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Carcinogenesis Advance Access originally published online on June 29, 2007
Carcinogenesis 2007 28(11):2274-2281; doi:10.1093/carcin/bgm140
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

ECRG2 inhibits cancer cell migration, invasion and metastasis through the down-regulation of uPA/plasmin activity

Ge Huang1, Zhi Hu1,3, Meining Li1, Yongping Cui1, Yuanyuan Li1, Liping Guo1, Wei Jiang1,2,* and Shih Hsin Lu1,*

1 Department of Etiology and Carcinogenesis, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China
2 The Burnham Institute, La Jolla, CA 92037, USA
3 Present address: Department of Laboratory Medicine and Comprehensive Cancer Center, University of California, San Francisco, CA 94115, USA

* To whom correspondence should be addressed. Tel: +86 10 87788408; Fax: +86 10 67712368; Email: shlu{at}public.bta.net.cn

Correspondence may also be addressed to Wei Jiang Email: wjiang{at}burnham.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The esophageal cancer-related gene 2 (ECRG2) is a novel gene that shows sequence similarity to KAZAL-type serine protease inhibitor. In this study, the migration and invasion of PG cancer cells were inhibited by ectopic expression of ECRG2 in vitro, and metastases decreased after injecting PG/pcDNA3.1-ECRG2 cells into the tail veins of nude mice. Control mice were injected with PG/pcDNA3.1 cells. To test the hypothesis that ECRG2 interacts with proteases and inactivates extracellular matrix degradation, binding affinity and co-immunoprecipitation experiments were performed using serum-free conditioned medium. The results showed that ECRG2 bound to two species of urokinase-type plasminogen activator (uPA) with molecular weights of 55 and 33 kDa. Furthermore, analysis of the uPA/plasmin activity showed that expression of ECRG2 reduced proteolysis of the plasmin substrate D-Val-Phe-Lys-p-nitroanilide, which was seen by a decrease of absorbance at 405 nm. Taken together, these results suggested that ECRG2 inhibits aggressiveness of cancer cell, possibly through the down-regulation of uPA/plasmin activity.

Abbreviations: ECM, extracellular matrix; ECRG2, esophageal cancer-related gene 2; MMP, matrix metalloprotease; PAI, plasminogen activator inhibitor; RECK, reversion-inducing cysteine-rich protein with kazal motif; SDS, sodium dodecyl sulfate; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Proteases that degrade the extracellular matrix (ECM) have long been viewed as essential for tumor progression and invasion into adjacent normal tissue. Production of proteases that degrade matrix barriers permits tumor cells to invade into surrounding connective tissues, as well as blood vessels, and both of these events are essential for metastasis to distant organs (1,2). Due to the multiplicity of ECM components, a variety of diverse proteases are likely required to participate in metastatic progression (3–5). Such proteases are subject to strict regulatory mechanisms including genetic regulation of their synthesis, secretion, catalytic activity and the presence of specific natural inhibitors. The most direct way to inhibit protease function is to block its enzymatic activity, and this has consequently led to intensive investigation into the therapeutic potential of protease inhibitors. However, while some well-known inhibitors, such as matrix metalloprotease inhibitors and plasminogen activator inhibitor (PAI)-1, have proven effective in experimental models, in the clinical setting, they exhibited a disappointing lack of efficacy in the treatment of human cancers (6–8). The innate cellular heterogeneity of tumors is the major cause of the therapeutic failure of these inhibitors. Moreover, this situation has also highlighted two urgent issues: which inhibitors are important in which cancers and precisely what are these inhibitors doing during tumor progression? In recent years, much research effort has focused on the cloning and identification of novel genes which may critically affect proteolytic degradation.

The esophageal cancer-related gene 2 (ECRG2) is a novel gene that contains kazal-like Pfam domains (9). The presence of Pfam domains is characteristic of protease inhibitors. Pancreatic secretory trypsin inhibitor, acrosin inhibitor and elastase inhibitor all contain Pfam domains (10–12). Recently, a growing body of evidence has indicated that some members of these inhibitor families may be involved in preventing tumor metastasis. For example, Takahashi et al. (13) showed that reversion-inducing cysteine-rich protein with kazal motif (RECK) is a protease inhibitor which has transformation suppressor activity. Northern blot analysis has detected RECK transcript in a wide variety of tissues and normal cell lines, but the number of transcripts is reduced in transformed and cancer cells. The RECK protein in vitro regulates at least three members of the matrix metalloprotease (MMP) family: MMP2, MMP9 and membrane-type 1 (MT1)-MMP. Restoring the expression of RECK in cancer cell lines results in a strong suppression of invasion, metastasis and tumor angiogenesis (13–17). Tumor-associated trypsin inhibitor, another protease inhibitor, inhibits the activity of MMP1, 2, 3, 8, 9 and 13. Tumor-associated trypsin inhibitor also results in reduced invasion and metastasis in many tumor types (18–22). The structural resemblance between ECRG2 and the other members of the kazal family suggests that this protein may have both similar as well as distinctive functions within cells.

Previous studies have suggested that ECRG2 is involved in the regulation of cell proliferation and the induction of apoptosis (23,24). The ECRG2 gene is located in 5q32–33, a chromosomal region which contains no other putative tumor suppressor genes, and which has additionally been reported, to be a region of frequent allelic loss (79%) in transformed cells (25). Reverse transcription–polymerase chain reaction analysis has shown that ECRG2 is down-regulated in the esophageal cancers (26). These data collectively suggest that down-regulation of ECRG2 may play a role in cell transformation and tumor progression.

In the work presented in this study, it is shown that the ECRG2 protein can bind to and down-regulate the activity of urokinase-type plasminogen activator (uPA)/plasmin system and provides evidence that the ECRG2 gene plays an important role in the prevention of tumor cell migration and invasion by regulation of plasmin-mediated proteolysis of the ECM.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture and transfection
The highly metastasic lung cancer cell line PG which has undetectable levels of endogenous ECRG2 were cultured in a humidified 5% CO2 incubator in RPMI 1640 medium containing 10% fetal calf serum, 1% penicillin and 1% streptomycin. Open reading frame of ECRG2 cDNA was amplified by polymerase chain reaction from pT-Adv-ECRG2 as described previously (23) and subcloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). Cells were transfected by LipofectamineTM 2000 (Invitrogen) according to the manufacturer's standard plasmid transfection instructions. Briefly, 1 day prior to transfection, 2 x 106 cells in 15 ml of growth medium without antibiotics were plated into 10 cm culture dishes for in vitro transfection. The cells were 80–90% confluent at the time of transfection. For each 10 cm dish of cell culture, 24 µg plasmid DNA was added to 1.5 ml serum-free medium, and 60 µl LipofectamineTM 2000 was gently mixed in further 1.5 ml serum-free medium in separated tubes. After a 5 min incubation, the DNA solution was combined with diluted LipofectamineTM 2000 and incubated for 20 min at room temperature. This mixture was then added to the cells. After a 4 h of incubation at 37°C, the medium was replaced with RPMI 1640 medium containing 10% fetal bovine serum. Evaluation of the gene transfer efficiency was performed by using ß-gal staining at 24 h after the transfection. Cells were incubated with serum-free medium for 48 h and thus mediums were concentrated ~10-fold in Centricon-10 units (Amicon, Billerica, MA) for further studies. For stable transfection, cells were selected with full growth medium containing G418 (400 µg/ml). Colonies of G418-resistant cells were pooled after 3 weeks, and ECRG2-positive cells were detected by western blots.

Wound closure assay
Confluent cell monolayers were wounded by manually scraping the cells with a piece of sterile film. Debris was removed from the culture by washing with phosphate-buffered saline (PBS) twice, and the cells were then incubated with serum-free medium. Images were captured immediately after wounding, and wound sizes were verified with an ocular ruler to ensure that all wounds were the same width at the beginning of the experiment. Wound closure was monitored with microscopy at various times after the wound was formed (27).

Boyden chamber assay
Migration and invasive potential of PG cells were measured by an in vitro Boyden chamber assay described as before (28). Briefly, cells (2.5 x 104 for transwell migration assay and 1 x 105 for Matrigel invasion assay) in 0.5 ml of serum-free RPMI 1640 medium were added to the wells of 8 µm pore membrane Boyden chambers, either coated with (BD Biosciences, Franklin Lake, NJ) or without (Corning, Corning, NY) Matrigel. Cells were allowed to migrate or invade for 24 h. Cells that had not penetrated the filters were removed by scrubbing with cotton swabs. Chambers were fixed in 100% methanol for 2 min, stained in 0.5% crystal violet for 2 min, rinsed in PBS and examined under a bright-field microscope. Values for invasion and migration were obtained by counting five fields per membrane and represent the average of three independent experiments performed over multiple days.

In vivo metastasis assay
PG cells were stably transfected with pcDNA3.1-ECRG2 or pcDNA3.1. Six-week-old female BALB/c nude mice (seven mice per group) were injected through the tail vein with 1.5 x 106 cells. Animals were observed every 2 days, and all animals were killed after 40 days. The lungs were removed and inflated with 2 ml of 15% India ink dye, washed in PBS for 5 min and fixed in Fekete's solution (70% ethanol, 3.7% paraformaldehyde and 0.75 M glacial acetic acid). Surface lung metastases were scored by eye. Lung, liver, kidney and metastases in other sites, dorsal subcuticle and lymph node, were fixed and embedded in paraffin for histological analysis (28).

Immunoblot analysis
Protein concentrations from serum-free conditioned medium were measured with the Bradford assay, and equal amounts of proteins were subjected to 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membranes at 100 V for 2 h. The membranes were then immunoblotted according to the standard protocol with antibodies as follows: anti-ECRG2 monoclonal antibody was generated in our lab (29); anti-MMP2, anti-MMP9 and anti-uPA were purchased from Merck (Whitehouse Station, NJ). Immunoreactive bands were visualized by chemiluminescence (Pierce, Rockford, IL).

Gelatin zymography
Equal amounts of proteins from conditioned medium were subjected to zymography to detect MMP2 and MMP9 activities. Samples were added to each lane and subjected to 10% SDS–polyacrylamide gel electrophoresis containing 1 mg/ml gelatin (Sigma). After electrophoresis, the gel was renatured in 2.5% Triton X-100 for 30 min at room temperature and incubated at 37°C for 20 h in 0.1 M glycine–sodium hydroxide, pH 8.3. The gel was stained with 1% Coomassie Brilliant Blue R-250 (Amersham Biosciences, Piscataway, NJ) and destained with destaining buffer (5% acetic acid and 10% methanol).

Binding affinity study
Recombinant purified ECRG2 protein, 5 µg per well (purified as GST fusion proteins in Escherichia coli described as previously), was coated on 96-well microtiter plates followed by bovine serum albumin blocking. Serum-free conditioned cell culture medium was then added to the wells and incubated for 2 h. After washing, anti-MMP2, anti-MMP9 or anti-uPA were added to the wells and incubated for 30 min at 37°C. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Santa Cruz, CA) were added to the wells and incubated for 20 min at 37°C. After incubation, the substrate o-phenylenediamine dihydrochloride was added to the wells, and the colored reaction product was quantified using a microplate reader at 490 nm.

Co-immunoprecipitation
Immunoprecipitation and western blot analysis was performed according to the standard protocol (Sigma). Briefly, equal amounts of serum-free conditioned medium proteins were incubated with ECRG2 antibody, immobilized onto protein A-Sepharose, overnight at 4°C with gentle rotation. Beads were washed and immunocomplexes were eluted in 2x Laemmli buffer (20% glycerol, 2% SDS, 250 mM Tris pH 6.8, 10% ß-mercaptoethanol and 0.1% bromophenol blue), boiled and microcentrifuged. Supernatant proteins were subjected to 10% SDS–polyacrylamide gel electrophoresis, immunoblot analysis for uPA was performed as described above. All the immunoprecipitation analysis studies were repeated on three separate experiments.

uPA/plasmin activity
uPA/plasmin activity was analyzed as follows: 140 µl of reaction mixture was added to a 96-well plate. The reaction mixture was composed of 1 mg/100 ml plasminogen and 0.5 mM plasmin substrate D-Val-Phe-Lys-p-nitroanilide (Chromogenix, Viale Monza, Milano, Italy) (30) dissolved in reaction buffer (50 mM Tris–HCl, pH 7.5, 20 ng/ml leupeptin, 20 ng/ml pepstatin A, 0.5 mM o-phenanthroline and 1 mg/ml fibrin). Ten microliters of serum-free conditioned medium was added to the mixture and incubated at 37°C. Absorption, A405, was measured after 0.5 h.

Statistical analysis
Statistical analysis was carried out with SPSS10.0 software. Statistical significance was determined using Student's t-tests and analysis of variance. Results were considered to be significant at P < 0.05 or P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The inhibition of migration and invasion of tumor cells in vitro
PG lung cancer cells were transiently transfected with pcDNA3.1-ECRG2 to allow expression of the ECRG2 protein (Figure 1A). The role of ECRG2 was tested in wound closure assay, which was used as an in vitro model for tumor cell migration during wound healing. Confluent cell monolayers were wounded by a piece of sterile film and wound closure was monitored at various times. After wounding, cells migrate from the edges of ‘scrape-wounded’ monolayers to cover the denuded surface. PG/pcDNA3.1 cells moved rapidly, growing and filling the denuded surface within 8 h. However, PG/pcDNA3.1-ECRG2 cells moved slowly and could not fill the wounded area completely until 10 h (Figure 1B). These results indicate that migration of PG cells is suppressed or slowed by ectopic expression of ECRG2.


Figure 1
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  		Fig. 1. Inhibition of the migration and invasion of PG cells by ECRG2 in vitro. (A) Western blot. ECRG2 protein was detected in PG/pcDNA3.1-ECRG2 cells 24 h after transient transfection. (B) Wound closure assay. Expression of ECRG2 increased the time required for PG cells to fill the wounded area completely. (C) Transwell migration assay. The average number of cells penetrating the filters decreased from 100.6 ± 7.1 per field (x10 objective) to 72.0 ± 5.6 per field (*P<0.05, Student's t-test) for PG cells transfected with ECRG2 cDNA. (D) Matrigel invasion assay. PG/pcDNA3.1-ECRG2 cells responded to expression of ECRG2 protein with a prominent decrease in the number of cells able to cross the Matrigel-coated filters. The number of invasive cells per field (x4 objective) was 67.7 ± 7.5 for PG/pcDNA3.1-ECRG2 cells and 743.5 ± 82.2 for PG/pcDNA3.1 cells. This difference approached statistical significance (**P<0.01, Student's t-test).

 
Since the Boyden chamber is a widely accepted method for assessing the migratory and invasive potential of tumor cells, Boyden chambers were used to measure the migration and invasion of PG cells in response to the expression of ECRG2. Cells were added to Boyden chambers coated with (for invasion assays) or without (for migration assays) Matrigel. The number of cells which moved through membrane was counted and analyzed after a 24-h incubation. The average number of PG/pcDNA3.1-ECRG2 cells was 72.0 ± 5.6 per field (x10 objective) compared with 100.6 ± 7.1 for the PG/pcDNA3.1 control cells (P < 0.05) (Figure 1C). PG/pcDNA3.1-ECRG2 cells also exhibited a marked decrease in the number of cells able to cross through the Matrigel-coated filters (67.7 ± 7.5 cells per field, x4 objective), and then compared with PG/pcDNA3.1 cells (743.5 ± 82.2) (P < 0.01) (Figure 1D). The expression of ECRG2 in PG cells thus significantly inhibits the migration and invasion of otherwise aggressive PG cells. Therefore, it was next determined if ectopic expression of ECRG2 affected the metastatic behavior of tumor cells.

Restraint of metastasis by ECRG2 in nude mice
In order to examine how ECRG2 affects the metastatic potential of tumor cells, PG cells were stably transfected with pcDNA3.1-ECRG2 to express the ECRG2 protein and with pcDNA3.1 as control (Figure 2A). Cells were injected into tail vein of nude mice. Mice were observed every 2 days and killed 40 days later. As Figure 2B shows, metastases on the lung surface were observed in two mice from PG/pcDNA3.1 group (2/7) compared with none in the PG/pcDNA3.1-ECRG2 group (0/7) (Figure 2E). Furthermore, histological analysis revealed the presence of micrometastases in more mice injected with PG/pcDNA3.1 cells (4/7), as compared with mice injected with PG/pcDNA3.1-ECRG2 cells (1/7). Metastases in the PG/pcDNA3.1 control group were more frequent, larger and exhibited many ‘break points’ where the cancer cells were able to invade the interstitial space (Figure 2C and D). In the PG/pcDNA3.1-ECRG2 group, the metastases were sparse, small, typically embolic and displayed a low-invasive phenotype (Figure 2F). Surprisingly, metastatic tumor nodules were visible on the dorsal flank in control mice (4/7) (Figure 2G–I), but no metastasis could be detected on the dorsal flank in the PG/pcDNA3.1-ECRG2 group. However, in one mouse in inguinal lymph node and in one mouse in axillary's lymph node were confirmed to contain cancer cells in the PG/pcDNA3.1-ECRG2 group (Figure 2J–L). Histological examination showed no evidence of metastatic cells in other organs (kidney and liver) in either group. This is the first demonstration that ECRG2 potentially suppresses tumor metastasis and changes metastatic tropism in vivo.


Figure 2
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  		Fig. 2. Reduction of metastasis by the expression of ECRG2 in a nude mouse model. PG cells were stably transfected with pcDNA3.1-ECRG2 and with pcDNA3.1 vector DNA as a control. ECRG2 protein was detected with western blot in PG/pcDNA3.1-ECRG2 cells before implanting them into nude mice (A). Six-week-old female BALB/c nude mouse (seven mice per group) was injected through the tail vein with 1.5 x 106cells. Arrow denotes metastasis. Row 2, lung metastases occurred in four of seven nude mice in the PG/pcDNA3.1 control group and could be observed on the lung surfaces (B). These appeared more aggressive, more frequent and larger (C and D). Row 3, lung metastases occurred in one of seven nude mice in the PG/pcDNA3.1-ECRG2 group, and was not detected on the lung surfaces (E). These tumors were sparse, small, typically embolic and displayed a low-invasive phenotype (F). Row 4, metastatic tumor nodules outside of the lung were visible on the dorsal flank in the PG/pcDNA3.1 control mice (4/7) (G), and they were composed of PG cells as confirmed by histological analysis (H and I). Row 5, tumors in the inguinal lymph node in one mouse and in the axillary lymph node in one mouse were confirmed as containing as cancer cells (J–L) in PG/pcDNA3.1-ECRG2 group.

 
Efforts were next made to learn how ECRG2 affects metastatic aggression in tumor cells. Because ECRG2 has structural similarities to other protease inhibitors, thus implicating protease inhibitory functions, it appeared plausible that the expression of ECRG2 in tumor cells might prevent protease-mediated destruction of matrix barriers which is critical for cell migration and invasion. Experiments were next designed to assess how ECRG2 affects proteases which are involved in matrix degradation.

Expression of MMP2, MMP9 and uPA and gelatinase activity
The activity of a protease in a tumor microenvironment may influence critical cell behavior such as proliferation, motility and invasion. MMP2, MMP9 and uPA are members of extracellular protease, which are up-regulated in many cancers and in transformed cells in culture (1–5,31–33). Gelatinases, MMP2 and MMP9, are known to degrade almost all basement membrane constituents, including type IV collagen, nidogen and laminin and gelatins (1–3,33), and uPA is thought to trigger this proteolytic cascade (34,35). Therefore, it was of interest to investigate whether the expression of MMP2, MMP9 and uPA was related to levels of ECRG2. Serum-free conditioned mediums from PG/pcDNA3.1-ECRG2 cells and PG/pcDNA3.1 cells were analyzed with western blots. ECRG2 was found to be secreted and diffused into the serum-free conditioned medium of PG/pcDNA3.1-ECRG2 cells. No detectable difference of latent MMP2, latent MMP9, active MMP9 or uPA was observed between PG/pcDNA3.1-ECRG2 and control cells, but a strong decrease in active MMP2 was observed in PG/pcDNA3.1-ECRG2 cells relative to controls (Figure 3A). Furthermore, conventional gelatin zymography was performed to assess whether expression of ECRG2 affects both latent and active gelatinases A (MMP-2) and B (MMP-9). The intensity of MMP9 and latent MMP2 gelatinolytic zones on the gels was similar between PG/pcDNA3.1-ECRG2 cells and control cells, but that of active MMP2 were dramatically decreased in response to expression of ECRG2 protein in the PG cells (P < 0.01) (Figure 3B). This result confirmed that expression of ECRG2 contributes to the inhibition of a specific activating reaction for MMP2.


Figure 3
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  		Fig. 3. Analysis of serum-free conditioned medium with western blots and gelatin zymography. (A) Western blots. ECRG2 protein was detected in PG cells transfected with ECRG2 cDNA. No detectable difference in latent MMP2, MMP9 and any type of uPA was seen between PG/pcDNA3.1-ECRG2 cells and control cells, but active MMP2 was significantly decreased in serum-free conditioned medium from PG/pcDNA3.1-ECRG2 cells (P<0.05). (B) Gelatin zymography. The intensity of MMP9 and latent MMP2 gelatinolytic zones on the gels are similar for PG/pcDNA3.1-ECRG2 cells and control cells, but those of active MMP2 dramatically decreased in response to expression of the ECRG2 protein in PG cells (P<0.01).

 
ECRG2 binds to uPA in vitro
A protease inhibitor must bind to a specific protease in order to interact with it. Extensive studies have been performed to learn more about the binding affinity between ECRG2 and MMP2, MMP9 or uPA. Recombinant ECRG2 protein was coated into the wells of a 96-well plate and incubated with serum-free conditioned medium from either PG/pcDNA3.1-ECRG2 cells or PG/pcDNA3.1 cells. Bound protease was detected using anti-MMP2, anti-MMP9 or anti-uPA antibodies. As a control for background protein binding, bovine serum albumin was coated into the wells of the 96-well plate and incubated with PBS instead of serum-free conditioned medium. As Figure 4A shows, uPA exhibited binding to recombinant ECRG2 protein, although a slight decrease in bound protein was detected in serum-free conditioned medium from PG/pcDNA3.1-ECRG2 cells when compared with medium from PG/pcDNA3.1 cells. No detectable binding of MMP2 or MMP9 to ECRG2 was observed.


Figure 4
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  		Fig. 4. Interaction between ECRG2 and uPA in a binding affinity study and in a co-immunoprecipitation assay in vitro. (A) Binding affinity study. uPA in serum-free conditioned mediums from PG/pcDNA3.1 cells (column 14) and PG/pcDNA3.1-ECRG2 cells (column 15) were bound to recombinant ECRG2 protein which had been pre-coated on the plate. The absorbance of these columns was significantly higher than those of controls (columns 12 and 13), **P<0.01, analysis of variance (Q-test). (B) Co-immunoprecipitation assay. uPAs, 55 and 33 kD, were confirmed to bind to ECRG2.

 
This result was further confirmed by a co-immunoprecipitation assay. uPAs, 55 and 33 kDa, bound to ECRG2 and precipitated from PG/pcDNA3.1-ECRG2 cells' serum-free conditioned medium by ECRG2 antibody immobilized beads (Figure 4B), and the binding of ECRG2 to uPA may be independent of the active site of uPA, because the active site is masked in precursor uPA (55 kDa). These findings suggest that ECRG2 may suppress cell migration and invasion by binding and inhibiting uPA-mediated matrix proteolysis.

ECRG2 reduces uPA/plasmin activity
Since it was observed that ECRG2 binds to uPA, the activity of uPA/plasmin was quantified in a further study by using a specific chromogenic substrate to assess the effect of ECRG2. Serum-free conditioned medium from PG/pcDNA3.1-ECRG2 cells reduced proteolysis of the plasmin substrate D-Val-Phe-Lys-p-nitroanilide, as indicated by a decrease in absorbance (1.436 ± 0.021) at 405 nm, when compared with control (2.738 ± 0.024), P < 0.001 (Figure 5). These findings, together with the observation that ECRG2 binds to uPA, indicate that ECRG2 inhibits the proteolytic activity of the uPA/plasmin system.


Figure 5
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  		Fig. 5. Detection of uPA/plasmin activity by proteolytic chromogenic substrate analysis. Expression of ECRG2 in PG serum-free conditioned medium reduced the proteolysis of the plasmin substrate D-Val-Phe-Lys-p-nitroanilide as shown by a decrease in absorbance (1.436 ± 0.021) at 405 nm when compared with control medium (2.738 ± 0.024), **P<0.001, Student's t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Tumor cell invasion is dependent on finely regulated extracellular proteolytic activity, which allows tumor cells to penetrate through the ECM (1–5). Among the proteolytic enzymes involved in this process are proteases, whose expression in cells is regulated by several types of growth factors and cytokines. Invasive tumor cells not only express cell-associated proteases but also secrete anti-proteases, to prevent excessive digestion of the ECM, which can lead to a loss of cell attachment. The finely regulated balance between proteolytic activity and inhibition is critical for the invasive and metastatic events in tumor cells (6–8). In the work shown here, a novel protein, ECRG2 is described. ECRG2 contains a characteristic serine protease inhibitor structure, which, when over-expressed in PG tumor cells, inhibits both migration and invasion. The inhibitory effects of ECRG2 on cancer migration and invasion led to the hypothesis that ECRG2 might be a protease inhibitor which affects the proteolytic activity of proteases involved in the degradation of the ECM. Therefore, the interactions of ECRG2 with major proteolytic effectors known for involvement in ECM degradation during migration and invasion were investigated.

uPA-mediated proteolysis is of great importance during the process of tumor cell invasion, metastases and angiogenesis (5,31–33). uPA is a serine protease with a mass 45–55 kDa (depending on the species analyzed) exists in a proenzyme form (pro-uPA), upon activation, activates plasminogen to active serine protease plasmin. Pro-uPA is converted from a single-chain protein to a two-chain protein, with a disulfide bond link. Chain A has motifs with homology to plasminogen, fibronectin and prothrombin and has an epidermal growth factor homology motif in its receptor-binding domain. The B chain contains the active site, which is homologous to the active site of other serine-proteases, such as trypsin, plasmin and thrombin (36). Plasmin is a broad-specificity protease, which degrades several ECM components, such as fibronectin, laminin and collagen (5,31–33). In addition, uPA triggers a proteolytic cascade which involves the activation of MMPs, which are responsible for collagen degradation (34,35). It has been also noted that uPA itself can degrade several ECM proteins. When the ECM is degraded, several growth factors are released, such as FGF, tumor necrosis factor and TGF, allowing tumor cells to become more aggressive (37–39). uPA binds to a specific urokinase-type plasminogen activator receptor (uPAR), which is anchored to the outer plasma membrane by a glycophosphatidylinositol chain. The uPA–uPAR complex, besides its proteolytic function as a zymogen, also functions as a vitronectin receptor and has been shown to participate in normal and tumor cell motility processes such as monocyte migration and tumor cell migration and invasion (40). The interaction between uPAR and integrin may be of functional importance in terms of providing a basis for integrin-mediated transmembrane signal transduction after the binding of ligands to uPAR, and also, the interaction between uPAR and integrin may provide a basis for the complex pattern of mutual modulations involving the affinity of receptor–ligand interactions (31,41). Thus, down-regulation of uPA or its receptor may lead to inhibited pericellular proteolysis, changes in signal transduction and decreased migratory behavior, which among other changes may result in the loss of the invasive phenotype by tumor cells.

In vivo, uPA catalytic activity can be inactivated by several inhibitors, including PAI-1, PAI-2 (42) and maspin (43). Of these three, PAI-1 is thought to be the primary inhibitor of uPA, and was expected to prevent invasion and metastasis. Indeed, in some model systems, over-expression of PAI-1 reduced the formation of metastases (44,45). Other studies, however, have shown that PAI-1 promotes, rather than inhibits, invasion and metastasis (46). In other studies, PAI-1 deficiency in mice decreased angiogenesis and prevented cancer cell invasion (47).

In this study, evidence has been provided that ECRG2 has a protease inhibitory function which was implicated in previous structural studies. Given the reduced metastatic potential in mouse model, as well as the reduced rate of cell migration during wound-healing assay, it appears that ECRG2 might be critically involved in cellular migration and invasion, most probably through interactions with proteases involved in ECM degradation. Investigations of the possible interactions between MMP2, MMP9 or uPA and ECRG2 showed that ECRG2 can directly bind and inhibit uPA/plasmin activity (Figures 4 and 5). While ECRG2 was not found to bind MMP2, it was found that, in vitro, ECRG2 could also strongly inhibit the activation of MMP2 (Figure 3). It is now generally accepted that the physiological activator of pro-MMP2 is MT1-MMP. MT1-MMP processes 72 kDa pro-MMP2 into a 68/66 kDa intermediate form, which may undergo autocatalytic processing into 64/62 kDa active form. Cooperation between MT1-MMP and plasma membrane-generated plasmin in MMP2 activation has not yet been clearly documented. However, some previous studies have suggested that the PA/plasmin system is able to activate pro-MMP2 (34,48). Monea et al. (48) reported that plasmin can activate pro-MMP2 in the presence of MT1-MMP. Baramova et al. (34) provided evidence that the PA/plasmin system may be involved in the second step of pro-MMP2 activation. Concerning the inhibition of both uPA/plasmin activity and MMP2 activation by ECRG2, the present data support the implication of the serine protease uPA/plasmin system in pro-MMP2 processing. Whether or not expression of ECRG2 directly contributes to the inhibition of MT1-MMP-mediated MMP2 activation remains to be established.

Taken together, the findings presented here have important implications for the regulation of the cell-surface plasminogen activation system and pericellular proteolytic activity. ECRG2 appears to play an important role in the control of uPA/plasmin proteolytic activity and thus influences cellular migration and invasion. Ultimately, protease inhibitors may play a major role in cancer therapeutics, however, further investigation is necessary to determine the exact mechanisms which are involved, and also more about the balance of activities involved in protease activation and inhibition.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The Chinese State Key Projects for Basic Research (2002CB513101 and 2004CB518701).


   Acknowledgments
 
We thank Dr Leon Neal Kapp for his critical revision and discussion.

Conflicts of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received January 29, 2007; revised May 17, 2007; accepted June 16, 2007.


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