Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase.

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

tissue remodeling, inflammation, and in the regulation of cancer cell migration, extracellular matrix (ECM) invasion, and metastasis by degrading the ECM proteins (2). Several reports (3,4) have indicated that increased levels of uPA correlate with the invasive properties of certain tumor cell types. A specific receptor for uPA, uPAR, forms a complex with both the inactive and active forms of uPA (5). Receptor-bound uPA can be activated on the cell surface. uPA binding to uPAR could enhance cellular invasiveness.
Bikunin is a Kunitz-type protease inhibitor and a heavily glycosylated protein (6). This protein inhibits trypsin and plasmin. In our previous experiments, cellular expression of several proteins known to be involved in invasion and metastasis, including uPA and uPAR, were significantly suppressed by exogenously added bikunin (7)(8)(9)(10). Furthermore, transfection of highly invasive human ovarian cancer cell line HRA with the bikunin gene reduced the expression of uPA and uPAR and diminished cellular invasiveness (11). Current investigations have focused on understanding the molecular mechanism(s) by which bikunin down-regulates uPA and uPAR expression both in vitro and in vivo and controls invasiveness and tumor growth in HRA cells (12)(13)(14)(15). We also found that several highly metastatic, transformed cells synthesize lower levels of bikunin compared with non-tumorigenic cells. 2 In addition, we showed that there is a direct correlation between bikunin overexpression and the reduced metastatic potential of primary tumor biopsies and tumor cell lines, as reflected by their in vitro potential to invade through a Matrigel barrier (16). In this invading model system, we have also shown that bikunin is part of the negative invasive program, as evaluated by invasion and uPA/uPAR synthesis.
For identifying the full repertoire of bikunin-regulated genes, we recently conducted a cDNA microarray hybridization screening, using mRNA from bikunin-treated or bikunin-transfected HRA cells (15). A number of bikunin-regulated genes were identified (15). This screen identified suppression of several genes, such as CDC-like kinase, LIM domain binding, Ets domain transcription factor, Rho GTPase-activating protein, tyrosine phosphorylation-regulated kinase, hyaluronan-binding protein, matriptase (also known as suppression of tumorigenicity 14, epithin, and membrane-type serine protease-1), pregnancy-associated plasma protein-A, and phosphoinositide 3-kinase (PI3K), which have been implicated previously in enhancing tumor promotion. These results show that bikunin alters the expression pattern of several genes in HRA cells leading to a block in cell invasion. Interestingly, Northern blot analysis confirmed that matriptase was markedly down-regulated by bikunin. Therefore, matriptase is considered to be an important bikunin target gene (15).
Matriptase, a type II membrane serine protease (17), may play important roles in cell migration and tumor cell metastasis (18,19). It has a multidomain structure, containing a putative amino-terminal transmembrane region, a sperm protein, enterokinase, and agrin (SEA) domain, two complement subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenic protein (CUB) domains, four low density lipoprotein receptor class A repeats, and a carboxyl-terminal serine protease domain (20 -23). Matriptase can convert hepatocyte growth factor/scatter factor to its active form, which can activate c-Met tyrosine phosphorylation (18). Furthermore, protease-activated receptor 2 (PAR2) and enzymatically inactive single-chain uPA (pro-uPA) were identified as substrates of matriptase (18,19). The identification of these molecules as putative in vivo substrates suggests that matriptase regulates the functions mediated by PAR2, such as the inflammatory response or cell adhesion, and by uPA, such as tumor cell invasion and metastasis. The expression of this protease is associated with a more invasive phenotype (24). Thus, studies with matriptase suggest that the protease may play important roles in cell migration as well as cancer invasion and metastasis.
There are no data indicating whether matriptase can activate receptor-bound pro-uPA on the surface of tumor cells. To clarify how reduced levels of matriptase may confer repressed invasiveness, we transiently transfected HRA cells with antisense (AS)-matriptase, and we compared the properties of the transfected clones to those of parental HRA cells and stable bikunin transfectants.
Cell Culture-The ovarian cancer cell line, HRA, was grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 g/ml) in a 5% CO 2 atmosphere with constant humidity (10). For all experiments in which TGF-␤1 was added, cells were incubated in the serum-free medium. Total RNA isolations were done using the Trizol reagent (Invitrogen). The protein concentrations in the supernatants of cell extracts were measured by the Bio-Rad protein assay.
Preparation of Conditioned Medium and Cell Lysate-For the preparation of conditioned medium, subconfluent cultures of ovarian cancer cell clones were washed three times with phosphate-buffered saline (PBS) and incubated with serum-free Dulbecco's modified Eagle's medium in the presence or absence of 10 ng/ml TGF-␤1 for 24 h. The conditioned medium was collected, centrifuged to remove cell debris, filtered, lyophilized, concentrated, and stored at Ϫ80°C until needed. In general, the supernatants concentrated 10-fold were used for zymography and Western blot.
The cell monolayers treated with or without various agents for indicated times were washed with PBS. 1 ϫ 10 6 cells were lysed in 750 l of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 12.5 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl 2 , 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 2 mM dithiothreitol, 1 mM sodium vanadate, and 1 mM phenylmethylsulfonyl fluoride at 4°C for 15 min and scraped with a rubber policeman. Cell extracts were then centrifuged at 3,000 ϫ g to remove cell debris. All samples were stored at Ϫ80°C until use. In parallel, cells treated in the same condition in different dishes were harvested and counted using a hemocytometer.
Northern Blot Hybridization with cDNA Probes-Northern blot hybridization was carried out as described previously (9). Samples of total RNA (10 g) were separated by electrophoresis through denaturing 1.2% agarose gels containing 1% formaldehyde and transferred onto nylon or nitrocellulose membranes using standard molecular biological techniques. Hybridization was carried out with [␣-32 P]dCTP by random oligonucleotide priming to specific activities of 0.4 -0.9 ϫ 10 9 cpm/g. The following cDNA sequences were used as probes: the full-length matriptase cDNA (pcDNA3/matriptase) ligated into the pcDNA3 vector (Invitrogen) under the control of a cytomegalovirus promoter (26). uPA cDNA (27) and uPAR cDNA (9) were prepared as described. For pSP64-hPAI-1, a 1.4-kbp EcoRI-BglII fragment (position 54 -1480 (28)) isolated from pPAI1-C1, a plasmid containing a 2.2-kbp human PAI-1 cDNA insert (29), was subcloned between the BamHI and EcoRI sites of pSP64 (30). Filters were reprobed with the cDNA for glyceraldehyde-3phosphate dehydrogenase (GAPDH) to correct for the amount of RNA loaded onto the filters (9,27). After hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at Ϫ70°C. Filters were quantitated by scanning densitometry using a Bio-Rad model 620Video Densitometer with one-dimensional Analyst software package for Macintosh.
Bikunin Transfection-The bikunin expression vector pCMVbikunin-IRES-bsr (bik ϩ ) and the control vector pCMV-luciferase-IRESbsr encoding luciferase (luc ϩ ) were transfected into HRA cells by the standard calcium phosphate precipitation method as described previously (11). Bik ϩ clones (clones 1-5) with inducible bikunin protein expression were confirmed by immunocytochemical staining and Western blot analysis. Bik ϩ clone 1 produces the highest concentration of bikunin protein, and clone 1 was mainly used throughout this study. Uninduced parental HRA cells or Luc ϩ cells were used as a control (11).
Antisense Matriptase Oligodeoxynucleotides and Cell Transfection-Antisense 18-base phosphorothioate oligodeoxynucleotides, corresponding to the human matriptase mRNA, were synthesized and consisted of the antisense sequences of 5Ј-AGC TGC TCA TCC TAG GCA-3Ј (ASmatriptase) (15). Oligonucleotides mixed with Lipofectin reagent were incubated for 15 min at room temperature. Thereafter, the oligonucleotides-liposome complexes were then added to cells and washed twice with medium (31). After 4 h, fresh normal growth medium containing 10% fetal bovine serum was added. The AS-matriptase clones were used for ECM invasion and in vivo metastatic experiments, as described below.
Antisense uPA cDNA Transfection-A 1020-bp PstI and EcoRI digest cDNA fragment of uPA was blunt-ended and cloned in antisense orientation in a HindIII cut ph␤APr-1-neo eukaryotic expression vector (32). The HRA cells were transfected with antisense uPA cDNA-containing vector (AS-uPA) or empty vector, using Lipofectin, as described (33,34) and according to the manufacturer's instructions.
uPA cDNA Transfection-The HRA cells were transfected with the expression plasmid pcUKLTR6, containing a human full-length uPA cDNA construct, and the plasmid pSVneo, encoding the neomycin resistance gene (33,35). The uPA expression plasmid contains a 1300-bp SmaI-SmaI fragment containing the enhancer-promoter element and the start of transcription from the Harvey sarcoma virus 5Ј-long terminal repeat. A separate transfection with expression plasmid pSVneo was performed in the HRA cell line to serve as a control.
Cell Surface Receptor Binding-Pro-uPA and diisopropylfluorophosphate (DFP)-treated high molecular weight (HMW)-uPA (150 g/100 l) were each radiolabeled with 1 mCi of Na 125 I in the presence of 10 g of chloramine T and 0.15 M sodium phosphate (pH 7.5), and after adding 83 g of sodium metabisulfite were purified on a PD-10 column (Amersham Biosciences) equilibrated and eluted with 0.2% BSA in PBS (33). Specific activities were typically 3.0 -4.1 Ci/g. For binding studies, 200-l aliquots of 5 ϫ 10 5 acid-treated cells in binding buffer (Dulbecco's modified Eagle's medium containing 0.1% BSA plus 0.2% sodium azide) were incubated with increasing concentrations of 125 I-DFP-uPA in the presence or absence of an excess (up to 1000-fold) of HMW-uPA. After 2 h at 4°C, the cells were washed by centrifugation twice with cold binding buffer and counted. For displacement assays, 200-l aliquots of 5 ϫ 10 5 cells in binding buffer were treated with 1.0 nM 125 I-DFP-uPA and differing concentrations of competitors (HMW-uPA, LMW-uPA, and amino-terminal fragment (ATF)). After 2 h, the cells were washed, and the bound radiolabel was quantitated by dissolving cells in NaOH. Specific binding was quantified using a multiwell ␥-counter.
Flow Cytometric Analysis-Cultured cells were harvested and washed with binding buffer. Because uPAR is trypsin-sensitive, cells were treated with hyaluronidase and used for further experiments. A single cell suspension (10 6 /ml) was incubated with affinity-purified antibodies or isotype control antibodies on ice for 1 h. Cells were washed three times with binding buffer, and 2 l of the primary antibody (3689 (uPA), 379 (PAI-1), and 3932 (uPAR)) and 3 l of fluorescein isothiocyanate-conjugated second antibody (Dako) were added for 1 h on ice, respectively. Cells were analyzed in a FACScan (BD Biosciences). At least 10,000 cells were analyzed per sample in all experiments.
Quantitation of uPA Level, Enzyme-linked Immunosorbent Assay-The amount of uPA was measured in the cell-conditioned medium and in the cell lysate using the Imubind uPA ELISA kit from American Diagnostics Inc. Lower detection levels were set at 10 pg/ml. The uPA assay measured all forms of uPA and its complexes.
Measurement of uPA Amidolytic Activity-The amidolytic activity of plasma membrane-bound uPA was monitored by the release of p-nitroaniline from Spectrozyme UK, measured by the rate of change in light absorbance at 405 nm as described (36) with a minor modification. The amidolytic activity induced by the binding of pro-uPA to its receptor was determined on cell surfaces. Confluent cultures of cells grown in 96-well tissue culture plates were washed twice with PBS, incubated for 5 min with glycine buffer (pH 3), to elute endogenous uPA, and washed two additional times. The cells incubated with 10 nM scuPA for 3 h at 4°C were washed and then incubated with PBS containing 300 M Spectrozyme UK and 1 M Glu-plasminogen in the presence and the absence of 10 g/ml anti-plasmin antibodies (3641 or 3644) or nonimmune IgG for 2 h at 37°C, and the A 405 was determined.
Western Blot Analysis-Conditioned media and centrifuged cell lysates (50 g) from each cell line were analyzed by SDS-PAGE and transferred to a PVDF membrane by semi-dry transfer. Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 2% BSA. Blots were probed with the following primary antibodies overnight at 4°C: monoclonal anti-uPA (3471 plus 3689) (each 1:500), anti-matriptase antibody (1:2000), anticathepsin B antibody (1:200), and anti-cathepsin L antibody (1:500). This was followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Dako) at a dilution of 1:50,000 for 1 h. Detection was achieved by enhanced chemiluminescence (Amersham Biosciences) and exposed to film.
Zymographic Analysis-Plasminogen activator activities were qualitatively analyzed by fibrin zymography or casein film fibrinolysis assay. SDS-PAGE zymographic analysis was performed using copolymerized plasminogen-rich fibrinogen. Equal volumes (20 l) of 10ϫ concentrated conditioned medium were mixed with 4ϫ nonreducing buffer (200 mM Tris-HCl (pH 7.4), 10% SDS, 4% sucrose, and 0.4% Coomassie Blue) for 30 min at 25°C. Each sample was then subjected to electrophoresis on polyacrylamide gel. After electrophoresis, gels were incubated in buffer containing 2.5% Triton X-100 (twice for 30 min at room temperature), rinsed in distilled water, and incubated overnight at 37°C in 100 mM Tris buffer (pH 7.4) containing 15 mM CaCl 2 .
Cleavage of Receptor-bound Pro-uPA-Unstimulated and TGF-␤1stimulated cells were washed twice with PBS containing BSA and 1000 kallikrein-inactivating units/ml of Trasylol (pH 7.4), followed by two washes in PBS/BSA. Subsequently, 1.0 ϫ 10 7 cells were resuspended in 0.5 ml of PBS/BSA in the presence of 5 nM 125 I-pro-uPA and then incubated for 2 h at 4°C. Cells were washed in PBS/BSA and then incubated for 80 min at 37°C in PBS/BSA. The reaction was stopped by washing with cold PBS twice and then treated with 50 mM glycine HCl, 0.5 M NaCl, pH 3.0 (acidic buffer). The supernatants were obtained by centrifugation and immediately adjusted to pH 7.4. The supernatants were concentrated, dissolved in electrophoresis sample buffer with or without 2-mercaptoethanol, and then boiled for 3 min. Proteins separated by SDS-PAGE were electrophoretically transferred to PVDF membrane and visualized using a PhosphorImager.
MTT Assay for Cell Proliferation-Cells were seeded in 96-well plates at a density of 1000 cells/well. The following day, the medium was changed to regular medium. Viable cells were measured using the quantitative colorimetric MTT assay kit according to the manufacturer's protocol.
In Vivo Peritoneal Carcinomatosis Model-In order to assess survival, three groups of mice were inoculated intraperitoneally with 0.1 ml of tumor cell suspension (5 ϫ 10 6 cells; parent HRA cells, bik ϩ clones, and luc ϩ clones) in sterile PBS. All animals receiving tumor cells intraperitoneally were monitored on a daily basis for tumor burden, abdominal distension, weight, cachexia, or other abnormalities. Once life-threatening symptoms became markedly manifest, the animal was killed by cervical dislocation.
Statistics-Data are expressed as mean Ϯ S.D. of at least three independent triplicate experiments. Statistical analysis was performed by one-way analysis of variance followed by the Student's t test. p Ͻ 0.05 was considered statistically significant. The log-rank test was used to analyze the survival of animals injected with each subline.

RESULTS
Invasive Potential of HRA Cells-Several studies point to a leading role of the PA and MMP systems in the development of tumor progression (37). The uPA up-regulation by TGF-␤ has been observed in a number of cell types and neoplastic cell lines including HRA cells (38 -40). Our previous observation (10) that HRA cells produce a significant amount of uPA but do not produce a detectable level of MMPs, as determined by Western blot and zymography, rules out the possibility that the MMP system has a functional role in invasiveness of these cells. In agreement with this assessment, we observed that invasiveness of HRA cells is sensitive to pretreatment with the neutralizing antibodies directed against uPA but resistant to pretreatment with the neutralizing antibodies to MMP-2/9 (data not shown). We therefore conclude that the TGF-␤-dependent invasiveness of HRA cells involve uPA-dependent and MMPindependent pathways.
Effect of AS-Matriptase Transfection on Matriptase Protein Expression-Matriptase is localized at the surface of epithelial cells and then released into conditioned medium. HRA cells were transfected with bikunin (bik ϩ ), AS-matriptase, and ASmatriptase (two independent transfection experiments), AS-uPA, or a luciferase (luc ϩ ) control. As shown in Fig. 1, when conditioned medium (A) and cell lysate (B) prepared from each cell were electrophoresed under nonreduced conditions and immunoblotted with anti-matriptase monoclonal antibody M32, two bands of polypeptides with apparent sizes of 75 and 70 kDa were detected in conditioned medium; three bands of polypeptides with apparent sizes of 100, 75, and 70 kDa were detected in cell lysate (Fig. 1). The 100-kDa band is consistent with a matriptase-HAI-1 complex (41). Matriptase expression in AS-uPA cells resembled that of parental HRA and luc ϩ cells. On the other hand, AS-matriptase transfection resulted in significant reduction of matriptase protein expression. Densitometric analysis showed 70 -80% reduction of matriptase protein expression by AS-matriptase transfection. Under our conditions, AS-matriptase or AS-uPA transfection did not induce any death signaling (data not shown). In a parallel experiment, we showed that bik ϩ clone 1 markedly reduced expression of matriptase protein, whereas luc ϩ transfection did not reduce the expression. In addition, other bik ϩ clones (clones 2-5) also significantly reduced the matriptase expression (data not shown). Therefore, Fig. 1 provides data on the extent to which matriptase protein production and release are specifically re-duced by the antisense strategy and bikunin gene transfection.
Effect of AS-Matriptase Transfection on Secretion of uPA, ELISA Analysis-As assessed by uPA ELISA, day 1-cultured HRA cells spontaneously released detectable amounts of uPA into the culture conditioned medium (Fig. 2). We reported previously (15) that TGF-␤1 specifically increased uPA mRNA in HRA cells, promoting cell invasion and metastasis, and that bikunin reduced the TGF-␤1-stimulated uPA mRNA expression. Here we show that uPA release in response to TGF-␤1 was dose-dependent, with maximal effects at 4.0 ng/ml. ASmatriptase cells did not reduce secretion of uPA in unstimulated and TGF-␤1-stimulated cells. In a parallel experiment, we found that bik ϩ clone 1 and AS-uPA transfection markedly reduced uPA secretion, whereas S-uPA transfection only marginally changed secretion of uPA protein.
Effect of AS-Matriptase Transfection on Expression and Activation of Pro-uPA, Binding of Pro-uPA to Its Cell Surface Receptor and Subsequent Activation-Based upon the fact that the activation of pro-uPA stems from its binding of pro-uPA to its receptor, we evaluated activation of receptor-bound pro-uPA on the surface of each cell. Unstimulated and TGF-␤1-stimulated cells (parental HRA cells, AS-matriptase cells, bik ϩ clone 1, and luc ϩ control cells), treated with acidic buffer, were incubated with radioiodinated pro-uPA for 2 h at 4°C, washed, and subsequently incubated for further 80 min at 37°C. After washing, uPA molecules were dissociated from each cell by acid treatment, analyzed by SDS-PAGE under nonreduced (A) and reduced (B) conditions, and visualized using a PhosphorImager. Receptor-bound uPA under nonreduced conditions showed a low basal expression of the 50-kDa uPA in HRA cells (Fig. 3A, lane 3) and AS-matriptase cells (lanes 5 and 7). TGF-␤1, at 10 ng/ml, significantly increased receptor-bound uPA in both HRA cells (lane 4) and AS-matriptase cells (lanes 6 and 8). Densitometric scans demonstrated that TGF-␤1 increased the binding of labeled pro-uPA in both cells by 4 -5-fold compared with unstimulated controls. In agreement with the flow cytometric analysis results (see below), TGF-␤1 increased the binding of labeled pro-uPA. Bikunin transfection significantly reduced the unstimulated (lane 9) and TGF-␤1-stimulated, receptor-bound uPA expression (lane 10), whereas the uPA expression was not affected by transfection of cells with AS matriptase (lanes 5-8 versus lanes 3 and 4). Furthermore, the amount of receptor-bound uPA was not significantly affected by transient transfection of cells with AS-uPA (lanes 13 and 14). In a parallel experiment, we evaluated the expression pattern of the cell-associated uPA protein in luc ϩ clones. Like parental HRA cells, similar receptor-bound uPA expression level was observed in luc ϩ clones (lanes 11 and 12).
As shown in Fig. 3B, under reduced conditions, the Phospho-rImager detected three bands corresponding to 50 (proenzyme), 30 (B-chain of uPA), and 20 kDa (A-chain of uPA), respectively, in TGF-␤1-stimulated HRA cells (lane 4). The 30-and 20-kDa bands result from pro-uPA activation and reflect the level of its activation. The levels of 30-and 20-kDa bands were reduced in bik ϩ clone 1 (lanes 9 and 10 versus lanes 3 and 4). Interestingly, the level of the 50-kDa form was enhanced by AS-matriptase, but very weak signals of the 30-and 20-kDa proteins were detected in these cells (lanes 5-8). As expected, activation of receptor-bound uPA was markedly suppressed in AS-uPA cells (lanes 13 and 14). These results demonstrate that bikunin mediates down-regulation of uPA protein expression and functional uPAR protein expression, whereas matriptase may be involved only in the activation process of pro-uPA but not uPAR protein expression.
Activation of Pro-uPA in the Surface of AS-Matriptase Cells-We monitored conversion of receptor-bound pro-uPA to enzymatically active two-chain HMW-uPA by fibrin zymography in these cells when cultured for 80 min. As shown in Fig.  4A, zymography showed the presence of a proteolytic activity at 50 kDa in the receptor-bound uPA of TGF-␤1-stimulated HRA cells (Fig. 4A, lane 4), consistent with the pattern of recombinant HMW-uPA used as a control (lane 2). Only low levels of the enzymatically active form of uPA were detected in unstimulated ( lanes 5 and 7) and TGF-␤1-stimulated AS-matriptase cells (lanes 6 and 8) compared with those of parental HRA cells (lanes 3 and 4). AS-uPA transfection significantly reduced activation of receptor-bound pro-uPA on the cell surface (lanes 13  and 14). A similar uPA activation pattern was observed in the luc ϩ clone (lane 12 versus lane 4). uPA fibrinolytic activity was detected at a very low level in the bik ϩ clone 1 (lanes 9 and 10). Therefore, enzymatically inactive pro-uPA was detected as the major species in AS-matriptase cells, suggesting matriptase specifically promotes activation of pro-uPA. Tissue plasminogen activator was undetectable in HRA cells under these culture conditions.
In order to directly demonstrate that matriptase affects uPA activation in living cells, we took advantage of the fact that uPA has the ability to degrade casein through plasminogen activation. We tested whether AS-matriptase might decrease caseinolytic activity in HRA cells (Fig. 4B). Cells were seeded in a pellet on a reconstituted casein film and incubated in serumfree medium containing plasminogen. Cells expressing luc ϩ control (lane 2) and parental HRA (lane 1) readily degraded the underlying casein film to a similar extent. However, neither AS-matriptase cells (lanes 3 and 4) nor bik ϩ clones (lanes 5 and 6) degraded as much the underlying casein matrix as the parental HRA cells.

Flow Cytometric Analysis of Expression of Plasma Membrane-bound uPA, PAI-1, and uPAR Proteins on Unstimulated and TGF-␤1-stimulated Cells (Parental HRA Cells, AS-
Matriptase Cells, Bik ϩ Cells, and Luc ϩ Cells)-Binding of antibodies directed against uPA B-chain, PAI-1, and uPAR was assessed by flow cytometry using the unstimulated and TGF-␤1 (10 ng/ml, 12 h)-stimulated cells (Fig. 5). Nonimmune mouse IgG was used as the control IgG. cells. In addition, TGF-␤1 also significantly enhanced cell-surface expression of these proteins in AS-matriptase cells. Interestingly, there are no significant differences in expression of uPA, PAI-1, and uPAR proteins between parental HRA cells and AS-matriptase cells, irrespective of whether these cells were stimulated with TGF-␤1. In contrast, following in vitro culture of bik ϩ cells with TGF-␤1, there were 1.8 (164 versus 91 (fluorescence mean channel))-, 1.6 (58 versus 36)-, and 1.5-fold (212 versus 141) increases in cell surface expression of uPA, PAI-1, and uPAR, compared with unstimulated bik ϩ cells. Bikunin transfection significantly reduced expression of uPA and uPAR, but not PAI-1, although TGF-␤1 also weakly enhanced cell-surface expression of uPA and uPAR proteins in bik ϩ cells. Control transfection (luc ϩ cells) did not affect expression of the PA system proteins.
Binding of uPA to uPAR on AS-Matriptase Cells-To characterize the binding of DFP-treated two-chain HMW-uPA (DFP-uPA) to the cells, possibly via uPAR, radiolabeled DFP-uPA was displaced by unlabeled competitors from each cell. As shown in Fig. 6A, unlabeled ATF and HMW-uPA displaced the binding of 125 I-DFP-uPA to the acid-pretreated HRA cells in a similar fashion. In contrast, LMW-uPA had no significant effect. The EC 50 , the concentration of unlabeled protein necessary for 50% displacement, was 2.1 nM for HRA cells (Fig. 6A) compared with 2.7 nM for AS-matriptase cells (Fig. 6B). Furthermore, we showed that the binding to HRA and ASmatriptase cells of 125 I-DFP-uPA was blocked in the presence of the anti-uPAR monoclonal antibody 3936, which completely blocked uPA binding to uPAR (data not shown).
In a parallel experiment, we tested direct binding of 125 I-DFP-uPA to HRA and AS-matriptase cells pretreated with acidic buffer. Fig. 6C shows that 125 I-DFP-uPA bound to the  These data suggest that half-maximal binding was apparent at ϳ2 nM with 125 I-DFP-uPA in both cells and that an interaction between uPA and uPAR is preserved in the AS-matriptase cells. Therefore, it is likely that AS-matriptase-mediated reduction of uPA activation does not coincide with suppression of uPA expression and secretion. Furthermore, AS-matriptase transfection does not affect uPAR expression nor its function.

Effect of Bikunin or AS-Matriptase Transfection on Expression of uPA, PAI-1, and uPAR mRNA-
The RNA was isolated from the untransfected cells and bik ϩ clone 1, or AS-matriptase cells. Expression of the PA system proteins was analyzed by Northern blot analysis. The GAPDH bands are shown as a control. As shown in Fig. 7, the levels of uPA, PAI-1 and uPAR mRNA expression were almost identical in the parental cells and AS-matriptase cells. The densitometric data indicated that there were no differences in the PA system expression in these cells (data not shown). In contrast, bikunin transfection significantly reduced expression of uPA and uPAR mRNA but not PAI-1 mRNA. We reported previously that TGF-␤1 increased uPA and uPAR mRNA in HRA cells and that bikunin reduced the TGF-␤1-stimulated uPA and uPAR mRNA expression. Therefore, we determined whether AS-matriptase transfection could regulate uPA and uPAR mRNA expression in TGF-␤1stimulated cells. Here we show that TGF-␤1, at a concentration of 10 ng/ml, significantly increased uPA and uPAR mRNA levels even in AS-matriptase cells and that a high basal uPA and uPAR mRNA was observed in cells stimulated with TGF-␤1, irrespective of whether cells were transfected with AS-matriptase.
Matriptase Is Needed for Receptor-bound Pro-uPA Activation-Plasmin and cathepsins (B and L) are proposed to be necessary components for uPA activation at the cell surface (37). We found that, in the HRA and AS-matriptase cells, plasmin activity was predominantly responsible for the cell membrane activation of pro-uPA, when Glu-plasminogen was exogenously added to these cells, and that AS-matriptase transfection does not affect expression of cathepsin B and L proteins (data not shown).
Because plasmin can still process pro-uPA in AS-matriptasetransfected cells, we tested whether matriptase is really needed for receptor-bound pro-uPA activation. We performed colorimetric assay in the presence of anti-plasmin antibodies (3641, which does not affect plasmin activity; and 3644, which competitively inhibits plasmin activity) or control nonimmune mouse IgG in the TGF-␤1-stimulated HRA cells and TGF-␤1stimulated AS-matriptase-transfected cells. As shown in Fig. 8, cell-associated uPA activity of these two cells was markedly inhibited by neutralizing anti-plasmin antibody 3644 but slightly but significantly enhanced by 3641. Antibody 3641 has been reported to promote the conversion of Glu-plasminogen to the enzymatically active plasmin (data sheet from American Diagnostics). Therefore, this seems to be in accordance with the 3641-induced enhancement of cell-associated uPA amidolytic activity. Furthermore, there was a dramatic suppression of cell-associated uPA activity (by ϳ75%) when AS-matriptase cells were treated with neutralizing anti-plasmin antibody 3644. Therefore, we can say that matriptase system is apparently responsible for receptor-bound uPA activation process.

HRA Cells Transfected with Bikunin, AS-Matriptase, AS-uPA, or S-uPA Exhibit Distinct Invasive Properties in Matrigel
Invasion Assay-Each transfected cell was further used for ECM invasion assay in order to establish their roles in ECM invasion. The cells tested were initially stimulated with 10 ng/ml TGF-␤1. To assess whether the uPA system is responsible for this process, we monitored cell invasion in the presence of an inhibitor for uPA, amiloride, as well as neutralizing antibodies. As shown in Fig. 9, invasion of HRA cells through Matrigel was markedly inhibited by amiloride, anti-uPA, or anti-uPAR-neutralizing antibodies. No enhancement of invasion was observed in S-uPA cells. There was a dramatic suppression of ECM invasion (by ϳ65%) when AS-uPA cells were used. As expected, the bik ϩ clone 1 resulted in ϳ50% reduction in cell invasion. We evaluated the biological effect of reduced matriptase expression by analyzing the ability of transfected HRA cells to invade Matrigel. Fig. 8 shows significant decreases in cell invasion by 35-40% when AS-matriptase cells were used in the upper chamber. Indeed, AS-matriptase cells had a decreased ability to invade the Matrigel layer, as compared with luc ϩ clone or parental HRA cells that had not been transfected at all. Cell invasive ability was not affected by luc ϩ transfection procedures.
In a separate experiment, chemotaxis assays were performed in the absence of Matrigel. Chemotaxis ability was not affected by transfection with bikunin or AS-matriptase (data not shown), suggesting that the result rules out the possibility that AS-matriptase cells fail to invade Matrigel because their ability to migrate is impaired. Our results support the notion that matriptase mediates cell invasion through activation of receptor-bound pro-uPA.
To examine whether matriptase has any role in the growth rate of these cells, we performed a proliferation assay using AS-matriptase cells. The MTT assay showed that ASmatriptase transfection does not affect cell growth (94 -107%) in these cells (data not shown).

Reduced Matriptase Expression Inhibits Intraperitoneal Tumor Growth in Nude
Mice-The in vitro results prompted us to examine whether matriptase has any role in tumor growth and regulation of uPA expression in an in vivo system. To demonstrate further the anti-tumoral activity of AS-matriptase in ovarian cancer, we evaluated the effect of reduced matriptase expression on the tumor development of HRA cells by intraperitoneal injection into nude mice. Tumor cells were inoculated intraperitoneally into mice, and tumor size was evaluated at day 8 (Fig. 10A). No significant tumor reduction or growth was observed for luc ϩ clone and S-uPA cells. The weights of the S-uPA-transfected tumors were almost identical to the tumors of the non-transfected mice. When AS-uPA cells were injected into mice, the size of the tumors was reduced drastically (Fig.  10A), compared with luc ϩ -or S-uPA-transfected mice. The intraperitoneal tumor growth of bik ϩ clone 1 and ASmatriptase cells was significantly inhibited, as compared with parental HRA cells (ϳ60% inhibition for bik ϩ , 30-40% inhibition for AS-matriptase). Expression and activity of uPA were confirmed by Western blot and zymographic analyses of the intraperitoneal tumors obtained at day 9, demonstrating that bik ϩ clones, AS-matriptase cells, and AS-uPA cells showed a weak signal for enzymatically active uPA (data not shown).
Survival of Tumor-bearing Mice-We ascertained the relative survival times of nude mice after intraperitoneal transfer of each subline (Fig. 10B). The mean survival times of nude mice receiving 5 ϫ 10 6 parental HRA cells (n ϭ 7) and the luc ϩ clone (n ϭ 7) were both 8 days. The mean survival times of animals receiving the bik ϩ clone (n ϭ 7), AS-matriptase cells (n ϭ 9), AS-matriptase cells (n ϭ 9), AS-uPA cells (n ϭ 7), and S-uPA cells (n ϭ 7) were increased or decreased to 12, 11, 10, 17, and 7 days, respectively. There was a significant increase in the mean survival times of the group receiving the AS-uPA cells, bik ϩ clone, AS-matriptase cells compared with the luc ϩ clone and the parental HRA cells (p Ͻ 0.05). Interestingly, AS-uPA transfection markedly prolonged survival times of mice. Consistent with the delayed growth of the intraperitoneal tumors, the survival time was prolonged. DISCUSSION Previously, our group reported that bikunin inhibits invasiveness and metastasis of the mouse and human cancer cell lines, possibly via suppression of uPA and uPAR expression at the mRNA and protein levels, without affecting in vitro cell proliferation (7)(8)(9)(10)(11)(12)(13)(14). A previous microarray study (15) showed that exposure of a highly invasive ovarian cancer cell line HRA to bikunin or bikunin gene overexpression markedly reduced the gene expression of a set of genes including matriptase, which had been shown by other investigators to activate uPA in an in vitro, uPAR-free, solution-based study (18,19). A previous microarray study by us identified down-regulation of ϳ30 genes (15). Thus, bikunin alters the pattern of gene expression in HRA cells leading to a block in cell invasion and metastasis. Among these genes, matriptase was markedly down-regulated by bikunin.
We initially analyzed whether HRA cells express uPA and uPAR. Zymography, ELISA, flow cytometry, ligand binding and competition assays, and Western and Northern blot analyses showed whether the HRA cells express any functional uPA and uPAR. The previous (8 -11) and present results clearly suggest a direct role for uPA in the invasion of HRA cells. Our data are supported by the facts that the localization of active uPA at the cell surface uPAR is essential for ECM invasion and tumor growth (2) and that AS-uPA expression in transformed malignant rat fibroblasts correlates with decreases in tumor growth (32)(33)(34)(35). In this study, we showed that transfection of HRA cells with AS-uPA caused pronounced inhibition of uPA protein expression and drastic suppression of ECM invasion, whereas transfection of cells with S-uPA gene does not markedly stimulate cell invasiveness and intraperitoneal disseminated metastasis. This suggests that the proteolytic cascade generated by a cell may be multiform, and solitary alterations in uPA may not modify the proteolytic capability for invasion and metastasis. Jarrard et al. (35) reported that uPA is necessary but not sufficient for cancer cell invasion. Another possibility is that the HRA cells do not require additional uPA expression for cell invasion.
We next tested the hypothesis that down-regulation of matriptase expression plays a role in inhibition of unstimulated or TGF-␤1-stimulated receptor-bound pro-uPA activation and decreased in tumor invasiveness and disseminated metastasis. TGF-␤1 addition to HRA cells elicited uPA expression and activation, as judged by Northern and Western blot analyses, as well as the appearance of enzymatically active uPA. Given the well established connection between uPA expression/ activation and cellular invasiveness, the hypothesis proposed is a reasonable one. In the current study, we employed both stable and transient transfection approaches to achieve overexpression of bikunin or antisense inhibition of matriptase expression in HRA cells. The binding of uPA to uPAR is thought to be a prerequisite of uPA activation. To this end, it is important to note that the earlier in vitro evidence that matriptase activates uPA was obtained from a uPAR-free, solution-based study (18). Because it is critical to investigate whether the matriptase-mediated activation depends on the interaction between uPA and uPAR, we attempt to address how matriptase can activate receptor-bound pro-uPA. The present study showed that reduced matriptase expression led to suppression of receptor-bound pro-uPA activation, decreased cell invasion in vitro, and decreased tumor growth in an intraperitoneal xenograft animal model.
In this experiment, HRA cells carrying an antisense to matriptase or to uPA, or overexpressing bikunin, proved to be impaired in Matrigel invasion. Matriptase can convert hepatocyte growth factor/scatter factor to its active form, which can induce scatter of Madin-Darby canine kidney epithelial cells and can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells (18). Matriptase is involved in the processing of pro-uPA and pro-hepatocyte growth factor, both being motility factors (the chemotactic activity of uPA is catalytically independent). Therefore, at least another interpretation may be considered is that cells do not invade Matrigel because their ability to migrate is impaired. However, ASmatriptase transfections were not significantly impaired for their chemotactic ability. These results allow us to conclude that matriptase predominantly mediates cell invasion, rather than chemotaxis, through activation of receptor-bound uPA.
Bikunin transfection reduced uPA expression at the mRNA and protein level (11). However, the levels of total uPA proteins (pro and active forms) in AS-matriptase cells were almost identical to non-transfected controls, suggesting that the decrease in uPA activity in AS-matriptase cells does not result from a decrease in the level of uPA mRNA and protein expression. Because bikunin down-regulates both matriptase and uPA expression, it is possible that the reduced matriptase expression by bikunin overexpression may contribute to the bikunin-repressed invasiveness. However, it is very unlikely that the results derived from matriptase silencing (inhibiting only the activation but not the expression of uPA) represent the action of bikunin. The present study, using the matriptase antisense strategy, cannot explain all of the biological functions of bikunin.
In addition, we have detected a decreased level of uPAR expression in bikunin clones, but not in AS-matriptase cells. Activation of uPA could be carried out at the cell surface through interaction with uPAR (42). Plasmin, plasma kallikrein, trypsin-like proteases from human ovarian tumors, a T cell-associated serine protease, cathepsins B and L, nerve growth factor ␥, human mast cell tryptase, prostate-specific antigen, and matriptase have been reported to activate pro-uPA in a uPAR-free solution-based study (18). Our previous report (43) showed that cathepsin B significantly activates both soluble and receptor-bound pro-uPA. In the present study, we now show for the first time that matriptase can act as an activator to convert enzymatically inactive pro-uPA to active uPA on the HRA cell surface.
More recently, we have established that PI3K p85 is also a candidate bikunin target gene (15). We transfected HRA cells with PI3K p85 AS-oligodeoxynucleotide and compared the properties of the transfected cells to those of parental cells. Our preliminary data showed that enforced expression of AS PI3K oligodeoxynucleotide significantly reduced TGF-␤1-stimulated uPA mRNA and protein expression. In addition, the AS PI3K cells had a decreased ability to invade the ECM layer, as compared with controls, and when the AS PI3K cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed a longer survival. We speculate that PI3K plays an essential role in promoting FIG. 10. Suppression of matriptase expression reduces intraperitoneal tumor growth and shows better prognosis. A, matriptase down-regulation inhibits intraperitoneal (i.p.) tumor growth in nude mice. Each HRA cell (5 ϫ 10 6 cells/200 l; parent HRA cells, bik ϩ clone 1, AS-matriptase and AS-matriptase, AS-uPA cells, S-uPA cells, and luc ϩ clone) was implanted intraperitoneally into the abdomen of female athymic nude mice. Four mice were used in each set of experiments. Mice were followed for tumor growth for 8 days. Data represent mean Ϯ S.D. tumor volumes at day 8. This experiment is representative of two independent experiments. B, survival of nude mice after intraperitoneal transfer of tumor cells. These groups of female nude mice were injected intraperitoneally with 5 ϫ 10 6 tumor cells on day 0 and monitored daily for morbidity. Survival of the animals was monitored, and the data were analyzed statistically by the log-rank test. Survival of AS-uPA, bik ϩ clone 1, AS-matriptase, and AS-matriptase inoculated mice was significantly longer than the parent HRA mice and luc ϩ transfectant mice. *, p Ͻ 0.05 versus HRA. There was no significant difference between the S-uPA cell group and the parental cell group. Results are representative of two separate experiments. the uPA-mediated invasive phenotype in HRA cells. These preliminary data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion. Bikunin will also have additional global effects on cancer cells by modulating the expression of a large number of cellular genes. Clearly, the genomic response to bikunin signaling is complex. Future studies focused on the regulation and functional significance of the target genes should increase our knowledge of the biological activity of bikunin in neoplastic cells.
In conclusion, we provide evidence for the first time that blocking matriptase expression by its antisense strategy abrogates activation of receptor-bound pro-uPA in ovarian cancer HRA cells. Decreased invasiveness, tumor growth, and peritoneal disseminated metastasis may correlate, at least in part, with decreased expression of matriptase, which may be one of the bikunin-target gene family.