The Kazal motifs of RECK protein inhibit MMP-9 secretion and activity and reduce metastasis of lung cancer cells in vitro and in vivo

RECK is a membrane-anchored glycoprotein which may negatively regulate matrix metalloproteinase (MMP) activity to suppress tumor invasion and metastasis. In this study, recombinant proteins corresponding to the residues 285–368 (named as CKM which contained cysteine knot motif), 605–799 (named as K123 which contained three Kazal motifs), 676–799 (named as K23 which contained the last two Kazal motifs) and full-length RECK were produced and their anti-cancer effects were tested. Full-length RECK and K23 but not K123 and CKM inhibited MMP9 secretion and activity. In addition, RECK and K23 inhibited invasion but not migration of metastatic lung cancer cells in vitro. Protein binding and kinetic study indicated that K23 physically interacted with MMP-9 and inhibited its activity by a non-competitive manner. Moreover, K23 reduced metastatic tumor growth in lungs of nude mice. Taken together, our results suggest that the K23 motifs of RECK protein can inhibit MMP-9 secretion and activity and attenuate metastasis of lung cancer cells.

Although the control of RECK gene transcription has been partially elucidated recently, the biological activities of different domains of RECK protein are largely unclear. The N-terminal of RECK protein contains five repeats of a putative cysteine knot motifs and five potential glycosylation sites [7]. In addition, a recent study demonstrated that RECK protein was glycosylated at Asn 86 , Asn 200 , Asn 297 and Asn 352 and glycosylation was critical for RECK to suppress tumor cell invasion by inhibiting MMP-9 secretion and repressing MMP-2 activation [17]. However, the functions of other domains are poorly understood. In this study, we investigated the anti-invasive activity of the Kazal motifs of RECK protein in cultured cells and tested their effect on the inhibition of tumor metastasis in vivo.

Materials and methods
Cell culture and experimental reagents A549 and CL1.5 human lung cancer cells were obtained from the cell bank of National Health Research Institute (Maoli, Taiwan) and Dr. Kuo ML (National Taiwan University, Taiwan) respectively. Cells were routinely cultured in DMEM/F12 medium containing 10% fetal calf serum (FCS) and antibiotics. OneStep RT-PCR kit was from Qiagen (Valencia, CA, USA). Human RECK cDNA was kindly provided by Dr. Noda M. (Kyoto University, Japan). Anti-RECK antibodies were purchased from MBL (Nagoya, Japan) or BD Biosciences (San Jose, CA, USA). Anti-Hisx6 antibody was obtained from BD Biosciences and anti-actin antibody was purchased from Chemicon (Temecula, CA, USA). Antibody against MMP-9 was obtained from Oncogene Science (Cambridge, MA, USA). EnzChek MMP assay kit was purchased from Molecular Probe (Eugene, OR, USA). Bovine serum albumin (BSA) was obtained from Sigma (San Louis, MO, USA). Active recombinant MMP-9 was purchased from Calbiochem (San Diego, CA, USA).

Construction of expression vectors and purification of recombinant proteins
pSecTag2 expression vector was obtained from Invitrogen (Carlsbad, CA, USA). This vector was designed for the production of recombinant protein in mammalian cells with the proteins were fused at the N-terminus to the murine Ig -chain leader sequence for protein secretion and at the C-terminus to a peptide containing c-myc epitope and Hisx6 tag for protein detection and purification. pSecTag2-RECK was generated by deletion of nucleotides corresponding to the C-terminal 23 amino acids of human RECK cDNA to remove the GPI-anchored region and subcloned into the pSecTag2 vector. CKM, K123 and K23 were constructed by amplifying the cDNA corresponding to the 285-368, 605-799 and 676-799 residues of RECK protein by PCR primers flanked with BamH I and Xho I or Hind III restriction enzyme sites. After PCR reaction, the amplified DNA fragments were subcloned into pSecTag2 vector. All of the constructed vectors were verified by DNA sequencing. Expression vectors were transfected into 293T cells and conditioned medium was collected from 48 to 96 hrs after transfection. The secreted recombinant proteins were purified by using a nickel-chelating column. The elute was further concentrated by using the 10K MWCO dialysis cassette (PIERCE) to remove the high concentration of imidazole and sterilized with a 0.22 µm filter. Purity of these recombinant proteins was checked by HPLC and was >85%. Recombinant proteins were stored at Ϫ80°C until use.

Invasion and migration assays
In vitro invasion assay was performed by using 24-well transwell units with poly-carbonate filters (pore size 8 µm) coated on the upper side with Matrigel (Becton Dickinson Labware, Bedford, MA). The lower part of the transwell unit was filled with 10% FCS medium. A total of 5 x 10 3 cells in 100 µl of medium containing different recombinant proteins were placed in the upper part of the transwell unit and allowed to invade for 24 hrs. After incubation, non-invaded cells on the upper part of the membrane were removed with a cotton swab. Invaded cells on the bottom surface of the membrane were fixed in formaldehyde, stained with Giemsa solution and counted under a microscope. Migration assay was performed by similar procedures except the polycarbonate filters were not coated with Matrigel.

MMP activity assay
Conditioned medium of A549 or CL1.5 cells was harvested and incubated with various concentrations of BSA, CKM, K123, K23 or full-length RECK (as a positive control). The MMP activity was studied by the EnzChek MMP assay kit using fluorescein-conjugated gelatin (DQ TM gelatin) as substrate and purified type IV collagenase as a positive control enzyme. Experiments were carried out according to the procedures of the manufacturer and the output signal (excitation wavelength, 495 nm; emission wavelength, 520 nm) was recorded for 2 min at 37°C by using a fluorescent reader (BMG LABTECH, Offenburg, Germany). Results of three independent experiments were expressed as mean ± SE.

Kinetics of K23 on MMP-9 activity
To study the kinetics of MMP-9 inhibition, active recombinant MMP-9 (5 ng) was mixed with various concentrations of a fluorescein-conjugated gelatin (DQ TM gelatin, Molecular Probe) and recombinant K23 protein (0, 41.7 or 125 nM). Experiments were performed similar to the procedures of MMP assay. The output signal (excitation wavelength, 495 nm; emission wavelength, 520 nm) was recorded for 2 min at 37°C by using a fluorescent reader (BMG LABTECH) and the dissociation constant was calculated.

Gelatin zymography
Zymography assay was performed as described previously [15]. BSA or recombinant proteins were added into the culture medium of A549 cells and incubated for 24 hrs. Conditioned medium was collected and concentrated by using Centricon YM-50 columns (Amicon, Bedford, MA, USA). Cell number was determined by using the hemocytometer. Conditioned medium from an equal number of cells was separated by 10% acrylamide gels containing 0.1% gelatin (Invitrogen). The gels were incubated in 2.5% Triton X-100 solution at room temperature with gentle agitation to remove SDS and were soaked in reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2 and 0.5 mM ZnCl2) at 37°C overnight. After reaction, the gels were stained for 1 hr with staining solution (0.1% w/v Coomassie Brilliant Blue, 30% v/v methanol and 10% v/v acetic acid) and were destained in the same solution, but without Coomassie Brilliant Blue. Gelatinolytic activity of MMPs was visualized as a clear band against a dark background of stained gelatin.

Western blot analysis and immunoprecipitation assay
Cells were harvested and equal amount of cellular proteins was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously [18]. Proteins were transferred to nitrocellulose membranes and the blots were probed with various primary antibodies. Enhanced chemiluminescence reagents were used to detect the proteins on the membranes. For study of the interaction between K23 and MMP-9, active recombinant MMP-9 protein (100 ng) was incubated with K23 (500 ng) at 4°C for 2 hrs. Anti-MMP-9 antibody was added and incubated at 4°C overnight with gentle rotation. The immunocomplex was precipitated by protein G-agarose beads and subjected to SDS-PAGE. K23 was detected by using anti-Hisx6 antibody.

Establishment of K23-expressing stable transfectants and experimental metastasis assay
A549 cell were transfected with pSecTag2 (control) or K23-expressing pSecTag2 (K23) vectors by Lipofectamine2000 reagent. Stable transfectants were selected by continuous treatment of hygomycin (400 µg/ml) for 4 weeks and expression of K23 was examined by Western blot analysis. 1 x 10 6 cells in 0.2 ml PBS were injected into the tail vein of the BALB/c nude mice. Eight weeks after inoculation, animals were sacrificed and the lungs of the animals were inspected for metastatic nodules as described in our previous study [19]. Each group contained five mice. Metastatic nodules were averaged and expressed as Mean ± SE. Animal studies were approved by the Animal Care and Ethics Committee of the National Sun Yat-Sen University.

Statistical analysis
Student's t-test was used to evaluate the difference between various experimental groups. Differences were considered to be significant at P < 0.05.

Production of full-length and various deletion polypeptides of RECK protein
To clarify the biological activity of Kazal motifs of RECK protein, we generated various deletion constructs and tried to produce recombinant proteins by using mammalian (pSecTag2) expression systems. Because RECK is a glycosylphosphatidylinositol (GPI)anchored protein and needs glycosylation for its activity, we deleted the C-terminal 23 amino acids and subcloned the cDNA into the pSecTag2 expression vector. The vector was transfected into 293T cells and the Hisx6-tagged full-length RECK protein secreted into the conditioned medium was purified by a nickel-chelating resin. Eluted proteins were further concentrated by using molecular weight cutoff (MWCO) dialysis cassette to remove the high concentration of imidazole and sterilized with a 0.22 µm filter. Purity of these recombinant proteins was checked by HPLC and was >85%. As shown in Fig. 1A, Western blotting indicated the transfected 293T cells produced high amount of recombinant full-length RECK protein with a molecular weight about 110 kD. Recombinant proteins corresponding to the residues 605-799 (named as K123 which contained three Kazal motifs) and 676-799 (named as K23 which contained the last two Kazal motifs) were produced similarly ( Fig. 1B and C). Because the recombinant proteins contained an N-terminal murine Ig -chain leader sequence and a C-terminal c-myc epitope and Hisx6 tag sequence, the molecular weights of K123 and K23 were estimated to be 40 and 22 kD, respectively. Western blot analysis demonstrated that specific bands representing the recombinant proteins were detected in the predicted positions. Another recombinant protein corresponding to the residues 285-368 (named as CKM which contained cysteine knot motif) was produced. Two protein bands were appeared in the Western blot because CKM was glycosylated on Asn 297 and Asn 352 (Fig. 1D). The glycosylation of CKM was confirmed by treatment of glycosidase and a reduction of molecular weight of the protein to 11 kD corresponding to the nonglycosylated form was observed (data not shown).

Kazal motifs of RECK protein inhibited cell invasion but not migration
We tested whether the recombinant proteins affected migration of two highly metastatic lung cancer cell lines A549 and CL1.5. Similar to the results reported in another highly invasive HT1080 fibrosarcoma cells, we found that full-length RECK did not inhibit the migration of A549 and CL1.5 lung cancer cells ( Fig. 2A and B).
Our data also showed that CKM, K123 and K23 did not affect the migration of A549 and CL1.5 cells. We next studied the effect of these recombinant proteins on cell invasive ability and our results demonstrated that CKM at 3 µg/ml showed marginal inhibition of invasion of A549 and CL1.5 lung cancer cells (Fig. 2C and D). On the contrary, full-length RECK and K23 inhibited cell invasion in a dosedependent manner (Fig. 2C and D). In addition, A549 cells were more sensitive to RECK and K23 than CL1.5 cells. Unexpectedly, K123 could not significantly repress the invasion of A549 and CL1.5 cells although a marginal reduction of invaded cell number was found when high concentration (3 µg/ml) of K123 was used.

K23 inhibited MMP-9 activity and secretion
Because K23 inhibited cell invasion but not migration, it seemed that K23 might affect the MMP activity to reduce the degradation of Matrigel and to attenuate cell invasion. Therefore, we examined the total MMP activity in the conditioned medium by using the EnzChek MMP assay kit. As shown in Fig. 3A and B, full-length RECK or K23 significantly reduced the MMP activity of A549 and CL1.5 cells. Conversely, CKM and K123 had no effect. Since the recombinant proteins were exogenously added, these data suggested that full-length RECK and K23 directly inhibited the enzymatic activity of MMP. RECK had also been demonstrated to reduce the secretion of MMP-9 via an unknown mechanism [7], so we tested whether K23 might affect MMP-9 release. A549 cells were used and recombinant proteins were added directly into the cultured medium for 24 hrs. Conditioned medium was collected and subjected to gelatin zymography assay. As expected, we found that full-length RECK reduced the pro-form and active form of MMP-9 in the conditioned medium (Fig. 3C). This data was consistent with the previous work showing that RECK inhibited MMP-9 secretion [7]. Secretion of pro-MMP-2 and active MMP-2 were not affected by RECK (data not shown). Under the same experimental condition, K23 also potently attenuated the amount of proform and active form of MMP-9 in the conditioned medium while K123 had no effect. These results were in agreement with the data of our MMP enzymatic assay and suggested that K23 repressed MMP-9 secretion and activity simultaneously. We also tested whether K23 affected epidermal growth factor (EGF)-stimulated MMP-9 gene transcription. Our data indicated EGF up-regulated MMP-9 mRNA by 2.5-3 folds and K23 did not significantly inhibit the increase of MMP-9 mRNA level by EGF in A549 cells (Fig.  3D). However, EGF-stimulated secretion and activation of MMP-9 was significantly attenuated by K23 (Fig. 3E). Therefore, K23 is effectively in the inhibition of basal-and growth factor-stimulated MMP-9 secretion and activation.

K23 functioned as a non-competitive inhibitor for MMP-9
Because K23 directly inhibited MMP-9 activity, we predicted that K23 might physically interact with MMP-9. We incubated K23 with active MMP-9 and immuno-precipitated MMP-9 with a specific antibody. Our data clearly demonstrated that K23 bound with active MMP-9 in vitro (Fig. 4A). This binding is specific because immunoblotting of MMP-9 verified that equal amount of MMP-9 was immunoprecipitated by the MMP-9 antibody and nonimmune Ig could not pull down the MMP-9/K23 complex (data not shown). We next investigated the mechanism by which K23 inhibited MMP-9. Kinetic analysis indicated that K23 inhibited MMP-9 activity in a non-competitive manner with the dissociation constant <15.5 nM (Fig. 4B).

Expression of K23 suppressed tumor metastasis in vivo
Our aforementioned results verified that K23 potently inhibited MMP-9 activity and invasion of lung cancer cells in vitro. We next studied whether K23 indeed functioned as a metastasis suppressor in vivo. A549 cells were transfected with control (pcDNA3.1) or K23 expression vector. Stable transfectants were selected by treatment of hygomycin (400 µg/ml) for 4 weeks. As shown in Fig. 5A, K23 transfectants indeed expressed high level of K23 protein in the culture medium while no signals were found in cells transfected with control vector. Stable transfectants were pooled and used for invasion assay. We found that expression of K23 in A549 cells reduced their invasion ability by about 45-50% (Fig. 5B). Pooled stable transfectants were also injected into mice via tail vein. After 8 weeks, mice were sacrificed and lungs were stained by intra-trachea injection of India ink. We found that metastatic tumor nodules were identified in the lungs (Fig. 5C).
The average number of tumor nodules in mice injected with control vector-transfected A549 cells was 23 ± 7 per mice (n = 5). Conversely, the average number of nodules was reduced to 10 ± 4 per mice (n = 5, Fig. 5D). Therefore, expression of K23 protein significantly induced a 57% of reduction of metastatic tumor nodules in vivo.

Discussion
In this study, we demonstrate that the K23 domain of RECK protein inhibits MMP-9 secretion and activity and reduces metas- tasis of lung cancer cells in vitro and in vivo. Three critical issues should be discussed. First, we have identified a new peptide inhibitor for MMP-9 in this study. Among MMPs identified in mammalian cells, MMP-2 and MMP-9 are strongly associated with tumor metastasis in various human cancers [20,21]. Therefore, MMP inhibitors either macromolecular inhibitors (TIMPs and monoclonal antibodies) or small chemical inhibitors (synthetic and natural products) are potential agents for the therapy of diseases in which excess MMP activity (like cancer and arthritis) has been implicated. However, results from clinical trials for cancer treatment were disappointing [22]. Previous studies suggested that invasion and metastasis, which MMPs contributed heavily during tumor progression were late events in cancer development. However, recent studies clearly indicated that invasion and metastasis could occur during early stages as well [23]. Therefore, the appropriate time for the application of MMP inhibitors should be re-evaluated. Recently, several hydroxamatebased MMP inhibitors including Marimastat, SC903, CGS27023A have been developed because hydroxamate has potent zincchelating activity that may block the active site of most MMPs [24][25][26][27]. However, these small inhibitors exhibit less substrate specificity and block a broad spectrum of MMPs [28]. Conversely, few peptide inhibitors have been developed until now. One peptide inhibitor Regasepin 1 with the amino acid sequence of PRCBGE has been identified recently [29]. Cell-based study shows that Regasepin 1 inhibits MMP-8 and MMP-9 with a 50% inhibitory dose (ID50) of 3 and 1 µm, respectively [30]. In this study, we demonstrate that K23 is a potent inhibitor for MMP-9. Kinetic analysis indicates that ID50 of K23 (<15.5 nM) is much lower than that of Regasepin 1 indicating K23 is a more potent MMP-9 inhibitor. In addition, K23 is a non-competitive inhibitor, which is different from most of the substrate-based inhibitors developed recently. Our animal study also verifies that K23 indeed represses the metastasis of lung cancer cells. Thus, we have identified a potential peptide inhibitor for MMP-9.
The second important issue is the potent in vivo efficacy and in vivo targets of K23 in lung metastasis. Our cell-based experiments demonstrated that only 30-40% of inhibition of cell invasion was found after treatment of K23. However, a 60% of inhibition of lung metastasis was observed in animal studies. Because MMP-9 has been verified to be a direct target for K23 in cultured cells, it is reasonable to hypothesize that inhibition of MMP-9 is one of the mechanisms for K23 to repress tumor metastasis. However, it is notable that there is a large contribution of noncancer cells in MMP-9 expression in tumors. MMP-9 can be produced by stromal fibroblasts [20,31]. In addition, a variety of immune cells including macrophages, monocytes, dendritic cells, natural killer cells, lymphocytes and neutrophils can express and produce MMP-9 [32]. Therefore, MMP-9 released by immune cells of the experimental animals may play some roles in the promotion of lung tumor metastasis, and K23 may inhibit all of MMP-9 produced by cancer and immune cells that causes a more profound effect in the inhibition of metastasis in vivo. Another possibility is there are some other in vivo targets of K23. Several proteases including MMP-2, MMP-3, MMP-7, MMP-10, MMP-13 and MMP-26 have been shown to be key regulators for the activation of MMP-9. So, K23 may repress the enzymatic activity of these MMPs to attenuate MMP-9 activation and to reduce metastasis. It is also possible that these MMPs are involved in the induction of lung metastasis and can be directly inhibited by K23. An important issue for further investigations is how many kinds of MMP are direct targets for K23.
The third issue is the inhibition of MMP-9 secretion by K23. Until recently, the molecular mechanism, which controls the secretion of MMP-9 is still unclear. By using human melanoma cells as a model, Schnaeker et al. demonstrated that MMP-9 was stored as small intracellular vesicles, which localized near the cell lamellipodia [33]. Double staining revealed that MMP-9 was strongly associated with ␣-tubulin and the MMP-9-containing vesicles were distributed along the microtubular network. In addition, most of the vesicles were also co-localized with another motor protein kinesin. Some intracellular or extracellular signals may stimulate the fusion of the vesicles and plasma membrane and then trigger the secretion of MMP-9. It seems possible that K23 may interact with specific membrane proteins and block the signal-induced fusion process to inhibit MMP-9 release. Identification of these membrane proteins will lead to the understanding of the mechanism by which K23 suppresses MMP-9 secretion.
The lack of inhibitory function of K123 on cell invasion is somehow surprising because K123 contains the amino acid Anti-MMP-9 antibody was added and the immunocomplex was precipitated by protein G-agarose beads. The binding of K23 to MMP-9 was detected by using anti-Hisx6 antibody and the blots were also probed with anti-MMP-9 antibody to confirm that the equal amount of MMP-9 was immunoprecipitated. (B) Active recombinant MMP-9 (5 ng) was mixed with various concentrations of a fluorescein-conjugated gelatin (DQ TM gelatin) and recombinant K23 protein (0, 41.7 or 125 nM). Experiments were performed similar to the procedures of MMP assay as described in Materials and methods. sequence completely matching to the Kazal motif at its N-terminal region. One possible explanation of this result is that K123 may exhibit some misfolding in the secondary or tertiary structure because many cysteine residues are existed in this recombinant protein, which may lead to mismatch pairing of disulfide bonds and distort its native conformation. Taken together, results of this study suggest that the Kazal motifs of RECK protein K23 is a potent inhibitor for MMP-9 secretion and activity and may inhibit the metastasis of lung cancer cells. The invasive ability of the stable transfectants was investigated as described in Fig. 2C. The invaded cell number of cells transfected with pSecTag2 (C) was defined as 100% (n = 3). *P < 0.05 when the results of K23-expressing group was compared with the results of control group. (C) A total of 1 x 10 6 stable transfectants were injected into the tail vein of the BALB/c nude mice. Eight weeks after inoculation, animals were sacrificed and the lungs of the animals were inspected for metastatic nodules (indicated by arrow sign). Typical figures of lungs from the two experimental groups were shown. (D) Metastatic nodules were averaged and expressed as Mean ± SE (n = 5). *P < 0.05 when the results of K23-expressing group were compared with the results of control group.