days in the presence of rTGF-β1. con, control oligonucleotide. Scale bar, 100 µm.

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We next investigated the relationship between TGF-β1 andthe expression of N-cadherin in human colon myofibroblasts.As shown in Fig. 3A, the N-cadherin protein is upregulated byrTGF-β1 in a dose-dependent manner, whereas the expressionlevels of the molecular partners of N-cadherin, such as β-catenin and α-SMA, remain unchanged upon rTGF-β1treatment. The stoechiometry of the molecular component ofthe N-cadherin-β-catenin-α-SMA complex was then analyzedin control and rTGF-β1-treated myofibroblasts by meansof immunoprecipitation (Fig. 3B). Approximately 2.5-foldhigher amounts of β-catenin and α-SMA were coprecipitatedwith antibodies recognizing N-cadherin in rTGF-β1-treatedmyofibroblast cultures, as compared with controls.

Role of MAPK in N-cadherin-dependent myofibroblastinvasion stimulated by colon-cancer-cell-derived TGF-β1TGF-β1 has been reported to signal through the MAPK

pathways (Mulder, 2000). We therefore, examined the possiblecontribution of the JNK, p38 and MEK1 as signalingcomponents in TGF-β stimulated invasion of myofibroblastsinto Matrigel. As shown in Fig. 4A, the JNK inhibitorSP600125 inhibited rTGF-β1-stimulated invasion ofmyofibroblasts in a dose-dependent manner, whereas thepharmacological inhibitors of MEK1 (PD98059 and UO126),and p38 (SB203580) were ineffective during the 72-hour assay.Similarly, only the JNK inhibitor SP600125 was effective toantagonize rTGF-β-stimulated myofibroblast invasion in thelong-term spheroid-invasion-assay performed for 7 days, usingcollagen type I as an ECM substrate (Fig. 4B). Interestingly,none of the MAPK inhibitors affected the baseline levels ofspontaneous invasion in untreated myofibroblasts (Fig. 4B).We performed experiments to determine whether JNK isinvolved in N-cadherin upregulation stimulated by rTGF-β1 inmyofibroblasts. As shown in Fig. 4C, only SP600125 reversedrTGF-β1 stimulated N-cadherin expression at concentrationsranging from 0.18 to 18 µM. The other MAPK inhibitorsSB203580, PD98059 and UO126 were ineffective atconcentrations that prevented p38 and MEK1 activity in otherstudies. None of the MAPK inhibitors had an effect on N-cadherin expression in untreated myofibroblasts (data notshown).

To validate our data obtained with the pharmacologicalinhibitors, we next examined the direct action of rTGF-β1 onthe activation status of the MAPKs in myofibroblasts. JNKactivity was determined by investigating the phosphorylationlevel of its direct and specific substrate Jun. We found that theaddition of rTGF-β1 led to phosphorylation of Jun at serineresidues 63 and 73, as shown in the western analysis withphosphospecific antibodies (Fig. 5). These activated(phosphorylated) forms of Jun, were increased 1.6-fold and 2-fold after rTGF-β1 treatment for 1 hour and 2 hours,respectively. By contrast, phosphorylation levels of ERK1/2(substrate of MEK-1) and p38 were either not affected or evendecreased following treatment with rTGF-β1.

Role of N-cadherin in the migratory phenotype ofmyofibroblastsSince N-cadherin plays a pivotal role in neurite outgrowth andin migration and invasion (Doherty and Walsh, 1996; Hazanet al., 2000; Williams et al., 2000), we investigated its role inthe migratory phenotype of myofibroblasts via a woundhealing assay. Confluent myofibroblast cultures were serum-starved for a minimum of 24 hours to establish quiescence,such that the presence of cells in the wounded area wouldowing to cell motility rather than cell proliferation.Myofibroblasts moved perpendicularly to the wound, in anirregular shaped front and not as a compact front like epithelialcells do (Suriano et al., 2003). Counting the number ofintersections of myofibroblasts – with marks at variousdistances from the original wound margin – showed that theN-cadherin-neutralizing GC-4 mAb and the decapeptide huN-CAD10, but not the control IgG1 or the control huN-CAD10scr

peptide, significantly inhibit the migration of myofibroblasts(Fig. 6A). Previous studies have shown that HAV peptides caninterfere with several cadherin-dependent processes, includingneurite outgrowth (Doherty and Walsh, 1996), hippocampallong-term potentiation (Tang et al., 1998), osteoclast

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Fig. 3.Effect of rTGF-β1 on N-cadherin expression and theorganization of N-cadherin–catenin–α-SMA complex inmyofibroblasts. (A) Treatment of myofibroblasts with rTGF-β1 for 7days stimulates N-cadherin protein expression in a dose-dependentmanner. Expression levels of β-ctn and α-SMA proteins are notsignificantly changed. Tubulin protein expression is used as loadingcontrol. (B) Immunoprecipitation (IP) on lysates of myofibroblaststreated or not for 7 days with rTGF-β1 (1 ng/ml) were performed toinvestigate the association of N-cadherin with β-catenin and α-SMA.Total lysates were stained for N-cadherin and tubulin (loadingcontrol).

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formation (Mbalaviele et al., 1995), myoblast fusion (Mege etal., 1992) and cancer invasion (Noë et al., 1999; Nawrocki-Raby et al., 2003). To confirm the role of N-cadherin inmigration, we used the siRNA knock-down method. As shownin Fig. 6B, silencing of N-cadherin expression significantlyinhibits myofibroblast migration.

F-actin staining showed filopodia-like cellular extensionsand actin stress fibers that were oriented towards the woundin control IgG1-treated myofibroblast cultures (Fig. 7A,B).The GC-4 mAb neutralizing N-cadherin, significantly reducedthe number of filopodia (Fig. 7A,B). A comparable decreasein filopodia formation was observed in myofibroblasts inwhich N-cadherin expression was knocked-down by siRNA(Fig. 7A,C). N-cadherin is extensively expressed at contacting

filopodia in control electroporated myofibroblasts. Bycontrast, myofibroblasts electroporated with siN-CADoligonucleotides do not show N-cadherin staining at all (Fig.7C).

We determined myofibroblast polarization by phase-contrastmicroscopy and analyzed the Golgi-complex orientationduring wound healing, using a specific and efficient mAb thatrecognized Golgi proteins originating from rat (Chicheporticheet al., 1984). Therefore, experiments were performed in N-cadherin-positive rat DHD-Fib myofibroblasts which migrateunidirectionally and develop cell protrusions perpendicular tothe wound that become visible after 4-6 hours after woundingand are prominent after 8 hours (Fig. 8A). The huN-CAD10,bearing the same HAV-flanking amino acids as the rat N-cadherin but not the same as its scrambled control huN-CAD10scr, caused loss of polarity. This was evidenced by theloss of unidirectional migration and the formation of cellprotrusions in a random direction. The GC-4 mAb, notrecognizing rat N-cadherin in western blots orimmunocytochemistry (data not shown), had no effect. Cellsalong the edge of monolayer wounds were consideredpolarized when the Golgi-complex was localized at the wound-side of the nucleus, (Nobes and Hall, 1999). We found thatsignificantly less huN-CAD10-treated cells (relative to huN-CAD10scr-treated cells) were polarized. In fact, polarization ofhuN-CAD10-treated cells at the edge of wounds was notsignificantly different from the random distribution observedin a confluent monolayer (Fig. 8B).

Role of MAPKs in migratory phenotype ofmyofibroblastsMAPKs are implicated in TGF-β stimulatedinvasion of myofibroblasts and we weretherefore interested whether MAPKs arealso implicated in wound-healing-inducedmigration of myofibroblasts (Fig. 9). The JNKinhibitor SP600125 inhibited wound-healing-induced migration of myofibroblasts in a

Fig. 4.Effect of MAPK inhibitors on rTGF-β1-stimulated N-cadherin expression and N-cadherindependent myofibroblast invasion. (A) 43105

single myofibroblasts were seeded on a Matrigelcoated filter, with rTGF-β1 in the lowercompartment to stimulate invasion. MAPKinhibitors were added in the upper compartmenttogether with the myofibroblasts. Incubation wasfor 72 hours. Bars indicate the mean number ofcells at the lower side of the filter (means of threeresults ± s.d.). The asterisk shows statisticallysignificant inhibition of invasion.(B) Myofibroblast spheroids were cultured for 7days in collagen gels in presence or not of rTGF-β1 and MAPK inhibitors. Medium was exchangedon day 3 and 5. A representative micrograph foreach condition is shown. Scale bars, 100 µm.(C) Western blot analysis of lysates frommyofibroblasts treated for 7 days with rTGF-β1 inpresence or not of MAPK inhibitors. The upperpart of the blot was probed for N-cadherin, thelower part for tubulin.

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dose-dependent manner. By contrast, the pharmacologicalinhibitors of MEK1 (PD98059) and p38 (SB203580) wereineffective during the 24-hour assay, as also observed in theinvasion studies (Fig. 4A).

DiscussionThere is accumulating evidence that non-cancer cells in thestroma of solid tumors provide critical information for cancercell growth, ectopic survival and invasion (De Wever andMareel, 2003). The interaction between the epithelial cancercells and the stromal compartment creates a local heterotypicinvasion field in which it is not always clear ‘who is invadingwhom’ (Liotta and Kohn, 2001). The importance of suchcancer-stroma interactions are becoming more and morerecognized, and this article describes a potential newmechanism how stromal myofibroblasts invade in vitro uponstimulation by colon-cancer-cell-derived factors. One can

argue that both cancer cells and stromal myofibroblasts areinterdependent for their migratory behavior, and can formcoordinated invasive layers during local invasion and tumorspreading. The 3-D spheroid-myofibroblast-collagen-type-I-assay enabled us to demonstrate myofibroblast invasion in atypical stellate pattern, radiating from the spheroid cells uponcontact with factors derived from cancer cells. Cancer cellsstimulate invasion of myofibroblasts and attract them to passMatrigel, which is a mimic of the basement membrane. Thus,myofibroblasts might also perform counter-current invasion,from the stroma into the epithelial cancer compartment,providing roads for cancer cell invasion, as has been describedfor inflammatory cells and immunocytes (Opdenakker and VanDamme, 1992). Alternatively, myofibroblasts might dock closeto accumulations of cancer cells and locally provide them withpro-invasive signals (De Wever et al., 2004).

Our in vitro ecosystem, consisting of colon cancer cells,ECM barriers such as collagen type I or Matrigel, and host cellssuch as myofibroblasts, does not perfectly mimic the naturaltumor tissue. It might, however, provide information about thereciprocal effects of one cell type upon another. In vitromethods widely make use of the 3-D collagen type I andMatrigel invasion assays to measure the ability of cells to attachto the matrix and migrate into it. Wound-healing-migration isa 2-D motility assay where myofibroblasts migrate as anirregularly shaped front towards the wound, a process thatdiffers from invasion into matrices. We used the wound-healing-migration assay as a tool to study the migrationresponse of myofibroblasts independently of cancer-cell-derived stimuli. Furthermore, 2-D analysis is an easy way toget information about defects in migratory cues such asfilopodia formation, Golgi-complex reorientation and polarity.

We have shown that TGF-β1 derived from colon cancer cellswas a necessary and sufficient signal to stimulate myofibroblastinvasion in collagen type I and Matrigel. Cancer cells ofdifferent origins produce TGF-β and are resistant to its growth-inhibitory effects, in contrast to their benign precursor cells,which are sensitive to these effects (Derynck et al., 2001).TGF-β accumulates in the ECM where it is sequestered andcan be processed into a bioactive ligand by myofibroblast-derived MMP-2 and MMP-9 (Yu et al., 2000). Accordingly,active TGF-β stimulates myofibroblasts to form a dendriticnetwork of extensions in the spheroid myofibroblast invasionassay.

We have shown by using three independent approaches that,N-cadherin plays a crucial role in TGF-β-stimulated invasionand wound-induced migration. Also, N-cadherin is implicatedin invasion of untreated myofibroblasts. This suggests thatTGF-β aggravates the invasion-properties of a spontaneouslyinvasive cell type by upregulating N-cadherin proteinexpression. Precipitation studies revealed that N-cadherin iscomplexed with β-ctn and the α-SMA cytoskeleton, suggestingthat N-cadherin might stimulate invasion by forming adherensjunctions linked to the cytoskeleton (Aberle et al., 1996). Theexpression of cadherin-11 in myofibroblasts is not affected bysilencing N-cadherin expression. This suggests that the siRNAapproach is very specific and that a reduction in N-cadherinprotein expression does not necessarily lead to changes incadherin-11 expression. Downregulation of one cadherin mightlead to the upregulation of another cadherin that functionallysubstitutes or differs from the one that is downregulated. The

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Fig. 5.Kinetics of rTGF-β1 activation of p38, ERK1/2 and JNKMAPK in myofibroblasts. Western analysis of lysates frommyofibroblasts that had been treated with rTGF-β1 for differenttimes (h=hours) and probed for the phosphorylated forms ofp38MAPK (p38MAPK-P), ERK1/2 (ERK-P), and the JNK substrateJun, mainly phosphorylated ser63 or ser73 (Jun-P63 and Jun-P73).Blots were stripped and re-probed for total amounts of p38MAPK,ERK1/2 and Jun and tubulin. The relative intensity of total ERK1/2,Jun and p38MAPK was normalized to tubulin levels. Consequently,the relative intensity of phosphorylated forms of ERK1/2, Jun andp38MAPK was normalized to total values of ERK, Jun andp38MAPK.

4699N-cadherin dependent myofibroblast invasion

latter was shown by Islam et al. in invasive squamouscarcinoma cells, where downregulation of N-cadherinexpression by an antisense construct increases the expressionof E-cadherin, leading to reduced invasion (Islam et al., 1996).

Intercellular contacts between fibroblasts, involvinginteractions of cadherins, actin and myosin, generate the forcesrequired for wound closure (Adams and Nelson, 1998).Therefore, N-cadherin has been implicated in intercellularmechanotransduction by activating Ca2+-permeable channels,consequently triggering the induction of actin assembly (Ko etal., 2001). The SK-N-SH neuroblastoma cell line, whichexpresses N-cadherin at the adherens junctions, is invasive intocollagen type I (Van Aken et al., 2003). Also, N-cadherin isexpressed in a punctuate pattern over the entire surface ofaxonal growth cones during neurite outgrowth (Riehl et al.,1996) and in Y79 retinoblastoma cells was shown to be

invasive into collagen type I (Van Aken et al., 2002). In allcases, invasion and neurite outgrowth depended on N-cadherinas evidenced by using the GC-4 mAb. These findings suggestthat the pro-invasive activity of N-cadherin cannot solely beattributed to its localization in the membrane and/or associationwith the cytoskeleton. As well the extracellular domain, thejuxtamembrane and the β-catenin binding domain of N-cadherin are involved in pro-invasive signaling pathways. First,the N-cadherin extracellular repeat 4 interacts with thefibroblast growth factor receptor and mediates increasedmotility in epithelial breast cancer cell lines (Kim et al., 2000).Second, the juxtamembrane domain which binds p120-catenin,is implicated in N-cadherin-dependent neurite outgrowth(Riehl et al., 1996). Third, the tyrosine phosphatase 1B mightfunction as a regulatory switch, controlling cadherin functionby dephosphorylating β-catenin and thereby maintaining cells

in an adhesion-competent state (Balsamoet al., 1998). We have shown thatneutralization of N-cadherin by the GC-4mAb or the huN-CAD10 peptide inhibitswound healing migration. Also, specificN-cadherin knock-down decreaseswound-healing migration. A scratchthrough the monolayer initiates anincrease in Cdc42-activity leading todirectional movement of themyofibroblasts (O.D.W., W.W., A.V.,N.B., M.B., C.G., E.B. and M.M.,unpublished data). Cell migration incontrol conditions is initiated by a changein morphology of myofibroblasts at thewound edge; they extend a longprotrusion into the space, generating anelongated shape with filopodia sensingthe environment. Movement becamedisoriented and leading edge cells losttheir filopodia after N-cadherin wasneutralized following treatment withantibody or peptide or after silencing.These findings suggest that homophilicbinding of N-cadherin at the trailing edgeof cells might provide cues in defining thedirection of invasion and migration. N-cadherin may also promote invasion andmigration by forming labile cellularadhesions that facilitate dynamic cell-cell

Fig. 6.Role of N-cadherin in wound-healingmigration of myofibroblasts. (A-B) Confluentmonolayers of serum-starved myofibroblastswere wounded with a razor blade. Migrationof myofibroblasts was quantified as numberof intersections/microscopic field at differentdistances after a 24-hour treatment with (A)N-cadherin neutralizing mAb or peptide and(B) electroporation in the presence of N-cadherin siRNA. Of the different conditionstested in A, representative pictures weretaken. The arrowhead indicates the scratch(time 0) made by the razor blade. Bar, 100µm.

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Fig. 7.Role of N-cadherin in filopodia formation of myofibroblasts.(A) Bars show mean numbers of filopodia revealed by F-actinstaining and counted on 50 myofibroblasts at the migration front(means of three results ± s.d.); asterisks indicated statisticallysignificant difference. (B) Representative pictures of F-actin-stainingof myofibroblasts at the migration front of cultures treated withcontrol IgG1 or the GC-4 mAb. Bar, 25 µm (C) Representativepictures of F-actin and N-cadherin-staining at the migration front ofmyofibroblasts electroporated with control (con), or siN-CAD3 andsiN-CAD4 oligonucleotides. Bar, 25 µm.

Fig. 8.Role of N-cadherin in the polarity of myofibroblasts.(A) Representative phase-contrast micrographs of DHD-Fibmyofibroblasts, taken 8 hours after wounding. Cells were treated withhuN-CAD10scror huN-CAD10 peptide. Loss of polarity is indicated byloss of unidirectional migration. Bars, 50 µm. (B) (upper panel) Golgi-complex immunostaining of DHD-Fib myofibroblasts in confluentconditions or 8 hours after wounding. Direction of movement is shownby arrows; (lower panel) bars show percentage of cells with their Golgicomplex facing the wound for 50 cells of the first 2 front rows or atrandom in wounded or confluent monolayers. Bar, 25 µm.

4701N-cadherin dependent myofibroblast invasion

interactions. Labile contacts would allow the release of N-cadherin at some adherens complexes while propulsive forcesare being generated at others. The engagement of cadherinsprobably has complex consequences for Rho-GTPase signalingthat might be influenced by the cellular and extracellularcontext and type of cadherin (Goodwin et al., 2003; Kovacs etal., 2002; Noren et al., 2003). Furthermore, cell polarity ischaracterized by (1) a perpendicular orientation of theprotrusion and the cell migration to the wound, and, (2) areorientation of the Golgi complex towards direction ofmigration. Although the relatively long time course suggeststhat Golgi-complex realignment is not essential for movement,it might facilitate movement and it clearly indicates thedevelopment of a polarized morphology. Cdc42 was shown toregulate filopodia formation and polarity in fibroblasts in an invitro wound assay (Nobes and Hall, 1999).

It is currently not exactly known, how N-cadherinexpression is regulated by TGF-β1. In mouse mammaryepithelial cells (NMuMG), TGF-β1 stimulates epithelial-to-mesenchymal differentiation and N-cadherin expressionthrough a RhoA-dependent pathway (Bhowmick et al., 2001).We have shown that upregulation of N-cadherin expressionby rTGF-β1 occurs via JNK activation with coincidentmyofibroblast invasion into collagen type I and Matrigel. BothMEK-1 or p38 MAPK blockade are inefficient in inhibitingTGF-β1-stimulated invasion into collagen type I and Matrigel.Accordingly, rTGF-β1 stimulates JNK activity causing JNK tophosphorylate the JNK substrate Jun at serine residues 63 and73. Jun is a member of the AP1 family of transcription factors.Interestingly, analysis of the chicken N-cadherin promoterrevealed several consensus Sp1 and AP2 elements, and an AP1binding site (Li et al., 1997). Alternatively, the stimulation ofJNK by TGF-β might regulate Smad activation and thereforeSmad dependent transcriptional activity (Engel et al., 1999). In

that way, TGF-β-dependent N-cadherin expression might bethe result of a convergence between Smad and JNK signalingpathways. Basal JNK, MEK1 or p38MAPK activity is notinvolved in spontaneous long-term spheroid-cell-invasion ofmyofibroblasts. However, JNK activity but not MEK1 orp38MAPK activity is necessary for wound-healing-inducedmigration of myofibroblasts. Consistent with this are datashowing that, JNK activity correlates with cell migration inseveral systems (Abassi and Vuori, 2002; Huynh-Do et al.,2002; Huang et al., 2003) and JNK is a common downstreamtarget of multiple signaling pathways that control cellmovement, such as Rac and Src (Minden et al., 1995; Oktay etal., 1999).

In malignant tumors, interactive cross signaling betweencancer cells and stromal host cells influences the invasivebehavior of both cell populations, leading to concertedinvasion. Neutralization of N-cadherin activity by severalindependent approaches inhibited the invasion response ofmyofibroblasts. Furthermore, TGF-β stimulated N-cadherinprotein expression via a JNK-dependent pathway inmyofibroblasts. By using an in vitro wound-healing assay wedemonstrated that neutralization of N-cadherin or the specificN-cadherin knock-down inhibit Golgi-complex reorientation,filopodia formation, polarization and finally wound-healing-migration. From a therapeutical point of view, it is interestingthat N-cadherin which is expressed by invasive cancer cells andmyofibroblasts might be targeted by one drug; and stablecyclic-peptide-antagonists of N-cadherin may be used toinhibit N-cadherin activity (Williams et al., 2000).

D. Vandekerkhove and G. De Bruyne are gratefully acknowledgedfor technical assistance, and J. Roels for preparation of theillustrations. This work was funded by ‘Kom Op Tegen Kanker, FWO-Vlaanderen’ (Brussels, Belgium). O.D.W. was supported by afellowship from the ‘Stichting Emmanuel van der Schueren’. Wethank Jean Willems (Kortrijk, Belgium) and P. Courtoy (Brussels,Belgium) for providing reagents.

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