Hepatocyte growth factor induces colonic cancer cell invasiveness via enhanced motility and protease overproduction. Evidence for PI3 kinase and PKC involvement

Stéphanie Kermorgant, Thomas Aparicio, Valérie Dessirier, Miguel J.M. Lewin and Thérèse Lehy,1

Unités INSERM U 10 and U 410, IFR Cellules Epithéliales, Faculté de Médecine Xavier Bichat, 16 Rue Henri Huchard, BP 416, 75870 Paris, Cedex 18, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumour progression to the metastatic phenotype is mainly dependent on tumour cell invasiveness. Cell migration is a crucial step in this process. Here we investigate the effect of hepatocyte growth factor (HGF) on the induction of in vitro invasiveness of poorly aggressive Caco-2 colonic cancer epithelial cells. Invasion assays through a Matrigel barrier were performed. Proteases were assessed by zymography, reverse transcription–polymerase chain reaction and immunoblotting. Caco-2 cells were found to express HGF receptor but not HGF and to secrete several proteases, namely matrix metalloproteinase-1 (MMP-1), MMP-2, possibly MMP-9 and urokinase plasminogen activator (uPA). Exogenous HGF promoted invasiveness of Caco-2 cells through an artificial basement membrane matrix and enhanced their production of proteases. In addition, analyses of media at the end of invasion assays indicated that anti-HGF antibody inhibited protease production in parallel with cell invasion. The involvement of proteases in the HGF-induced invasion process was further investigated using either a synthetic general MMP inhibitor or neutralizing antibodies against MMPs or uPA. All components significantly inhibited HGF-promoted cell invasion. Moreover, specific inhibitors of PKC{alpha}/ß1 and PI3 kinase also decreased both HGF-promoted cell invasion and protease expression in invasion assay media. Thus, our findings provide evidence that the process of HGF-activated invasiveness of Caco-2 cells involves PI3 kinase and PKC and results from close association of two events, stimulation of cell motile activity and concomitant overproduction of proteases, which permits cell migration through a degraded extracellular matrix.

Abbreviations: HGF, hepatocyte growth factor; MMP, matrix metalloproteinase; PKC, proteine kinase C; uPA, urokinase plasminogen activator.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumoural invasion and metastatic processes require the loss of cell adhesion, degradation of the extracellular matrix as well as the basement membrane and concomitant induction of cell movement (1). Various molecules promoting motility, such as proteases and growth factors, have been implicated in these processes.

Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are two major classes of proteases that play a role in cancer evolution. Matrix metalloproteinases are known to degrade extracellular matrix proteins that constitute connective tissues. They are classified into four groups on the basis of sequence homology and substrate specificity: (i) collagenases including interstitial collagenase or MMP-1; (ii) gelatinases including gelatinase A or MMP-2 and gelatinase B or MMP-9; (iii) stromelysins and (iv) membrane-type metalloproteinases. Expression of some MMPs has been reported in colorectal cancers (24). Matrix metalloproteinase regulation is a complex mechanism involving multiple molecules, among them growth factors. Thus, hepatocyte growth factor (HGF) has been reported to stimulate the production of MMPs in some colon cancer cell lines (5).

Hepatocyte growth factor is a mesenchymal-derived cytokine. Its receptor, c-Met, is a transmembrane tyrosine kinase encoded by the c-met proto-oncogene. HGF and c-Met are found in normal human digestive tissues (6,7) and c-Met is overexpressed in gastric, colonic and pancreatic cancers (810). Hepatocyte growth factor induces pleiotropic biological activities. Particularly, it exerts in vitro a motogenic effect on various target cells, which is displayed either by cell scattering, simple locomotion, notably migration during the wound repair process of cultured cells, or invasiveness through the extracellular matrix. Interestingly, these three types of motile events are induced by HGF in some epithelial cell lines of the digestive tract (5,1119), indicating that HGF may be a major factor in healing of digestive tissues injury and gastrointestinal cancer progression. The mechanisms by which HGF triggers motility are dependent on the activation of several intracellular molecular pathways. The role of phosphatidyl inositol triphosphate (PI3) kinase (20), Ras (21), 41/43 mitogen-activated-protein kinase (22) in the HGF-mediated cell scattering activity has been established. HGF-mediated cell invasion requires the activation of c-Src kinase (23), that of PI3 kinase and/or Grb2 (19,24).

Despite the fact that HGF is able to stimulate protease production and cell invasion, it is not clear whether both these two processes are induced in all target cells and whether they are linked together. Some of the overexpressed proteases may not play a role in invasiveness. Investigations aimed at establishing a direct relationship between these two processes are just emerging and yet none have concerned gastrointestinal tumour cells. On the other hand, since a recent work has shown the implication of PKC{alpha} in intestinal cell invasion (25), it is possible that the HGF-invasive signal is mediated by this molecule.

The aims of the present study were therefore to investigate further the capability of HGF to induce motogenesis and invasiveness of the Caco-2 human colon cancer cell line reputed as poorly aggressive (26), and to analyse the mechanisms accounting for these potential HGF effects. Our results show (i) that HGF activates cell motility through a Matrigel barrier in an invasion assay model, (ii) that HGF promotion of cell invasiveness is closely associated with increased production of proteases in media in the course of the invasion process and (iii) the involvement in PI3 kinase and PKC in these two responses to HGF.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell line and culture
Caco-2 cells, derived from a human colon carcinoma, passages 79–86, were cultured in 25 cm2 plastic flasks (Corning-Costar, Cambridge, MA) at 37°C in a humidified atmosphere containing 5% CO2. They were grown in DMEM medium (Gibco BRL, Eragny, France) supplemented with 2 mM glutamine, 1% non-essential amino acids, 20% fetal calf serum (FCS) (Gibco BRL), 100 U/ml penicillin and 100 µg/ml streptomycin. The medium was changed daily.

Growth factors, antibodies and inhibitors
Human recombinant HGF was purchased from R&D Systems (Abingdon, UK). Primary antibodies used were goat polyclonal neutralizing antibody against recombinant human HGF (Sigma, St Louis, MO), rabbit polyclonal antibody against the intracellular domain of human c-Met (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibodies against human MMP-1, MMP-2 and MMP-9 (the latter two being neutralizing) (Calbiochem, La Jolla, CA), rabbit polyclonal anti-human MMP-9 antibody (Triple Point Biologics, Forest Grove, OR), mouse monoclonal anti-human MMP-2 antibody (R&D Systems), goat polyclonal anti-catalytic uPA antibody (Chemicon, Temecula, CA). Secondary antibodies were peroxidase-labelled-sheep (Amersham, Les Ulis, France) or -goat (Dako, Copenhagen, Denmark) anti-mouse IgG, -monkey anti-rabbit IgG (Amersham) or rabbit anti-goat IgG (Dako). Normal mouse IgG and wortmannin, a PI3 kinase inhibitor, were purchased from Sigma; herbimycin A, a tyrosine kinase inhibitor, LY 294002, another PI3 kinase inhibitor and GÖ 6976, a selective inhibitor of PKC{alpha} and PKCß1, were from Calbiochem. Bisindolylmaleimide-1 (Bis-1), a second PKC inhibitor, was from Alexis Biochemicals (San Diego, CA). Batimastat or BB-94, a synthetic hydroxamic acid-based inhibitor of MMPs, was a gift from British Biotech (Oxford, UK).

Zymography
Proteases were detected using a modified technique (27). Serum-free conditioned media from normal cultures or from upper Transwell compartments after invasion assays were collected, centrifuged at 4000 g for 20 min and then concentrated if necessary. Laemmli's buffer without reducing agent was added to media. The latter lacked phenol red, this allowed measurement of protein concentration using the BioRad protein assay (BioRad, Hercules, CA). Proteins (40 µg loaded onto each lane) were separated by 10% SDS–PAGE containing 1 mg/ml gelatin or casein. Gels were then stained for 1 h in 30% methanol/10% glacial acetic acid solution containing 1.5% (w/v) Coomassie blue and destained in the same solution in the absence of dye as previously described (28). Unstained areas corresponded to zones of MMP proteolytic activities.

Western immunoblotting
For protease detection, conditioned media from normal cultures or from Transwells after invasion assays were prepared as for zymography. For detection of HGF and c-Met, harvested cells were lysed in the presence of protease inhibitors at 4°C for 15 min. Proteins were solubilized and separated by 7.5% SDS–PAGE after loading onto each lane 40 µg for protease detection or 10 µg for HGF or c-Met detection. Equivalence of loading across the gel lanes was verified with Ponceau red staining. Proteins were then transferred onto nitrocellulose sheets. Blots were probed with the different protein antibodies at appropriate dilutions, then with the corresponding secondary antibodies diluted 1:1000 to 1:10 000. The immune complexes were revealed by an enhanced chemiluminescence detection system (Amersham).

RT–PCR
Total RNA was extracted from Caco-2 cells using Trizol-Reagent (Gibco BRL) according to the manufacturer's protocol. First-strand cDNA was synthesized from 1.6 µg total RNA using murine reverse transcriptase and the First-strand cDNA Synthesis kit from Pharmacia Biotech (Uppsala, Sweden). Oligonucleotide primers were synthesized by Genosys (Cambridge, UK). Primers for HGF and c-Met previously described (7) produced DNA amplified sequences of 539 and 484 base pairs (bp), respectively. For MMP1, MMP2 and MMP9, specific primers (29) produced DNA amplified sequences of 786, 605 and 243 bp, respectively. The cDNA mixture was amplified by RT–PCR in a 50 µl volume with 25 pmol each primer. Thirty-five cycles were performed except for MMP-9 (45 cycles). Each amplification cycle consisted of denaturation at 95°C for 1 min, annealing at 55–60°C (depending on primers) for 1 min and polymerization at 72°C for 2 min. The amplification was terminated by a final extension step at 72°C for 10 min. PCR samples (10 µl) were electrophoretically separated on 1% agarose gel previously stained with ethidium bromide and visualized under UV light. After QIA quick gel purification (Qiagen, Courtaboeuf, France), amplified products were sequenced. For negative controls, reactions were performed without reverse transcriptase or polymerase or by omitting specific primers.

In vitro cell invasion assays
Assays were performed according to Albini et al. (30). Briefly, 12 µm-pored Transwell filter chambers (Corning-Costar) were used. The upper side of polycarbonate membranes were coated with Matrigel, a reconstituted basement membrane matrix (Becton Dickinson, Bedford, MA), diluted to 1.5 mg/ml. Cells (1x105) were seeded in the upper compartment of each Transwell unit containing FCS-free medium with 0.1% BSA (Sigma). The medium plus BSA was introduced in the lower compartment with or without 20% FCS and with or without HGF at different concentrations, i.e. 0 (control), 50 or 100 ng/ml. To analyse the mechanisms associated with the HGF-promoted cell invasiveness, we used HGF + FCS-supplemented medium, as other authors for other cell types (18,31,32), and the following substances were added, in separate experiments, either in the lower compartment: HGF antibody (40 µg/ml), or in the upper compartment: herbimycin A (0.5 µg/ml), uPA antibody (diluted 1:100), MMP-1, MMP-2 and MMP-9 antibodies (30 µg/ml), batimastat (diluted in PBS-Tween at 50 µg/ml), PKC or PI3 kinase inhibitors (diluted 10–6 M or 10–7 M, respectively, in 0.1% DMSO). After 72 h of culture, the non-migratory cells on the upper surface were removed with a cotton swab and the membranes fixed in methanol for 5 min. The cells which had migrated through Matrigel + membrane were attached to the lower surface of the membrane. They were stained with toludine blue and their density estimated by cell counting at x400 magnification, using a calibrated ocular grid, in 15 representative areas per well. Experiments were carried out in duplicate or triplicate and several independent sets of experiments for each type of invasion assays were performed.

[3H]Thymidine incorporation
Caco-2 cell proliferation was examined to verify the absence of direct toxicity of batimastat. Cells were cultured in the presence or absence of 50 µg/ml batimastat for 3 days. [3H]Thymidine (0.1 µCi/ml) was added to cells during the last hour before harvesting. Cells were washed twice with phosphate buffer saline and trypsinized. DNA was precipitated with 5% trichloroacetic acid at 4°C for 30 min, washed with 95% ethanol, then dissolved in 10% Triton. Incorporated radioactivity was analysed in a ß scintillation counter and expressed in c.p.m.

Statistical analysis
Values were expressed as the mean ± SEM. Comparisons between all groups were analysed by one-way ANOVA followed by the t-test for comparison between two groups. The level of statistical significance was set at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caco-2 cells express c-Met but not HGF
First of all, we determined whether Caco-2 cells express HGF and its receptor. Neither HGF mRNA nor HGF protein was found in these cells, at least at the passages examined, whereas fetal human liver, used as a positive control (7), expressed HGF. However, RT–PCR and western immunoblots indicate that Caco-2 cells express both c-Met transcript and protein (Figure 1Go).



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Fig. 1. (A) RT–PCR and (B) western immunoblots for HGF and its receptor c-Met in Caco-2 cells on day 3 of culture. Molecular weight markers are indicated on the left. The expected 484 bp c-Met DNA amplicon and the 145 kDa c-Met protein band (c-Met ß-subunit) were detected, whereas no HGF mRNA nor HGF protein was identified in Caco-2 cells. The expected 539 bp HGF DNA amplicon was seen in human fetal liver used as a positive control: lane 1, Caco-2 cells; lane 2, fetal liver.

 
Caco-2 cells express proteases
Although proteases are thought to be produced mainly by mesenchymal cells, we examined whether Caco-2 cancer epithelial cells could express proteases. Cells were seeded in 6-well plates. At different days after plating, cells from three wells were rinsed twice in serum-free medium then cultured for 48 h in the same medium. The expression of MMPs and uPA was examined in conditioned media at the end of this culture period by zymography or western immunoblotting. Analysis of gelatin zymograms indicated the presence of two proteolytic activities in media from days 2–3 after plating, at 92 and 62 kDa, which correspond to the molecular masses of pro-MMP-9 and the active form of MMP2, respectively. Their expression decreased after confluence which was reached on day 5 after plating. In contrast, analysis of casein zymograms indicated the absence of protease activity in the media first days post-plating and the detection, subsequently, of a band at about 50 kDa, which corresponds to the molecular mass of MMP1 (Figure 2AGo). RT–PCR using specific primers confirmed the presence of MMP-2 and MMP-1 in Caco-2 cells and their different behaviour over culture time, but no MMP-9 mRNA was detected (Figure 2BGo). Expression of uPA was also detected by western blotting in the culture media of Caco-2 cells (Figure 2CGo).



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Fig. 2. Detection, at different time-points of culture, of MMPs and uPA in conditioned culture media of Caco-2 cells. Before media collection , cells were cultured in serum-free medium for 48 h (3 wells per time-point). (A) Zymograms (40 µg protein/lane). Detection of a band at 50 kDa in the casein gel, molecular mass corresponding to that of MMP1 (upper panel), and of two bands at 92 and 62 kDa in the gelatin gel, molecular masses corresponding to those of the latent form of MMP-9 and active form of MMP-2, respectively (lower panel). The 92 and 62 kDa bands appeared in the first days of culture and decreased after confluence—reached on day 5 post-plating—whereas appearance of the 50 kDa band was delayed but its detection maintained subsequently. (B) RT–PCR from extracted cell RNA with MMP-specific primers. MMP-1 mRNA was expressed later than MMP-2 mRNA. MMP-9 mRNA was not detected. This discrepancy raises the question of real MMP-9 production (see Discussion). (C) Detection by western immunoblotting of uPA, indicated by a 52 kDa protein band.

 
HGF stimulates MMP production by Caco-2 cells
Given that Caco-2 cells express MMPs, we wished to investigate whether HGF could influence MMP secretion in Caco-2. Cells at 80% confluence were FCS deprived for 24 h, then cultured for 48 h in serum-free medium in the presence of 20 ng/ml HGF, followed by media collection and the examination of MMP expression both by zymography and western immunoblotting. After scanning, the surface and densitometry of the digestion bands and blots were measured with the NIH Image 1.61/ppc program. A discrete but clear stimulation in the relative activity and expression of MMP-1, MMP-2 and the putative MMP-9 was observed in the presence of HGF (Figure 3A and BGo).



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Fig. 3. Stimulation of MMP-1, MMP-2 and MMP-9 production by HGF. Cells at 80% confluence were FCS deprived for 24 h, then cultured for 48 h in serum-free medium in the presence of 20 ng/ml HGF. (A) Casein (upper panel) and gelatin (lower panel) zymograms and (B) western blots from conditioned culture media collected on day 6 post-plating (40 µg protein loaded per lane).

 
HGF stimulates Caco-2 cells invasiveness
First of all, we confirmed that HGF was indeed able to enhance migration of Caco-2 cells after monolayer wounding (data not shown). Then the ability of HGF to induce matrix invasion by these poorly aggressive cells was studied in Transwells covered with Matrigel. First, the effect of HGF was assessed under conditions previously used for Madin–Darby canine kydney epithelial cells (pores 8 µm, Matrigel dilution 5 mg/ml) reported as invasive under HGF (33) and used as controls. We confirmed that HGF stimulated the invasiveness of these cells (data not shown), whereas Caco-2 cells were repeatedly not invasive. Secondly, we changed the experimental conditions, using a higher Matrigel dilution (1 mg/ml), larger membrane pores (12 µm) as for other intestinal cells (17) and a 3 day incubation time. Under these conditions, cells were not able to pass through the Matrigel + membrane barrier when FCS-free medium was introduced into the lower compartment. There was a small number of invading cells when FCS-supplemented medium was used in place of FCS-free medium (Figure 4aGo) or when HGF was added to FCS-free medium (Figure 4bGo). However, when 50 or 100 ng/ml HGF were added to the FCS-supplemented medium, there was a 7–20-fold increase in the number of invading cells as compared with the corresponding concentration of HGF alone, indicating that there was more than an additive effect of HGF and FCS (Figures 4c and d and 5AGoGo). Whatever the HGF concentration, the additional introduction of HGF antibody significantly reduced the number of invading cells by 65–90%, depending on the experiment, P < 0.02 to P < 0.001. Similarly, inhibition of tyrosine kinase by herbimycin A, used at a concentration found efficient by others in inhibiting HGF stimulation (34), significantly decreased the HGF-induced cell invasion by 42–75% (P < 0.005 to P < 0.001) (Figures 4e and f and 5BGoGo).



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Fig. 4. Effect of HGF on invasion of Caco-2 cells in Matrigel invasion chamber; 105 cells were seeded in the upper compartment. Representative photographs showing at low magnification, after 3 days of culture, the cells having invaded Matrigel and passed across the 12 µm-pored membrane. a–c, Same experiment; d–f, same experiment. In the lower chamber, there were (a) FCS, (b) 50 ng/ml HGF, (c) FCS + 50 ng/ml HGF, (d) FCS + 100 ng/ml HGF, (e) idem d + anti-HGF 40 ng/ml (~ 90% inhibition), (f) idem d + herbimycin A 0.5 µg/ml (upper side) (~75% inhibition).

 


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Fig. 5. (A) Graphical representation of the number of invading Caco-2 cells under the conditions corresponding to Figure 4a–dGo. The number of wells in which cell measurements were performed is indicated within each column and represents the results from three to five independent experiments. Area corresponds to the surface occupied by the ocular grid at x400 magnification. (B) Graphical representation of mean numbers of invading cells under the conditions corresponding to Figure 4e–fGo, expressed as a percentage of the FCS+HGF (50 or 100 ng/ml)-stimulated value. *P < 0.02; **P < 0.005; ***P < 0.001. The number of wells is indicated within columns. (C) and (D), MMP and uPA profiles in media from the upper chamber at the end of invasion assays under the conditions corresponding to Figure 4a–fGo and after loading 40 µg protein per lane. (C) Western blots with anti-MMP-1, MMP-2 and uPA antibodies; (D) gelatin zymogram. There was a decrease in HGF-stimulated expression of MMP and uPA when anti-HGF antibody or herbimycin A was introduced in Transwells.

 
We speculated that proteases previously shown to be secreted by Caco-2 cells might be implicated in this HGF-promoted invasiveness. Therefore, we next performed western blots and zymographies in gelatin and casein gels from the conditioned culture media collected from the upper Transwell compartments, after loading equal quantities of protein per lane. The secretion of MMPs by Caco-2 cells was enhanced when HGF was added to the FCS supplemented-medium. When the anti-HGF antibody or the tyrosine kinase inhibitor was furthermore introduced, MMPs and uPA expression decreased or apparently disappeared from the culture media (Figure 5C and DGo), supporting the hypothesis that HGF favours tumour cell invasion by promoting protease production.

MMPs and uPA are involved in HGF-induced cell invasion
In an effort to analyse further the potential implication of proteases in the HGF-stimulated Caco-2 cell invasiveness, we performed additional experiments under HGF + FCS-supplemented medium and in the presence or absence of MMP-1 antibody, anti-catalytic MMP-2 or uPA antibodies, since protease antibodies have been previously shown to inhibit matrix invasion by other tumour cells (35,36). We also performed invasion assays in the presence of a general MMP inhibitor, batimastat. Incubation with each of these MMP antibodies as well as with batimastat resulted in significant decreases, ranging from 25 to 55%, in the number of invading cells stimulated by HGF (P < 0.05 to P < 0.005), without apparent cell damage by these molecules (Figure 6AGo). In contrast, normal mouse IgG has no influence on cell invasion. We also verified, as we have already shown in other colon cancer cells (28), that batimastat at the concentration and under the conditions used here did not significantly alter Caco-2 cell proliferation as compared with controls (1711 ± 125 versus 1268 ± 212 c.p.m., respectively; NS), indicating that batimastat has no direct toxicity on these cells. In addition, analyses of conditioned Caco-2 cell media showed that, in the presence of anti-uPA antibodies, there was a concomitant reduction in HGF-stimulated MMP levels, as shown by zymography and western blotting (Figure 6B and CGo). All these results indicated that proteases such as MMP-1 and -2 and uPA are involved in the cell invasiveness process induced by HGF. In contrast, MMP-9 antibody gave contradictory results in three independent experiments and therefore no conclusions could be drawn as to the putative part played by MMP-9 in the HGF-induced invasion in this cell line.



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Fig. 6. Effect of anti-MMPs and anti-uPA antibodies (30 µg/ml) and of the synthetic general MMP inhibitor batimastat (50 µg/ml) on HGF-induced invasion of Caco-2 cells. (A) Graphical representation of the numbers of invading Caco-2 cells expressed as a percentage of the FCS+HGF (50 or 100 ng/ml)-stimulated value. The number of wells is indicated within each column.*P < 0.05; **P < 0.01; ***P < 0.005. (B) and (C) MMPs profiles in media from the upper chamber at the end of invasion assays (40 µg protein loaded). (B) Gelatin zymogram, (C) western blots with anti-MMP-1 and anti-MMP-2. HGF-stimulated MMP activity/expression decreased in presence of anti-uPA antibody.

 
PI3 kinase and PKC are involved in HGF-stimulated Caco-2 cell protease expression and invasiveness
Caco-2 cells have been shown to express PKC{alpha} (37) and it has recently been reported that transfection of an activated form of PKC{alpha} into the HT29-M6 intestinal cell line results in induction of cell invasiveness in vitro (25). On the other hand, PI3 kinase has been shown to mediate the HGF-promoted invasion in c-src-transformed intestinal cells (19). It is possible, therefore, that these two kinases are implicated in HGF-induced Caco-2 cell invasiveness? To investigate this further, wortmannin, a specific inhibitor of PI3 kinase and GÖ 6973, a specific inhibitor of two PKC isoforms, PKC{alpha} and PKCß1, were added. The GÖ 6973 concentration used has been previously shown to be effective on Caco-2 cells (38). First, we verified that the solvent of inhibitors (0.1% DMSO) had no effect on HGF-stimulated cell invasiveness and, also, that the viability of cells, examined by trypan blue exclusion, was not altered in the presence of these inhibitors after 3 days. Wortmannin and GÖ 6973 inhibited HGF-stimulated Caco-2 cell invasion by 60 (P < 0.005) and 28%, respectively (Figure 7A and BGo) and a concomitant decrease in HGF-stimulated MMP activity was seen as depicted in zymograms from cell culture media (Figure 7CGo). In an additional experiment, we examined MMP levels in conditioned culture media of HGF-stimulated cells, cultured for 3 days onto Matrigel in the presence of two other inhibitors, LY 294002 for PI3 kinase and Bis-1 for PKC. Again, a decrease in MMP activity was noted as compared with HGF alone (Figure 8Go). These results indicate that PI3 kinase and PKC are involved in the two responses to HGF.



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Fig. 7. Effect of GÖ 6976, a PKC{alpha} and -ß1 inhibitor, and of wortmannin, a PI3 kinase inhibitor, on HGF-induced invasion of Caco-2 cells. (A) Representative photographs of invading cells; a–c, same experiment; (a) FCS + HGF 100 ng/ml; (b) idem a + GÖ 6976; (c) idem a + wortmannin. (B) Graphical representation of the number of invading cells expressed as a percentage of the FCS + HGF (100 ng/ml)-stimulated value. ***P < 0.005. (C) Gelatin zymograms of MMPs from culture media of the upper chamber at the end of invasion assays (40 µg protein/lane). There was a decrease in the HGF-stimulated expression of MMP-2 and the 92 kDa band in presence of GÖ 6976 (two independent experiments) or wortmannin (two wells of the same experiment).

 


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Fig. 8. Examples, from two experiments, of gelatin zymograms from conditioned culture media of Caco-2 cells cultured for 3 days onto Matrigel in presence of HGF without or with 10 µM LY 294002, a PI3 kinase inhibitor or Bis-1, a PKC inhibitor. Inhibitors decreased MMP activity, notably the 92 kDa band.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The metastatic characteristics of tumour cells are dependent mainly on the degradation of the extracellular matrix by proteases and concomitant induction of cell movement, the latter being the determining step in the process of tumour cell invasiveness. The present investigation provides the first evidence that HGF stimulates motility of Caco-2 cells through the extracellular matrix due to overproduction of a specific subset of proteases and the involvement of PI3 kinase and PKC in this process.

At variance with previous data (17) and in spite of the poor Caco-2 cell aggressiveness (26), HGF, at the two concentrations tested, was indeed able to trigger their invasiveness across a Matrigel barrier. Nevertheless, this process was greatly facilitated when HGF was added to FCS-supplemented medium as compared with FCS-free medium, the number of invading cells being roughly HGF dose-dependent. Obviously, our current results indicate synergism between HGF and one or several components of FCS. One of these components could be uPA, which is known to activate HGF (39). We have shown that invasion was mainly induced by the complex HGF/c-Met, since the number of invading cells under HGF greatly decreased by blocking HGF with a neutralizing anti-HGF antibody and by inhibiting tyrosine kinase activity, including that of c-Met.

MMPs and uPA have been shown to be required for optimum invasiveness of ovarian and lung cells, respectively (35,36). In the present work, we found that Caco-2 cells produced MMP-1, MMP-2 and uPA. Expression of MMP-1 mRNA had been evoked in these cells (40). A relatively weak proteolytic activity at 92 kDa, which might correspond to that of MMP-9, was also consistently found on zymograms, but we were unable to confirm the presence of MMP-9 mRNA by RT–PCR. There are two possible explanations: (i) the level of MMP-9 mRNA was too low to be detected in cells at a given time-point, whereas zymograms revealed the enzymatic activity of the secreted protein that had accumulated in the culture medium over 2–3 days. Consistent with this hypothesis, western blots with anti-MMP-9 antibody detected a discrete 92 kDa band. (ii) Alternatively, this band corresponded to a protease different from MMP-9 but with a similar apparent molecular mass.

We have further shown that Caco-2 cell MMP production increased in the presence of HGF and that, conversely, neutralizing anti-HGF antibody strikingly reduced, or apparently abolished, the expression of MMP and uPA in the culture media of invasion assays. Two possible explanations for the disappearance of protease with HGF-antibody (as seen in Figure 5CGo) are as follows. (i) FCS is a natural fluid containing a variety of molecules, among them growth factors, whose concentration is variable. So, the production of some protease(s) by FCS alone may be due, at least in part, to HGF. Actually, western blots performed from one FCS batch with anti-HGF antibody showed a band at 70 kDa, corresponding to HGF (data not shown); this does not exclude other FCS components from stimulating protease production. (ii) Alternatively, it is possible that a more prolonged exposure with the chemiluminescent reagent would reveal a weak protein band. This reduction in protease expression accompanied the diminution in cell invasion and implicated proteases in HGF-induced Caco-2 cell invasiveness. The involvement of each protease in this process was then investigated using MMP antibodies and a general MMP inhibitor. Our findings provide evidence that MMP-1, MMP-2, as well as uPA were actively involved. The magnitude of inhibition of cell invasion observed in the presence of batimastat was consistent with the sum of inhibitions noted in the presence of anti-MMP-1 and anti-MMP-2 antibodies and addition of the inhibitions noted in the presence of batimastat and uPA antibody theoretically accounted for complete inhibition of HGF-promoted cell invasion. There was a concomitant diminution in MMP expression in culture media in the presence of anti-uPA antibody as compared with experiments with FCS + HGF alone, consistent with the postulated role that uPA may play in pro-MMP activation. Indeed, plasmin, generated from plasminogen by uPA, has been proposed as a possible activator of pro-MMPs in vitro but also in vivo (41). Reduction in plasmin generation by uPA antibody must consequently reduce the MMP levels. In the present work, the lack of clear-cut inhibition with anti- MMP-9 antibody should suggest that the 92 kDa band actually did not represent MMP-9. Alternatively, it is possible that Caco-2 cells express MMP-9 without implication of the latter in cell invasion. In that way, it has been shown that squamous cancer cells secrete uPA which does not play a role in the invasiveness of these cells (42). Recently, two groups have investigated in parallel the HGF-induced stimulation of invasiveness and protease activities in gallbladder and squamous carcinoma cell lines (32,42). However, to our knowledge, our study is the only one which examines the relationships between these two processes by studying systematically the profile of protease production in the media of cell invasion assays under all the above-mentioned conditions. Moreover, our results concern another type of cells, i.e. colon cancer cells.

Finally, in addition to the activation of tyrosine kinase, other interesting findings described herein suggest that HGF apparently requires, at least in part, the involvement of PKC{alpha} and PI3 kinase pathways to activate the invasive behaviour of Caco-2 cells. The sum of effects noted in the present study with inhibitors of these two signalling molecules roughly accounted for 90% inhibition of HGF-induced cell invasion. However, for cell invasion assays, we used a single inhibitor for PKC or PI3 kinase, commonly used by investigators, and their specificity might be questionable as they may inhibit other protein kinases. GÖ 6976 is known to be reasonably selective for two PKC isoforms; and very recently, from 28 commercially protein kinase inhibitors tested, wortmannin was ranked among the eight compounds having the most impressive selectivity profile (43). Although our results would need to be reinforced by using other structurally unrelated inhibitors of these kinases, as such, they confirm that PI3 kinase is a transducer of HGF-promoted cell invasion (19,24) and strongly suggests for the first time that PKC ({alpha} or ß1) is also involved in this process. In addition, although HGF regulation of MMP-1 expression has already been reported to require PKC activity (34), herein, it is the first time that PI3 kinase and PKC have been shown to participate in the HGF stimulation of MMP production by cells in the course of their invasion process. The involvement of these signalling molecules in this latter process was confirmed by using two other inhibitors, LY 294002 and Bis-1, respectively. Further studies should determine whether in HGF-stimulated cell invasiveness, elements other than MMP(s), such as cytoskeleton components and adhesion molecules, are possible targets for PI3 kinase and PKC.

In conclusion, our findings support the hypothesis that HGF favours invasion of Caco-2 cells through the basement membrane by close association of two concomitant mechanisms, stimulation of their motile activity and activation of proteases, possibly PI3 kinase/PKC-dependent, which permit cell migration through the degraded extracellular matrix.


    Notes
 
1 To whom correspondence should be addressed Email: tlehy{at}bichat.inserm.fr Back


    Acknowledgments
 
This work was supported by the Institut de la Santé et de la Recherche Médicale (INSERM) and by IRMAD (to S.K.). The authors thank Dr P.J.Parker (Protein Phosphorylation Laboratory, London) for kind advice and British Biotech for the generous gift of batimastat. S.Kermorgant was a recipient of the Ministère de l'Enseignement Supérieur et de la Recherche, of the Association pour la Recherche contre le Cancer (ARC) and of Sanofi-Synthelabo (Plessy Robinson, France).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 12, 2000; revised March 12, 2001; accepted March 21, 2001.