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[Cancer Research 66, 4734-4741, May 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

ß-Catenin/Wnt Signaling Regulates Expression of the Membrane Type 3 Matrix Metalloproteinase in Gastric Cancer

Andrew M. Lowy1, Wilson M. Clements1, John Bishop2, Ling Kong1, Tera Bonney1, Karena Sisco1, Bruce Aronow3, Cecilia Fenoglio-Preiser2 and Joanna Groden4

1 Department of Surgery, Division of Surgical Oncology; 2 Department of Pathology and Laboratory Medicine; 3 Cincinnati Children's Hospital Medical Center; and 4 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, University of Cincinnati, Cincinnati, Ohio

Requests for reprints: Andrew M. Lowy, Division of Surgical Oncology, Barrett Cancer Center, University of Cincinnati, Cincinnati, OH 45219. Phone: 513-584-8900; Fax: 513-584-0459; E-mail: andrew.lowy{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of Wnt signaling through ß-catenin dysregulation occurs in numerous human tumors, including gastric cancer. The specific consequences of Wnt signaling in gastric cancer, however, are not well characterized. This study shows that the introduction of mutant ß-catenin into gastric cancer cell lines by adenoviral infection enhances invasiveness and proliferation and up-regulates the expression of the gene encoding the matrix metalloproteinase (MMP) family member membrane type 3 MMP (MT3-MMP). Up-regulation of MT3-MMP is critical to the invasive phenotype as shown by small interfering RNA (siRNA) studies. Immunohistochemical staining also showed that MT3-MMP was highly expressed in gastric cancers with activating ß-catenin mutations. These observations suggest that Wnt activation may contribute to gastric cancer progression by increasing the invasiveness of neoplastic cells in the stomach via up-regulation of MT3-MMP expression. (Cancer Res 2006; 66(9): 4734-41)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Wnt/ß-catenin signaling is now well recognized as a critical pathway in the regulation of growth and development. Aberrant Wnt signaling is a characteristic shared by numerous human tumors and may occur as a result of gene mutation in APC, ß-catenin, axin/conductin, or by dysregulation of Wnt ligands. The central downstream effector of Wnt signaling is ß-catenin, a 92 kDa protein, which also functions directly in cell-to-cell adhesion. The critical event in Wnt pathway activation is the elevation of the cytoplasmic pool of ß-catenin and its resultant transport to the nucleus. There, ß-catenin, partnered with TCF/LEF transcription factors, causes changes in gene transcription (1, 2). Numerous targets of ß-catenin/TCF transcription have now been described, including genes important in proliferation, tumor invasion and metastasis, angiogenesis, and drug resistance (3–7).

Several lines of evidence support the importance of Wnt pathway activation in gastric carcinogenesis. Persons with germ line mutations of the APC tumor suppressor gene have a 10-fold increased risk of developing gastric cancer compared with normal persons (8). Mutations in APC and ß-catenin have been found in sporadic gastric adenocarcinomas, as well as in tumors from carcinogen-based animal models of gastric cancer (9–11). Recently, our group reported a 29% rate of Wnt pathway activation in gastric adenocarcinoma as defined by the presence of nuclear ß-catenin and the occurrence of activating ß-catenin mutations in these tumors (12). Other groups have also found ß-catenin mutations in gastric cancer, but no clear relationship to stage or histology has been established. Thus, despite these studies demonstrating activating Wnt pathway mutations in gastric cancer, the effects of activated Wnt signaling on gastric cancer cells have not been directly examined.

A common characteristic of gastric cancer is early invasiveness into surrounding tissues and the peritoneal cavity. Several genes important for tumor invasion and metastasis have been previously identified as targets of the Wnt pathway, including matrix metalloproteinase (MMP)-7, membrane type 1 (MT1)-MMP, MMP-26, urokinase plasminogen activator, laminin-5{gamma}2, and CD44 (5, 13–17). We were therefore interested in the effects of Wnt signaling on the invasiveness of gastric cancer cells and asked which transcriptional targets might mediate changes in this property. Here, we show that activation of Wnt signaling increased invasiveness in gastric cancer cell lines and that a member of the MMP family, MT3-MMP (also known as MMP-16), is a ß-catenin target gene in human gastric cancer. We further show that induction of the MT3-MMP protein product is critical to the Wnt-mediated invasive phenotype in gastric cancer cells. Finally, our work suggests that up-regulation of MT3-MMP by Wnt signaling occurs by an indirect mechanism. We conclude that activation of Wnt signaling may contribute to gastric cancer progression by effecting an increase in cell invasiveness and that these changes are mediated, in part, by up-regulation of MT3-MMP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The gastric cancer cell lines used in these studies were Hs746T and NCIN87. The colon cancer cell line SW480 and Cos1 cells were also included as controls. All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained according to ATCC recommendations. The Wnt pathway is inactive in each of these cell lines as measured by localization of ß-catenin (data not shown) with the exception of SW480 in which Wnt signaling is activated due to mutations in the APC tumor suppressor gene.

Expression vectors and gene transfer. The adenoviral expression vectors were created using the Adeno-X kit (Clontech, Palo Alto, CA). One form of oncogenic ß-catenin contains a serine to alanine mutation at codon 37 (S37A). An S37A ß-catenin gene was PCR-amplified with a hemagglutinin (HA) tag and directionally cloned into the pShuttle plasmid using AflI and NotI restriction enzyme sites. The pShuttle expression cassette was then cloned into an adenoviral backbone; fidelity of the amplified transcript was confirmed by DNA sequencing. The adenoviral expression plasmid was then used to infect HEK-293 cells to amplify the virus (AdS37A). Expression was confirmed by Western analysis using a monoclonal mouse antibody to ß-catenin (Transduction Laboratories, Lexington, KY) and a monoclonal antibody to HA (Covance, Berkeley, CA). Once an adequate viral titer was obtained, unpurified virus was used at a multiplicity of infection (MOI) of 1,000 for infections of gastric cancer cell lines. The AdLacz virus was created similarly by PCR cloning the ß-galactosidase cDNA into the pShuttle plasmid using the same AfII and NotI sites. pAPC-FLAG and pEGFP were cloned and sequenced as previously described (18). TOPFLASH assays were conducted as previously described (19).

S37A-ß-catenin was separately cloned into the EGFP expression vector for use in electroporation. siRNAs for MT3-MMP were obtained from Ambion (Austin, TX). Sequences were as follows:

Sense, 5'-CGUGAUGUGGAUAUAACCAtt-3'.
Antisense, 5'-UGGUUAUAUCCACAUCACGtt-3'.

Control missense small interfering RNAs (siRNA) were obtained from Upstate (Chicago, IL).

Electroporation was done using the Nucleofector II, program 20 (Amaxa, Gaithersburg, MD). For electroporation, 106 cells NCIN87 were electroporated with 1 µg pS37A-ß-catenin or pEGFP, with or without 10 to 30 pmol/L siRNA. Activation of Wnt signaling following pS37A-ß-catenin electroporation was confirmed using the TOP/FOP assays previously described (15).

SW480 cells were plated at a density of 1 x 107 per 100 mm2 plate. After 16 hours, the cells were inspected and transfected with 0.5 µg each of pAPC-FLAG and pEGFP, or 1 µg of pEGFP alone using FuGENE6 (Roche Applied Science, Indianapolis, IN) according to the instructions of the manufacturer.

Western analyses. After an incubation time of 48 hours following transfection, cell plates were washed in cold PBS and 1 mL lysis buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.5% NP40, 1.0 µg/mL aprotinin, 1.0 µg/mL pepstatin, 1.0 µg/mL leupeptin, 1.0 mmol/L EDTA, and 0.2 mmol/L phenylmethylsulfonyl fluoride] was added directly to the plates. Cells were then harvested and incubated at 4°C for 30 minutes with shaking. Cell lysates were spun at 15,000 rpm in a microcentrifuge for 15 minutes to pellet the debris. The supernatant was collected and quantified using the Lowry protein assay (Bio-Rad, Hercules, CA).

Twenty micrograms of total protein from each sample were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Immunoblotting was done using monoclonal antibodies to ß-catenin (1:500; Transduction Laboratories), HA (1:500; Covance), MT3-MMP (1:500; Neomarkers, Freemont, CA), actin (1:500), and p21 (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were blocked in TBS with 0.1% Tween 20 and 5% nonfat dry milk for 30 minutes while shaking. Primary antibodies were added to the blocking solution, incubated at room temperature for 2 hours, and then washed four times for 10 minutes in TBST. Secondary antibody was incubated in blocking solution for 1 hour and then washed six times for 10 minutes in TBST and developed with the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

Bromodeoxyuridine staining. Cells were plated on coverslips in six-well plates at 200,000 per well. After 24 hours, the cells were infected with either the AdS37A or AdLacZ constructs at a MOI of 1,000. After 48 hours, the cells were cultured for 4 hours with bromodeoxyuridine (BrdUrd) at a 1:1,000 dilution of the stock BrdUrd solution. Cells were then fixed for 15 minutes in 3.7% formaldehyde in PBS at room temperature, rinsed with PBS, and permeabilized in 0.3% Triton X-100/PBS for 15 minutes at room temperature. After rinsing, the cells were placed in a humidified chamber and stained with primary antibody for 45 minutes at 37°C in a staining solution containing 250 µL of 0.5% NP40, 5 mg/mL bovine serum albumin (BSA)/PBS, 5 µL of 1 mol/L MgCl2, 0.5 µL rat anti-BrdUrd, and 0.5 µL DNase I. After incubating in primary antibody, the coverslips were washed thrice, for 5 minutes each, in PBS at room temperature. Secondary antibody staining (1:200 rhodamine donkey anti-rat in 0.5% NP40, 5 mg/mL BSA in PBS) was then carried out for 45 minutes at 37°C in a humidified chamber. Cells were then washed thrice, for 5 minutes each, in PBS at room temperature. 4',6-Diamidino-2-phenylindole (DAPI) was included in the PBS at a dilution of 1:20,000. Coverslips were then mounted on slides and examined. Cells staining positive for BrdUrd and DAPI were counted with a fluorescent microscope using a Zeiss Axioplan 2. Experiments were done in triplicate. Student's t test was used to compare the difference between means. P < 0.05 was considered statistically significant.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assays were done using the MTT cell proliferation kit I from Roche Applied Science (Indianapolis, IN). Four thousand cells from each cell line (Hs746T, NCIN87) were infected with AdS37A or AdLacZ at a MOI of 1,000 and plated in 96-well plates for 24 hours at 37°C in 5% carbon dioxide in the appropriate medium. After 24 hours, the medium was changed and cells were incubated an additional 24 hours. Forty-eight hours following infection, MTT labeling reagent was added to the wells and incubated for 4 hours at 37°C. The solubilization reagent was then added and the cells were incubated overnight at 37°C. The 96-well plate was then read in a microplate reader at 595 nm wavelength. Experiments were done in triplicate and Student's t test was used to compare the difference between means. P < 0.05 was considered statistically significant.

Invasion assays. The ability of cells to migrate through Matrigel-coated membranes was measured in a modified Boyden chamber. Cells were initially plated in a 24-well plate. Twenty-four hours after plating, the cells were infected with AdS37A or AdLacZ at a MOI of 1,000, and transferred to a 24-well plate containing chemoattractant. For invasion assays using siRNAs, cells were plated onto polyethylene teraphthalate membranes 24 hours following electroporation. For each invasion assay, 50,000 cells were plated in triplicate in the top chamber of Matrigel-coated polyethylene teraphthalate membranes (24-well insert, 8 µm pore size; Becton Dickinson Labware, Franklin Lakes, NJ). 3T3 conditioned medium was used as the chemoattractant in the lower chamber. After 24 hours, cells that did not migrate through the membrane were removed from the upper surface with a cotton swab. Cells that had migrated to the lower surface were stained with Diff-Quick (Dade, Deerfield, IL); three random fields were counted at x200. Values for invasion were expressed as the average number of cells per microscopic field and expressed as averages for three independent experiments. Student's t test was used to compare the difference between means. P < 0.05 was considered statistically significant.

Expression profiling with Affymetrix GeneChips. The gastric cancer cell lines were plated at a density of 1 x 107 cells per 100 mm2 plates. After 16 hours, the cells were inspected and infected with the AdS37A and the AdLacZ at a MOI of 1,000. After an incubation time of 48 hours, total RNA was extracted from AdS37A-infected and AdLacZ-infected control cell lines, using Trizol (Life Technologies, Carlsbad, CA). Biotinylated target cDNA was generated from total sample RNA and hybridized to human oligonucleotide probe arrays (U133A, Affymetrix, Santa Clara, CA) according to established protocols (20). Data analysis was done as follows: For the first filter criteria, genes were subject to analysis using the Affymetrix Gene Suite 5.0 algorithm such that the gene was called by the algorithm to be present in at least one sample in the study. This resulted in the identification of 13,066 genes. These genes were normalized to the mean of their expression level in the AdLacZ-infected control cells and then further filtered for most-regulated by AdS37A criteria.

Quantitative reverse transcription-PCR. Total RNA was extracted from AdS37A and AdLacZ infected cells. cDNA was generated using reverse transcription by random hexamers. Quantitative PCR was then done by monitoring in real-time, the increase in fluorescence of SYBR green dye (Qiagen, Chatsworth, CA) with an ABI 7700 (Applied Biosystems, Foster City, CA) sequence detection system. The relative gene copy number was calculated by the standard curve quantitation Ct method (21). Primers were designed using Primer Express (Applied Biosystems).

MT3-MMP forward: TGTAATCCAAGATGGTTGGCTCTA.
MT3-MMP reverse: GGCACTGACTGCCTTTGTTCT.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: ATGGAAATCCCATCACCATCTT.
GAPDH reverse: CGCCCCACTTGATTTTGG.

Reporter assays. To construct reporter plasmids, 1.9 kb of the MT3-MMP 5' untranslated region (UTR) and 1.5 kb of MT3-MMP intron 1 were separately PCR cloned from BAC clone RP11-586k2 (BACPAC Resource Center at Children's Hospital Oakland Research Institute) into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). PCR primers and conditions were as follows:

MT3-MMP promoter forward: ATTGGTACCGTCTTGGGTCAAGTTGGGTG.
MT3-MMP promoter reverse: TATCTCGAGGGAACGTGGCGCCTAAGTTC.
MT3-MMP intron 1 forward: AGGTACCGTAAGAGTGCGAGGGCTTTCT.
MT3-MMP intron 1 reverse: CCTCGAGCAGCCTATGAACTAATCCTTG.

PCR conditions are as follows: 94°C 30 seconds, 62°C 1 minute, 72°C 2 minutes, x30 cycles.

Fragments were then recloned into the pGL3-basic luciferase vector (Promega, Madison, WI) using Sac1/Xho1. Both constructs were verified by DNA sequencing in the University of Cincinnati DNA core laboratory.

For reporter assays, SW480 cells were plated at 1 x 105 per well in 12-well culture plates using Leibovitz medium supplemented with 10% fetal bovine serum (FBS). The cells were transfected 24 hours after plating. Transfection mixtures were prepared in Opti-MEM (Invitrogen), and consisted of 0.95 µg MMP-16 promoter/intron-1 firefly luciferase vector, 0.05 µg pRL-TK (Promega), and 2 µL LipofectAMINE 2000 reagent (Invitrogen). The Dual-Luciferase Reporter Assay (Promega) was done 48 hours posttransfection using the passive lysis method per instructions of the manufacturer. Luminescence was measured on a Turner BioSystems 20/20n Luminometer.

Tumor samples and immunohistochemistry. Gastric cancers previously collected as part of Southwestern Oncology Group Study 9008 and stored in the Southwestern Oncology Group Gastrointestinal Tumor Repository at the University of Cincinnati comprised the tissues used in this study. Seventeen of these tumors had previously been shown to contain mutations in exon 3 of the ß-catenin gene (11). Formalin-fixed, paraffin-embedded tissue blocks from these tumors were cut into 4-µm-thick sections and placed onto positively charged slides. Slides were incubated overnight, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol/water. Epitope unmasking was accomplished by microwaving in 10 mmol/L citrate buffer (pH 6.0) for 15 minutes, followed by cooling for 30 minutes at room temperature. The sections were incubated with an anti-MT3-MMP rabbit polyclonal antibody (Neomarkers) at a dilution of 1:20 for 2 hours at 37°C. Immunohistochemical staining was done using an indirect biotin streptavidin 3,3'-diaminobenzidine method and the Ventana ES automated immunostainer (Ventana Medical Systems, Tucson, AZ). Slides were lightly counterstained with Mayer's hematoxylin. Adjacent normal gastric tissues were used as controls. Staining was scored in a semiquantitative manner by two independent observers (J. Bishop and C. Fenoglio-Preiser). Staining was scored for intensity (0; 1, weak; and 2, strong) and the percentage of cells stained (0; 1,= 1-49%; and 2, >50%). MT3-MMP staining was considered positive if protein expression was present in >50% of cells. Statistical comparisons were done using {chi}2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell proliferation and invasion are increased in gastric cancer cell lines following activation of Wnt signaling. In our experiments, BrdUrd incorporation was used to measure the proliferative status of the cell lines. This assay revealed that both gastric cancer cell lines, when infected with AdS37A, significantly increased cell proliferation compared with cells infected with AdLacZ (Fig. 1A ). For NCNI87 cells, BrdUrd incorporation increased from 23% to 44% (P < 0.05); for the Hs746T cells, BrdUrd incorporation increased from 21% to 43% (P < 0.05). In addition to BrdUrd incorporation, we examined proliferation using the MTT assay, which measures the ability of viable cells to reduce a tetrazolium salt to an insoluble form. This assay also showed significant increased proliferation in both NCIN87 and Hs746T cells following infection with oncogenic ß-catenin (Fig. 1B).


Figure 1
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  		Figure 1. Wnt signaling increases proliferation of gastric cancer cells. A, HS746T and NCIN87 gastric cancer cell lines were infected with AdS37A or AdLacZ at a MOI of 1,000 for 48 hours and then labeled with BrdUrd. Results reflect the average of three independent experiments. Proliferation was significantly increased in both cell lines after AdS37A infection (P < 0.05). B, MTT assay also shows increased proliferation of gastric cancer cells 3 days following gene transfer of oncogenic ß-catenin (S37A) versus LacZ control (P < 0.05 for both cell lines).

 
We next asked whether Wnt activation affects changes in the invasiveness of gastric cancer cells. For invasion studies, the ability of cells to invade through polyethylene teraphthalate membranes coated with Matrigel was evaluated. After infection with AdS37A, invasiveness was increased in both the Hs746T (P < 0.01) and the NCIN87 line (P < 0.01) compared with AdLacZ-infected cells (Fig. 2 ).


Figure 2
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  		Figure 2. Wnt signaling increases the invasiveness of gastric cancer cells. After infection with AdS37A or AdLacZ, Hs746T and NCIN87 cells were assayed using the Matrigel invasion assay. Invasiveness was significantly increased in Hs746T cells (P < 0.01) and NCIN87 cells (P < 0.01).

 
Transcriptional profiling of gastric cancer cells with activated Wnt signaling identifies increased MT3-MMP expression. NCIN87 cell cultures were infected with AdS37A or AdLacZ in three independent experiments. RNA was purified and subjected to GeneChip gene expression analysis. To identify the most reproducibly and fold-regulated genes, we filtered the microarray data using the Affymetrix Gene Suite 5.0 algorithm such that genes were required to be present in at least one sample in the study. This resulted in the identification of 13,066 genes. From the 13,066 genes, we then sought to identify genes that were elevated in all AdS37A samples relative to AdLacZ using the parametric t test with a Benjamini Hochberg false discovery rate of 5%. This identified a total of 107 genes up- or down-regulated. Without false discovery testing at P < 0.01, an additional 87 genes were identified. This created a total pool of 194 genes. Twenty-nine additional genes not meeting the prior criteria were also identified as up-regulated in all three experiments, and at least 2-fold, in two of three experiments. This created a final pool of 213 genes (Fig. 3A ). Given our findings that Wnt activation increased gastric cancer cell invasiveness, we were specifically interested in genes known to have a role in extracellular matrix maintenance or degradation. Interestingly, whereas several previously identified Wnt target genes, including MMP-7 and MT1-MMP, were not differentially expressed, one MMP family member, MT3-MMP, was up-regulated an average of 2.6-fold following AdS37A infection compared with AdLacZ. Seven other differentially expressed genes with a putative role in maintenance or metabolism of extracellular matrix were identified including thrombospondin (Fig. 3B). Because the Wnt pathway regulates expression of other MMPs, we selected MT3-MMP for further analysis and validation using quantitative reverse transcription-PCR (RT-PCR). Quantitative RT-PCR experiments confirmed up-regulation of MT3-MMP by AdS37A, with mRNA levels ranging from 3.5- to 5.5-fold that seen after AdLacZ infection (Fig. 3C).


Figure 3
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  		Figure 3. Wnt signaling up-regulates MT3-MMP in gastric cancer cells. GeneChip and quantitative RT-PCR data is depicted. GeneChip uses the term MMP16 for the identical gene MT3-MMP. A, GeneChip data depicting the pattern of differentially expressed genes from three separate cell cultures of NCIN87 cells infected with AdS37A or AdLacZ. B, eight differentially expressed genes with a putative role in maintenance or metabolism of extracellular matrix. C, data for quantitative PCR on MT3-MMP expression levels compared with GAPDH for AdS37A-infected and AdLacZ-infected cells.

 
Down-regulation of Wnt signaling abrogates MT3-MMP expression. To test the idea that Wnt signaling regulates MT3-MMP expression, we examined whether down-regulation of Wnt signaling could abrogate MT3-MMP expression. As a gastric cancer cell line with constitutively active Wnt signaling was not available, we used the SW480 colon cancer cell line. SW480 is characterized by an active Wnt pathway due to the loss of a functional APC tumor suppressor. Western analysis verified that MT3-MMP is highly expressed in SW480 cells. Wild-type APC was then transfected into SW480 cells and lysates prepared to measure MT3-MMP expression. Introduction of APC efficiently down-regulated Wnt signaling as measured by a TOPFLASH reporter assay (data not shown) and reduced expression of MT3-MMP, thus further providing evidence that MT3-MMP expression is regulated by the Wnt pathway (Fig. 4 ). Analysis of the 5' UTR of the MT3-MMP gene reveals two putative TCF binding within 2 kb of the transcriptional start site and 10 putative TCF sites within the first intron. Previous studies have shown the presence of functional TCF sites within the first intron of the ß-catenin/TCF target gene (22).


Figure 4
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  		Figure 4. Down-regulation of Wnt signaling abrogates MT3-MMP expression. Western analysis for MT3-MMP in SW480 cells transfected with the APC tumor suppressor gene or control. Expected protein sizes for ß-catenin, MT3-MMP, and actin are indicated on the right, as determined by protein standards (not shown). Protein lysates were loaded in lanes 1 to 4 as follows: 1, pAPC-FLAG; 2, pEGFP; 3, mock transfectant; 4, mouse placenta (positive control). Note that transfection of APC down-regulates ß-catenin levels and results in reduced expression of MT3-MMP.

 
MT3-MMP up-regulation occurs by an indirect mechanism. To determine if ß-catenin/Wnt signaling activates and up-regulates MT3-MMP expression directly or indirectly, we created two reporter constructs, containing putative promoter regions for MT3-MMP. The first construct contained the first 1.9 kb of the MT3-MMP 5' UTR and the other contained the first 1.5 kb of MT3-MMP intron 1. The 1.9 kb fragment contains two TCF consensus sites, whereas the intron 1 fragment contained five TCF sites. Following verification of the correct sequences, we transfected the reporter plasmids into SW480 cells and determined luciferase activity. Despite the presence of TCF consensus sites in both constructs, we found no evidence of reporter activity (data not shown). These results suggest that it is likely that ß-catenin/Wnt signaling up-regulates MT3-MMP via an indirect mechanism.

Up-regulation of MT-3-MMP protein product by Wnt signaling regulates invasion in gastric cancer cells. Having shown that Wnt signaling induced an increase in the invasiveness of gastric cancer cells and that MT3-MMP expression is regulated by Wnt signaling, we next assessed whether induction of MT3-MMP was critical to the invasive phenotype. In this series of experiments, we first directly examined expression of MT3-MMP in NCI-N87 cells after high-efficiency electroporation with constitutively active ß-catenin by Western analysis. Overexpression of pS37A-ß-catenin induced expression of MT3-MMP at levels comparable that present within the SW480 colon cancer cell line that has constitutively active Wnt signaling. We then showed that this expression could be abrogated by coelectroporation of siRNAs specific for MT3-MMP message (Fig. 5A ). Following these preliminary experiments, we examined the ability of NCIN87 cells to invade through Matrigel after activation of the Wnt pathway with and without siRNA-mediated down-regulation of MT3-MMP. As in previous experiments, Wnt signaling induced a significant increase in the ability of NCI-N87 cells to invade through Matrigel (P < 0.01). Coelectroporation of siRNAs specific to MT3-MMP along with pS37A-ß-catenin resulted in near-complete abrogation of the effects of Wnt signaling on invasiveness. Mismatch siRNA controls showed the effect to be specific for MT3-MMP (Fig. 5B). These experiments show that invasiveness induced by activated Wnt signaling is mediated by up-regulation of the MT3-MMP protein product.


Figure 5
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  		Figure 5. A, Wnt signaling–mediated up-regulation MT3-MMP expression is abrogated by MT3-MMP-specific siRNAs. Western analysis of MT3-MMP expression in cells electroporated with S37A-ß-catenin, alone and in combination with siRNAs specific to MT3-MMP or control siRNA. Expected protein sizes for MT3-MMP and p21 loading control are indicated on the right, as determined by protein standards (not shown). Protein lysates were loaded in lanes 1 to 9 as follows: 1, SW480 cell line (untransfected, positive control); 2, NCIN87 (untransfected); 3, NCIN87 + S37A-ß-catenin; 4, NCIN87 + S37A-ß-catenin + 10 pmol/L siRNA MT3-MMP; 5, NCIN87 + S37A-ß-catenin + 20 pmol/L siRNA MT3-MMP; 6, NCIN87 + S37A-ß-catenin + 30 pmol/L siRNA MT3-MMP; 7, NCIN87 + S37A-ß-catenin + 20 pmol/L control siRNA; 8, NCIN87 + S37A-ß-catenin + 30 pmol/L control siRNA; 9, NCIN87 + empty vector transfectant. Note that MT3-MMP expression is up-regulated after activation of Wnt signaling by S37A-ß-catenin, and that MT3-MMP expression is abolished by siRNAs specific to MT3-MMP. B, Wnt-mediated invasiveness of gastric cancer cells is dependent on MT3-MMP up-regulation. Coelectroporation of MT3-MMP-specific siRNAs abolishes the enhanced invasiveness of NCIN87 cells that is observed after introduction of S37-A ß-catenin alone as assessed by assayed using the Matrigel invasion assay. Invasiveness was significantly increased in NCIN87 cells following introduction S37A-ß-catenin or S37A-ß-catenin + control siRNAs (P < 0.001) but returned to baseline levels following cointroduction of siRNAs specific for MT3-MMP with S37A-ß-catenin (P < 0.001).

 
Human gastric cancers with activated Wnt signaling overexpress MT3-MMP. In a previous report, nuclear expression of ß-catenin was observed in 90 of 311 (29%) of human gastric cancers. Of 73 cancers with nuclear ß-catenin, ß-catenin exon 3 mutations were identified in 19 (26%). These findings showed that Wnt pathway activation occurs in a subset of gastric cancers. In this study, the presence of ß-catenin mutation did not correlate with either Lauren classification (diffuse or intestinal histology) or the stage of disease. Material from 17 of the 19 gastric cancers found to have ß-catenin mutations was available for further study. Immunohistochemical staining to examine MT3-MMP expression was done on these gastric cancers and on normal-appearing gastric mucosa from 76 tumors. MT3-MMP expression was positive in 12 of 17 (71%) tumors containing oncogenic mutations in ß-catenin versus 29 of 76 (38%) cases of benign gastric mucosa (P < 0.05). Expression of MT3-MMP was present both at the cell membrane and in the cytoplasm (Fig. 6A and B ). Costaining studies revealed expression of MT3-MMP in 12 of 13 tumors with ß-catenin nuclear localization. Examples of costaining within a lymphatic channel and at the invasion front are depicted in Fig. 6C to F.


Figure 6
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  		Figure 6. MT3-MMP is highly expressed in human gastric cancers with activated Wnt signaling. A (low power) and B (high power), gastric cancer with a known mutation in ß-catenin. MT3-MMP immunostaining reveals high expression only in gastric cancer cells. C, immunostaining for ß-catenin. Note the prominent nuclear staining within gastric cancer cells present within a lymphatic channel. D, adjacent section stained for MT3-MMP revealing high expression within the same lymphatic channel. E, invasion front (dark line) of a gastric cancer stained for ß-catenin. F, adjacent section revealing high MT3-MMP expression at the same invasion front.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well accepted that dysregulation of the Wnt pathway plays a critical role in the development of human cancer. Numerous malignancies harbor mutations in the APC or ß-catenin genes and these mutations are mutually exclusive (23, 24). Each of these genetic events results in accumulation of nuclear ß-catenin and subsequent changes in gene transcription, as well as perhaps other nontranscriptionally directed changes in cellular phenotypes. Interestingly, although the contribution of Wnt/ß-catenin signaling to carcinogenesis can be surmised from the identification of transcriptional target genes, studies demonstrating Wnt-induced changes in other cellular processes are relatively few. It is now becoming increasingly clear that Wnt target genes may be specific to both tissue and cellular microenvironment. The identification of target genes dysregulated specifically in certain tumor types holds increasing importance as the design of biologically targeted therapies becomes increasingly precise.

Our laboratory has been interested in the effects of activated Wnt signaling in the initiation and progression of gastric cancer. Previous work has shown that Wnt activation occurs frequently in gastric carcinoma, often as a result of activating mutations in ß-catenin (12). This study directly investigated the effects of an oncogenic form of ß-catenin on the proliferation and invasive phenotype of gastric cancer cells. Early invasion and metastasis is a hallmark of gastric cancer and thus we were interested in the contribution of Wnt signaling to these clinical characteristics of the disease. Our results reveal that Wnt activation results in significant increases in invasiveness in gastric cancer cells.

Our findings show that in the MMP family member MT3-MMP, expression is regulated by Wnt signaling and is highly expressed in human gastric cancers containing an activated Wnt pathway. MT3-MMP is a membrane-anchored MMP normally expressed in human placenta, brain, and lung (25). It is a known activator of the zymogen of MMP-2 and can degrade type III collagen, gelatin, fibronectin, and laminin-1 (26). MT3-MMP has previously been shown to enhance the growth and invasiveness of Madin-Darby canine kidney cells and it is highly expressed in renal cell carcinoma and melanoma (27, 28). Our work corroborates the results of Liu et al. (29) who found that MT3-MMP mRNA levels were elevated by 2-fold in 16 human gastric cancers compared with matched noncancerous gastric epithelial tissue. The status of the Wnt pathway in these tumors, however, was not evaluated. Regulation of MMP expression is complex and it is certain that Wnt signaling is not the sole method of regulating MT3-MMP expression in gastric cancer and these tumors express other MMPs, including MMP-2. Up-regulation of MT3-MMP expression could therefore be synergistic as it is a potent activator of MMP-2 activity. Our results did not reveal the previously described Wnt target genes MMP-7, MT1-MMP, or MMP-26 to be up-regulated in the gastric cancer cell lines tested. Furthermore, our siRNA experiments suggest that MT3-MMP is largely responsible for the increase in invasiveness of gastric cancer cells after Wnt signaling is activated. This may simply be a finding particular to these cell lines or could reflect the tissue specificity of Wnt target genes. Cyclin D and c-myc have been long held as Wnt-induced mediators of proliferation in the colon, yet transgenic mice expressing activated ß-catenin in the liver displayed hepatomegaly without up-regulation of either gene (30).

Our work suggests that MT3-MMP expression is not regulated via direct binding of ß-catenin/TCF to the MT3-MMP promoter. Although we cannot rule out the presence of functional TCF sites in sequence outside the area we have examined, this seems unlikely. It is more probable that activation of MT3-MMP is occurring via Wnt-induced activation of another transcription factor or that ß-catenin itself is binding a partner other than TCF and activating transcription directly (31). In either case, alternative mechanisms such as this lend support for the tissue-specific nature of MT3-MMP regulation by ß-catenin/Wnt signaling. Confirming the existence of tissue-specific Wnt targets could be particularly relevant as the design of more specific MMP inhibitors continues. Interestingly, the only phase III clinical trial suggesting any clinical benefit for MMP inhibitors has been in patients with gastric cancer (32).

It remains unclear whether activated Wnt signaling contributes to the initiation or progression of gastric cancer. In a rat model of gastric cancer, mutations in ß-catenin contribute to tumor progression rather than initiation (11). Human studies of Wnt pathway mutations in gastric cancer have failed to show a clear relationship to stage. It has been hypothesized that Wnt/ß-catenin signaling could induce increases in invasiveness as several MMPs (MT1-MMP, MMP-7, and MMP-26) are transcriptional targets of the pathway. In addition, one member of the Wnt ligand family, Wnt 5a, increases motility and invasiveness in melanoma, although this phenotype is likely mediated via a noncanonical Wnt pathway (33). The fact that Wnt signaling induced changes in proliferation and invasiveness in gastric cancer cell lines that contain a host of other genetic alterations suggest it is a powerful effector of carcinogenesis.

In conclusion, the activation of Wnt signaling results in increased proliferation and invasiveness of gastric cancer cells. MT3-MMP mRNA expression is up-regulated following Wnt activation and MT3-MMP protein expression specifically enhanced the invasiveness of gastric cancer cells. MT3-MMP protein expression is up-regulated in gastric cancer specimens containing mutant ß-catenin. These results argue that Wnt activation may contribute to tumor progression by increasing the proliferation and invasiveness of gastric cancer cells and that this increase in invasiveness is likely mediated, in part, by MT3-MMP.


   Acknowledgments
 
Grant support: NIH grants CA89403 (A.M. Lowy), CA63507 (J. Groden), U10CA32102 (C. Fenoglio-Preiser), and U10CA38926 (C. Fenoglio-Preiser); and a grant from Ohio Cancer Research Associates (A.M. Lowy).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/29/05. Revised 1/26/06. Accepted 2/ 9/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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