INVOLVEMENT OF MATRIX METALLOPROTEINASE TYPE-3 IN HEPATOCYTE GROWTH FACTOR-INDUCED INVASION OF HUMAN HEPATOCELLULAR CARCINOMA CELLS Arnaud MONVOISIN, Christe `le BISSON, Karim SI-TAYEB, Charles BALABAUD, Alexis DESMOULI ` ERE and Jean ROSENBAUM * GREF, INSERM E9917, Universite ´ Victor Segalen Bordeaux 2, Bordeaux, France Intra-hepatic invasion is a key feature of hepatocellular carcinom a (H C C ) progression.W e have show n that hum an liver m yofibroblasts induce invasion ofHCC cells through M atrigel, via the secretion of hepatocyte grow th factor (H GF).In ourstudy,we investigated the role ofm atrix m et- alloproteinases (M M P)in HGF-induced HCC cells invasion. M arim astat,a synthetic M M P inhibitor,dose-dependently de- creased H GF-induced invasion ofH epG2 cellswith a m axi- m um of 82.7  13.3% at20  M .T IM P -2,a naturalinhibitor, decreased invasion up to 51.2  11.2% at 200 ng/m l.To determ ine the target for these inhibitors,w e exam ined M M P expression using RT-PCR. M M Ps 1, 7–9 and 10 were not expressed in HepG2 cells either in the absence or in the presence ofH G F.M M P-2 and M M P-13 transcriptswere de- tected in unstim ulated cells but their expression was un- changed after exposition to H G F. M M P -3 transcripts w ere undetectable in unstim ulated HepG2 cells. They becam e clearly expressed in H G F-stim ulated cells,how ever,and this was confirm ed by N orthern blot.By W estern blot,H GF dose-dependently stim ulated the secretion of pro-M M P -3 in the culture m edium .The role ofM M P-3 in HGF-induced invasion was directly confirm ed by using an antibody to M M P-3, that blocked invasion. Finally, R T -PC R dem on- strated M M P-3 expression in 10/16 hum an HCCstested,but not in norm alliver.In conclusion,our data dem onstrate that M M Ps,m ostlikelyM M P-3,m ediateHGF-induced invasion of H CC cells.The i n v i v o expression ofM M P-3in HCC suggests a role for this protease in H C C progression. © 2002 Wiley-Liss, Inc. Key words: liver; myofibroblast; marimastat; TIMP; stromelysin Tumor invasion and metastasis are complex processes that re- quire interactions between the invasive cells and the extracellular matrix (ECM). Among the enzymes responsible for ECM degra- dation, several studies have shown a critical role played by matrix- metalloproteinases (MMPs). 1 MMP-3 (or stromelysin-1) is able to degrade type III, IV and V collagens, in addition to non-collagenous matrix components such as proteoglycans, fibronectin and laminin. 2 Moreover, MMP-3 is considered to play a central role in the activation of various pro-MMPs including pro-MMP-1, 3,4 pro-MMP-7, 5 pro-MMP-8, 6 pro-MMP-9 7 and pro-MMP-13. 8 MMP-3 expression can be induced by numerous factors includ- ing cytokines and growth factors 9 –12 and notably by the hepatocyte growth factor (HGF). 13 Previous studies have shown that MMP-3 expression can be associated with the invasive phenotype of head and neck carcino- ma, 14 gastric carcinoma 15 and pancreatic carcinoma. 16 Abundant expression of MMP-3 in breast cancer has also been reported. 17 Transfection of non-transformed mammary epithelial cells with a MMP-3 expression vector leads to the acquisition of an invasive phenotype. 18 Furthermore, transgenic mice that overexpress an autoactivating mutant of MMP-3 targeted to the mammary gland spontaneously develop breast cancer. 19,20 Finally, MMP-3 anti- sense treatment blocks invasion of mouse metastatic mammary carcinoma cells in vitro. 21 We have previously shown that human liver myofibroblasts strongly increased the invasion of several human hepatocellular carcinoma (HCC) cell lines through Matrigel-coated filters. 22 This was due to HGF secreted by myofibroblasts and recombinant HGF could substitute for myofibroblasts to induce invasion. 22 The effect of HGF was at least in part due to the upregulation of the expres- sion of urokinase-type plasminogen activator (u-PA) in HCC cells. 23 u-PA is a serine protease that can activate plasminogen to plasmin. Because HGF has been shown to up-regulate MMP-3 expres- sion in some cell types and because pro-MMP-3 is a substrate for plasmin, we investigated the expression of MMP-3 in HCC cells, its regulation by HGF and its role in invasion of these cells. We also examined the expression of MMP-3 in human liver. MATERIAL AND METHODS Material Culture medium and additives, Moloney murine leukemia virus- reverse transcriptase (MMLV-RT) and library efficient DH5 competent cells were from Gibco-BRL (Life Technologies, Cergy- Pontoise, France). Human AB serum was from Centre Re ´gional de Transfusion Sanguine (Bordeaux, France). The RNeasy minikit was from Qiagen (Les Ulis, France), Hybond N+™-membrane and ECL kit from Amersham (Les Ulis, France). [ - 32 P]dCTP and [ - 32 P]dATP were from ICN Biomedicals (Orsay, France). The Ready-To-Go random priming kit was from Boehringer-Mann- heim (Meylan, France). Taq polymerase and the pGEM-T Easy Vector System I were from Promega (Charbonnie `res, France). Polyvinylidene difluoride membranes were from Millipore (Saint- Quentin en Yvelines, France). Polycarbonate filters were from Falcon (Becton Dickinson, Le Pont de Claix, France) and Matrigel basement membrane matrix from Collaborative Biomedical Prod- ucts (Bedford, MA). Chemicals were from Sigma Aldrich (Saint Quentin Fallavier, France). Recombinant human HGF was a kind gift from Dr. Vande Woude (NCI, Frederick, MD). Recombinant human pro-MMP-3 was from Valbiotech (Paris, France). The sheep polyclonal antibody against MMP-3 and the control sheep IgG were from The Binding Site (Birmingham, UK). According to the supplier, this antibody does not cross react with MMP-1, MMP-2, or MMP-9. The specific MMP inhibitor Marimastat (BB- 2516) was a gift from Dr P.D. Brown (British Biotech Pharma- ceuticals Ltd, Oxford). A stock solution was prepared extempora- neously at 1 mM in DMEM. Recombinant human TIMP-2 (rhTIMP-2), prepared from CHO cells transfected with a human TIMP-2 cDNA and purified as described, 24 was a kind gift from Dr J.M. Foidart (Lie `ge, Belgium). MMP-3 substrate II was from Calbiochem (Darmstadt, Germany). Grant sponsor: Comit´ e de la Dordogne from the Ligue Nationale Fran- c ¸aise contre le Cancer; Grant sponsor: Association pour la Recherche sur le Cancer; Grant sponsor: Conseil R´ egional d’Aquitaine. *Correspondence to: Groupe de Recherches pour l’Etude du Foie, IN- SERM E9917, Universit´ e Victor Segalen Bordeaux 2, 146 rue L´ eo Saignat, 33076 Bordeaux cedex, France. Fax: +33-556-514-077. E-mail: jean.rosenbaum@gref.u-bordeaux2.fr Received 4 May 2001; Revised 25 June 2001; Accepted 13 July 2001 Int. J. Cancer: 97, 157–162 (2002) © 2002 Wiley-Liss, Inc. Publication ofthe InternationalU nion A gainstCancer