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Clinical Cancer Research Vol. 11, 6479-6488, September 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Possible Involvement of RUNX3 Silencing in the Peritoneal Metastases of Gastric Cancers

Chouhei Sakakura1, Kazunori Hasegawa3, Koji Miyagawa1, Susumu Nakashima1, Tetsuji Yoshikawa1, Shuichi Kin1, Yuenn Nakase1, Shujiro Yazumi3, Hisakazu Yamagishi1, Takeshi Okanoue2, Tsutomu Chiba3 and Akeo Hagiwara1

Authors' Affiliations: 1 Surgery and Regenerative Medicine, Surgery and Physiology of Digestive System; 2 Division of Gastroenterology, Graduate School of Internal Medicine, Kyoto Prefectural University of Medicine; and 3 Division of Gastroenterology and Hepatology, Graduate School of Internal Medicine, Kyoto University, Kyoto, Japan

Requests for reprints: Chouhei Sakakura, Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kawaramachi-dori, Kyoto 602, Japan. Phone: 81-75-251-5527; Fax: 81-75-251-5522; E-mail: sakakura{at}koto.kpu-m.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray.

Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-ß (TGF-ß)–induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of ~21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified ~28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-ß signaling pathway.

Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

The prognosis of gastric cancer invading as deep as the gastric serosa remains poor, with a 5-year survival of <35% (1). Peritoneal dissemination is reported to be the most frequent type of recurrence after curative resection in such cases (2, 3). Although coordination of multiple genes is involved in metastasis, only one or a few genes have been characterized in most previous reports examining this process. Peritoneal metastases is thought to consist of several steps in host-tumor interaction: malignant cells detach from the primary cancer, invade surrounding tissue, penetrate into the peritoneal cavity, adhere to the peritoneal surface lined by mesothelial cells along a basement membrane, migrate into submesothelial tissue, and finally proliferate at these metastatic sites. Differences in metastatic potential are expected to result from differences in expression in a combination of genes related to cell adhesion, apoptosis, drug metabolism, signal transduction (4, 5), and other processes. Yet, details of the molecular basis of peritoneal metastasis remain unclear.

Recently, we described how the RUNX3 gene participated in gastric carcinogenesis through disruption of normal gastric mucosal development and differentiation (6). Whereas additional roles of RUNX3 in immune responses, gastric epithelial development, and neurologic development are well known, how RUNX3 might be involved in progression and metastasis of established gastric cancers has not yet been clarified. We previously reported an analysis of RUNX3 down-regulation showing progressive silencing according to cancer stage, especially in stage IV, almost 100% of cases showed silencing of RUNX3 (6). RUNX3 is a transcription factor that regulates expression of many downstream genes, but many details remain to be elucidated. Many genes would be likely to contribute to peritoneal metastases. As RUNX3 is related TGF-ß-dependent apoptosis, resistance to apoptosis involving to loss of RUNX3 expression is suspected in cases showing metastasis.

In the present study, we examined expression of RUNX3 in clinical tissue samples from peritoneal metastases. We also examined changes in metastatic potential in animal experiments using RUNX3-stable transfectants produced in gastric cancer cells obtained from malignant ascites. Furthermore, we globally analyzed expression profiles of ~21,000 genes in parent cells and stable RUNX3 transfectants using a cDNA microarray.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, gastric cancer specimens, and RNA preparation. Gastric cancer cell lines KATO-III were maintained at 37°C in a humidified atmosphere of 5% CO2, in high-glucose RPMI 1640 (Sigma, St. Louis, MO), with 10% fetal bovine serum, penicillin, and streptomycin. When they reached 80% to 90% confluence, cells were washed with ice-cold PBS and homogenized immediately in Isogen reagent (Nippon Gene, Osaka, Japan), and total RNA was extracted. mRNA extracted from each cell line was extracted by FAST track kit Ver.2 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

The study population is consisted of patients with gastric cancer undergoing surgery at Kyoto Prefectural University of Medicine during 1999 to 2003. Written informed consent was obtained from each patient before tissue acquisition. All experiments with clinical samples were conducted under institutional guideline from the Ministry of Health and Welfare.

Northern blot analysis. Northern blot was done as we described previously (7, 8). In brief, total cellular RNA was prepared by the guanidine isothiocyanate-phenol-chloroform procedure. Selection of polyadenylated RNA was done by an oligo dT column, then fractionated on 1% agarose/2.2 mol/L formaldehyde gels. Probes were labeled with 32P by random priming. Each blot was hybridized with probes for RUNX3 and ß-actin, as described previously (7, 8). We analyzed signals with a BAS 2000 image analyzer and calculated the degree of expression compared with control.

Western blot analysis. Western blot analysis was done as described previously (9). Cells were washed twice with PBS and lysed in 1% Triton X-100, 0.15 mol/L NaCl, and 10 mmol/L Tris-HCl (pH 7.4), with 50 µg/mL phenylmethylsulfonyl fluoride at 4°C for 60 minutes. Lysates were centrifuged at 10,000 x g for 10 minutes. Samples were boiled in SDS sample buffer for 5 minutes before running on a 10% to 20% SDS-polyacrylamide gel after overnight transfer of SDS-polyacrylamide gel to nitrocellulose membrane. After blocking the blot in 3% BAS for 2 hours at room temperature, immunodetection of RUNX3 was done with monoclonal antibody (mouse monoclonal antibody, kindly donated by Prof. Ito, Shingapore Institute) at 1:250 dilution for 24 hours at room temperature. Horseradish peroxidase–conjugated anti-mouse IgG antibody was incubated with the blot at 1:1,000 dilution in PBS with 1% nonfat dry milk. The blot was visualized with enhanced chemiluminescence kit (Amersham, Pittsburgh, PA).

Transforming growth factor-ß1 treatment. TGF-ß1 from porcine platelets (R&D Systems, Minneapolis, MN) was added at concentrations of 0.01 to 5 ng/mL to the human KATO-III cell cultures 24 hours after cell seeding, unless otherwise indicated. After addition of TGF-ß1, no medium change was carried out.

Measurement of [3H]thymidine incorporation and cell growth. For measurement of DNA synthesis, cells on 24-well plates were labeled for 2 to 24 hours with 1 µCi/mL per well [methyl-3H]thymidine (71 Ci/mmol; ART-178B; American Radiolabeled Chemicals, Inc., St. Louis, MO) before cell harvest. After radiolabeling, the cells were washed in situ twice each with ice-cold PBS, 5% trichloroacetic acid, and 95% ethanol. The cells were then lysed with 200 µL of 0.3 N NaOH. Aliquots of the cell lysates were neutralized with HCl, and the radioactivity was measured in a liquid scintillation counter.

Cell growth was also assessed directly by cell counting. Cells were treated with or without TGF-ß (5 ng/mL), and sequential cell-counting experiments were done every 24 fours over the periods of 5 days.

Luciferase assay. The effect of RUNX3 on RUNX3 mediated transcriptional activity was evaluated with the use of a dual-luciferase assay as described previously (10). Cells were plated with at 60,000 cells per well in 6-well plates in complete growth medium, incubated for 24 hours, and transfected with pEF-BOS-RUNX3 or neomycin according to the manufacturer's protocol. The next day, cells were treated for 16 hours with TGF-ß (5 ng/mL). Firefly luciferase activities in cell lysates were determined with use of the Dual Luciferease Reporter Assay system (Promega, Pittsburgh, PA) according to the manufacturer's protocol. Firefly luciferase activity normalized to Renilla reniformis luciferase activities is presented as fold activation changes. All assays were done in triplicates; each experiments was repeated four times.

Measurement of cell apoptosis. To determine the apoptotic effect of RUNX3/TGF-ß, cells were treated with TGF-ß of 5 ng/mL for 24 hours with or without TGF-ß in serum-free medium. APOPercentage Apoptosis Assay (Biocolor, Belfast, Northern Ireland) was used to assess the effect of RUNX3 overexpression on apoptosis. Vector or RUNX3-expressing KATO-III cells were seeded on a 96-well plate at a density of 2 x l03 cells per well. Apoptosis was induced by the addition of 5 ng/mL of TGF-ß, in RPMI 1640 supplemented with 5% fetal bovine serum. Cellular dye uptake was analyzed according to the protocol by the manufacturer.

Real-time quantitative reverse transcription-PCR. cDNA was produced from total RNA by using a Superscript preamplification system (Bethesda Research Laboratories, Bethesda, MD) and following the procedures suggested by the manufacturer. RNA was heated to 70°C for 10 minutes in 14 µL of diethylpyrocarbonate-treated water containing 0.5 µg oligo (dT). Synthesis buffer (10x), 2 µL of 10 mmol/L deoxynucleotide triphosphate mix, 2 µL of 0.1 mol/L DTT, and reverse transcriptase (Superscript RT; 200 units/µL) were added to the sample. The resulting reaction mixture was incubated at 42°C for 50 minutes, and reaction was terminated by incubating the mixture at 90°C for 5 minutes.

Quantitative PCR was done using real-time Taqman technology and analyzed on a Model 5700 Sequence Detector (Applied Biosystems Corp., Foster City, CA) as described previously (11).

Methylation-specific PCR. Methylation-specific PCR was done as reported previously (12, 13). Briefly, 2 µg of DNA was diluted in 50 µL of distilled water and 5.5 µL of 2 mol/L NaOH was added. After incubating the mixture at 37°C for 10 minutes, 30 µL of freshly prepared 10 mmol/L hydroquinone (Sigma) and 520 µL of 3 mol/L sodium bisulfite (pH 5.0) were added. The mixture was incubated at 50°C for 16 hours and the DNA was purified by Wizard DNA purification resin (Promega). The DNA pellet was dissolved in 20 µL H2O and 2 µL was used for PCR using following primers. The primer set used for untreated DNA: Rx3-5W (5'-GAGGGGCGGCCGCACGCGGG-3') and Rx3-3W (5'-CGGCCGGCGCGGGCGCCTCC- 3'). The primer set used for detecting methylated DNA: Rx3-5M (5'-TTACGAGGGGCGGTCGTACGCGGG-3') and Rx3-3M (5'-AAAACGACCGACGCGAACGCCTCC- 3'). The primer set used for detecting unmethylated DNA: Rx3-5U (5'-TTATGAGGGGTGGTTGTATGTGGG-3') and Rx3-3U (5'-AAAACAACCAACACAAACACCTCC-3'). Methylated nucleotides were verified by sequencing the PCR products.

Plasmids and stable transfection. To obtain RUNX3-expressing KATO-III cells, stable transfection of pEF-BOS-RUNX3 into KATO-III cells was done by the LipofectAMINE method as specified by the manufacturer (Life Technologies, Carlsbad, CA). G418 (600 µg/mL) resistant colonies were selected and subcultured as described previously (6). Independent clones that strongly expressed RUNX3 were screened by Northern blotting. KATO-III cells stably transfected with pEF-BOS-neo were used as controls.

Experimental model in nude mouse. Four-week-old female BALB/c nu/nu mice (Clea Japan, Inc., Osaka, Japan) were inoculated i.p. with 5 x 106 gastric cancer cells KATO-III (four clones of 7.stable transfectant of RUNX3 or neomycin) in 0.5 mL PBS. Four weeks later, the presence of disseminated foci or ascites was determined. All animal experiments were conducted in accordance with institutional guidelines for animal welfare. Representative whole mount specimen of a tumor in abdominal cavity where the stable transfectant of RUNX3 or contra vector in KATO-III were calculated.

DNA microarray analysis. One microgram of mRNA extracted from each gastric cancer cell line was labeled by incorporating Cy3 during random-primed reverse transcription. cDNA derived from the reference pool, which we labeled with Cy5, was used as the expression reference as described previously (14, 15).

The RIKEN human cDNA that comprised the target was hybridized in a final volume of 30 µL; the entire array consists of three multiblocks, and each multiblock required 10 µL of hybridization solution. Before hybridization, probe aliquots were heated at 95°C for 1 minute and cooled at room temperature. Coverslips were hybridized overnight at 65°C in a Hybricasette (obtained from ArrayIt.com, Sunnyvale, CA). After hybridization, slides were washed in 2x SSC/0.1% SDS until the coverslips dropped off, and the slides were then transferred into 1x SSC, shaken gently for 2 minutes, and rinsed with 0.1x SSC for 2 minutes. After washing, slides were spun at 800 rpm in a Sorvall centrifuge (RC-3B plus; H6000A/HBB6 rotor). These slides were scanned on a ScanArray 5000 confocal laser scanner, and the images were analyzed by using IMAGENE (BioDiscovery, Los Angeles, CA).

To improve the accuracy of the data, we did the experiment twice, labeling the same RNA template in two separate reactions. Data were normalized to the reference standard by subtracting (in log space) the median observed value if it were other than zero. We used only data points that were reproducible. To this end, we developed a filtering program, PRIM (16). We applied hierarchical clustering to both axes, using the weighed pair-group method with a centroid average as implemented by the program CLUSTER (17). The results were analyzed by using TREEVIEW (17).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Down-regulation of RUNX3 in peritoneal metastases of gastric cancers analyzed by quantitative reverse transcription-PCR. Relative values for mRNA encoding RUNX3 were determined as ratios to ß-actin mRNA (Fig. 1A). RUNX3 mRNA/ß-actin mRNA ratios (x103) in normal gastric mucosa (46 cases, mean ± SD) were 23.3 ± 7.6. In primary tumor of gastric cancers (48 cases), these were 5.8 ± 2.3, whereas in peritoneal metastases (21 cases), they were 2.0 ± 1.0. The ratios showed significant decreases in peritoneal metastases from that of normal gastric mucosa or primary main tumor, respectively (P < 0.01). Clinical data concerning cases with malignant ascites are presented in Table 1.



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  		Fig. 1. RUNX3 expression in gastric mucosa, primary cancer, and peritoneal metastasis. A, relative mRNA expression for RUNX3 in primary gastric cancers and normal mucosa measured by real-time reverse transcription-PCR with the Light Cycler. Expression of RUNX3 in normal mucosa was significantly greater than in primary gastric cancers (P < 0.01), and much greater than those of for peritoneal metastases (P < 0.001). B, methylation-specific PCR of remnant gastric cancer and adjacent mucosa. Abbreviations: M, methylated sequence–specific PCR; U, unmethylated sequence–specific PCR; DW, distilled water; SM, size marker. Methylated PCR products are present in cases 1, 3, 7, 12, and 14 of peritoneal dissemination and unmethylated PCR products in all cases. C, methylation status of the C residues between –218 and –69 relative to the translation initiation site of the RUNX3 exon 1 region. The nucleotide sequences of the products of MSP of the DNA samples from remnant gastric cancer that do not express RUNX3 (Exp–, cases 3 and 7) and noncancerous mucosa that express RUNX3 (Exp+, case 1 and 2 of peritoneal washes of benign disease) together with wt RUNX3 sequence (top). The red C indicates that it was resistant to bisulfite treatment due to methylation. The blue T indicates that it was converted from C by bisulfite treatment (i.e., not methylated).

 

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  		Table 1. Clinical features of peritoneal dissemination of gastric cancers

 
Promoter methylation status of RUNX3. We did methylation-specific PCR in 21 peritoneal metastases. Typical data for methylation-specific PCR of i.p. metastases from gastric cancers and peritoneal lavage specimens in benign disease are shown in Fig. 1B (M, methylated sequence–specific PCR; U, unmethylated sequence–specific PCR). Methylation of RUNX3 was present in all cases (100%, 21 in 21 cases) of peritoneal dissemination of gastric cancer. A significant negative relationship between methylation status of RUNX3 and RUNX3 expression was observed. In samples negative for RUNX3 expression, C residues in the CpG dinucleotide within the region were methylated (Fig. 1C). In contrast, in samples positive for RUNX3 expression (peritoneal washes of benign disease, cases 22 and 23), none of the C residues were methylated.

Characterization of RUNX3 stable transfectants in KATO-III. Morphologic changes of stable transfectants of RUNX3 in KATO-III cells were more rounded and easily separated than the parent cells or neomycin transfected KATO-III cells as shown in Fig. 2A.



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  		Fig. 2. Changes in RUNX3-transfected KATO-III cells. A, morphologic changes in RUNX3-transfected cell. B, Northern blot analysis for RUNX3 shows increased expression relative to parent cells of KATO-III. The blot was hybridized sequentially with the indicated probes to compare RUNX3 expression with that of ß-actin. C, Western blot analysis of RUNX3 shows increased expression relative to parent cells of KATO-III. The blot was hybridized sequentially with the indicated probes to compare RUNX3 expression with that of ß-actin. D, growth curve of each stable transfectant of RUNX3 and control cells. E, thymidine uptake of each stable transfectant of RUNX3 and control cells.

 
RUNX3 expression was examined in four different kinds of stable transfectant of the gastric cancer cell line KATO-III by Northern blotting and quantified relative to ß-actin expression. Stable transfectants showed 4- to 10-fold overexpression of RUNX3 compared with parent cells (Fig. 2B). RUNX3 protein was also examined in four independent clones of KATO-III cells stably transfected with RUNX3 (clones 1, 2, 3, and 4) by Western blot analysis. As shown in Fig. 2C, stable transfectants of RUNX3 showed strong expression of RUNX3 protein compared with control cells into which only the neomycin-resistant gene was introduced. Anti-ß-actin antibody was used to eliminate from loading variation. As shown in Fig. 2D, KATO-III cells transfected with RUNX3 showed slight appreciable change in their growth rates, whereas KATO-III cells transfected only neomycin showed no difference in their growth rates (P < 0.05; clones 2, 3, and 4). Expression of RUNX3 slightly but significantly prolongs the doubling time from 27 to 34 hours. As an independent confirmation of slightly changed growth rate, thymidine uptake differed between RUNX3-stable transfectants and cells transfected only with the neomycin resistance gene (Fig. 2E; P < 0.05; clones 1, 2, 3, and 4).

As shown in Fig. 3A, parent KATO-III cells displayed no inhibition of cell proliferation, when transfected with mock vector. In contrast, KATO-III transfection of RUNX3 followed by treatment with TGF-ß resulted in a >30% decrease in the rate of proliferation compared with the rate in the vector-transfected control cells treated with TGF-ß (P < 0.05). Parent KATO-III cells did not undergo apoptosis. In contrast, transfection of RUNX3 and treatment of TGF-ß induced cell apoptosis (Fig. 3B). Only RUNX3 expression did not induce cell apoptosis. Clones 1, 3, and 4 slightly showed apoptosis after the TGF-ß treatment but not in clone 2 compared with the KATO-III-neo-stable transfectant (P < 0.05; clones 1, 3, and 4).



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  		Fig. 3. TGF-ß induced growth inhibition and apoptosis in KATO-III cells by expressing RUNX3. A, restoration of growth inhibitory effect of TGF-ß in KATO-III cells by expressing RUNX3. *, P < 0.05. B, responsiveness of TGFß apoptosis. *, P < 0.05. C, luciferase assay. Abbreviation: RLU, relative luciferase unit. *, P < 0.01.

 
With luciferase assay, transcriptional activity involving the RUNX-binding site was examined in stable transfectants of RUNX3 and neomycin transfectants among KATO-III cells. As shown in Fig. 3C, basal luciferase activity was up-regulated by RUNX3-binding site-dependent transactivation in the absence of any stimulation. In contrast, TGF-ß treatment at 5 ng/mL for 16 hours dramatically enhanced activity in examined in stable RUNX3 transfectants (P < 0.01). Firefly luciferase and Renilla reniformis luciferase activities in cell lysates were determined using of the Dual Luciferase Reporter Assay System. Firefly luciferase activity normalized to Renilla reniformis luciferase activities is presented as fold activation changes.

Peritoneal dissemination potential of RUNX3 transfectants in KATO-III. For inspection (Fig. 4A), extensive peritoneal disseminated lesions were found in nude mice inoculated with the stable KATO-III neo transfectants, whereas far less metastatic lesions were found in mice inoculated with KATO-III-RUNX3.



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  		Fig. 4. Inhibition of peritoneal metastases of gastric cancers by RUNX3 expression. A, macroscopic findings of peritoneal dissemination of stable transfectants of RUNX3 in KATO-III cells and control cells. B, quantification of metastatic nodules in mouse peritoneal cavity. C, survival curve of mice after inoculation of each RUNX3-stable transfectants and control cells.

 
Viable stable RUNX3 transfectant in KATO-III and KATO-III-neo transfectant (1 x 106) in HBSS were injected into the peritoneal cavity of nude mice (n = 9). Six weeks after inoculation of transfectants, mice were sacrificed for examination concerning peritoneal dissemination. Figure 4B represents aggregate i.p. tumor weight per animal (n = 9 per each transfectant, P < 0.01). All mice of four groups that were injected with RUNX3 transfectants survived for >35 weeks. In contrast, all mice injected with KATO-III-neo cells (mock transfectant) died within 50 days (Fig. 4C; P < 0.001).

Microarray results. Among the 10,860 to 14,252 genes (known genes and expressed sequence tags) expressed, 22 genes showed a differential expression more than double that of KATO-III neo cells and 32 showed more than half that of other cell lines. To verify these findings, we did Northern blot to analyze all these genes that showed >2-fold difference in terms of cDNA array. When >2-fold changes in expression level by Northern blot analysis were considered significant, consistency with cDNA microarray analysis were 52% (28 of 54), whereas no significant change was seen in 37% (20 of 54) and discordant results were obtained in 11% (6 of 54). Genes identified from both analyses are listed in Tables 2 and 3.


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  		Table 2. Down-regulated genes in RUNX3-stable transfectant of gastric cancer cell line KATO-III

 

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  		Table 3. Up-regulated genes in RUNX3-stable transfectant of gastric cancer cell line KATO-III

 
Expression of selected genes in stable transfectant of RUNX3. As described above, expression of selected genes by microarray to exhibit up-regulation or down-regulation in stable RUNX3 transfectant in KATO-III cells was confirmed by Northern blotting. Typical data are shown in Fig. 5A, indicating that MET and amphiregrin were down-regulated after transfection of RUNX3, whereas caspase 9 was up-regulated in these stable transfectants.



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  		Fig. 5. Expression of selected genes. A, Northern blot analysis of some genes selected by cDNA microarray in stable transfectants of RUNX3 in KATO-III cells and control cells. B, relative mRNA values of MET and IRF in primary gastric cancers, normal mucosa, and peritoneal metastasis measured by real-time reverse transcription-PCR with the Light Cycler.

 
Expression of selected genes in peritoneal metastases of gastric cancers analyzed by quantitative reverse transcription-PCR. Expression of selected genes showing up-regulation or down-regulation by microarray in stable RUNX3 transfectant KATO-III cells was also examined by quantitative reverse transcription-PCR in clinical samples. Some of them showed up-regulation with RUNX3 expression; some showed apparent silencing by RUNX3; and others showed no significant correlation between their degree of expression and that of RUNX3. Typical data from these samples are shown by scatter plots in Fig. 5B, where expression of MET or IFN regulatory factor (IRF) was determined as the MET or IRF mRNA/ß-actin mRNA ratios and plotted for peritoneal deposits, primary tumors, and normal mucosa. Respective MET and IRF mRNA/ß-actin mRNA ratio (x103 and x104, respectively) in these three specimens types were 9.2 ± 6.3, 15.3.1 ± 6.3, and 29.2 ± 8.3 for MET and 28.2 ± 6.3, 13.1 ± 1.3, and 7.2 ± 1.3 for IRF (average ± SD). For both genes, expression was significantly more in primary gastric cancers than in normal gastric mucosa and still more in peritoneal dissemination (P < 0.01, two-tailed t test).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Peritoneal dissemination is the most frequent pattern of gastric cancer recurrence and carries a uniformly poor prognosis (3). Intensive analyses have been carried to clarify mechanisms responsible for these peritoneal metastases. Expression changes for genes in adhesion molecules, signaling molecules, growth factor receptors, drug-metabolizing enzymes, and immune mediators are likely to contribute in a complex manner to peritoneal dissemination of gastric cancer. Overexpression or down-regulation of these genes in RUNX3-stable transfectants without metastatic potential, which we have shown in this study, is in good concordance with findings of previous reports (4, 5) and indicates that gene expression changes in these cells accurately reflects certain characteristics of metastasis. Nevertheless, many details have not been clarified.

In the present study, transfer of RUNX3 gene to gastric cancer cells established from malignant ascites completely inhibited peritoneal metastases in a nude mouse model. Cell proliferation showed little decrease, but more importantly, tumorigenicity with i.p. injection was completely lost. Taken together with complete silencing of RUNX3 in all clinical samples of gastric cancer peritoneal metastases, we believe that RUNX3 expression in gastric cancer could result in loss of potential for peritoneal metastases. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest TGF-ß-induced antiproliferative and apoptotic effects were observed. Interestingly, transfection into gastric cancer cells strongly inhibited peritoneal metastases in nude mouse. A previous study by Hanai et al. indicated that RUNX3 interacts with smads (18) and suggests that transfection of RUNX3 would exert effects related to restored sensitivity to TGF-ß. However, restoration of this sensitivity to TGF-ß was weak; thus, loss of metastatic potential would likely be involved in both the TGF-ß signaling pathway and changes of expression in genes downstream from RUNX3 based on our global analysis of gene expression in RUNX3-stable transfectants of KATO-III cells.

Previous studies showed that Runx proteins have essential functions in both cell proliferation and differentiation factors, and in mammals, they were shown to act as both proto-oncogenes and tumor suppressors. Genes downstream from RUNX family genes have been examined intensively (19). Recent work indicates that RUNX1/AML1 and CAAT/enhancer binding protein factors regulates CD11a integrin expression in myeloid cells through overlapping regulatory elements (20). RUNX2 induces maturation of osteoblasts by regulation of osteocalcin (21), whereas RUNX3 regulates MDR1 in leukemia cells, germ line IgC{alpha} in T cells, and Trk-C in dorsal root cells (22–24). Nonetheless, relatively few reports have characterized the genes downstream from RUNX3. RUNX expression is specific for each cell type and regulates a number of different target genes. In addition, knowledge is limited concerning its downstream genes in gastric epithelial cells and gastric cancer cells. Accordingly, we presently examined global gene expression changes in stable RUNX3 transfectants compared with expression in parent cells.

We used cDNA microarray to screen >21,000 genes for expression changes. This identified many genes regulated by RUNX3 that were not previously shown to undergo such regulation in gastric cancers. We confirmed their expression pattern in clinical samples from malignant ascites, suggesting that they could be important in peritoneal metastases of gastric cancer. Microarray analysis identified ~28 candidate genes under the possible downstream control of RUNX3. Selected genes listed in Tables 2 and 3 are highlighted in this discussion according to their function. Some of them have been reported to be involved in gastric cancer metastases, and many genes showed major increases or decreases in expression directly or indirectly induced by RUNX3. The genes variously are involved in signal transduction, apoptosis, drug metabolism, and cell adhesion. We screened for RUNX-binding sites on these genes in their upstream (promoter) region. Among the selected genes, MET (hepatocyte growth factor receptor) has a RUNX-binding site.

MET is known to act importantly in cancer cell migration, invasion, and metastasis, and its expression carries a poor prognosis in gastric cancers (25). As MET has a binding site of RUNX3 in the promoter region, it may be regulated by RUNX3 transcriptionally, although it also is regulated by many other transcriptional factors (26). Further examinations are necessary to clarify this with luciferase assay, and we are now seeking to do so.

Several genes related to cell adhesion, invasion, and adhesion to the extracellular matrix and growth factor activity were down-regulated or up-regulated in our cell lines derived from stable transfectants of RUNX3. Annexin A1 has been reportedly involved in lymph node metastases of scirrhous gastric cancer (27), but participation of Annexin A5 and A7 is still unclear. Sialyltransferase 1 is reportedly involved in cancer metastases (28), as has galectin 4 (29). Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Type IV collagen, the major structural component of the basement membrane, is a polyvalent ligand possessing sequences bound by the {alpha}1ß1, {alpha}2ß1, and {alpha}3ß1 integrins, as well as by cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan. However, to our knowledge, no other findings related to collagen 13 and 16 in gastric cancers have been reported.

Although growth factors, their ligands, and downstream molecules are frequently overexpressed in cancer cells, to the best of our knowledge, several genes related to signaling listed in Tables 2 and 3 have not been reported previously as overexpressed. Amphiregulin has been implicated to be involved in liver metastasis of colon cancers (30), but its role in gastric cancers is still unknown. The LIM domain-only genes LMO1 and LMO2 are translocated in acute T-cell leukemia and have been shown to act as oncogenes in T lymphocytes (31, 32). LMO4, the fourth member of this family, is overexpressed in >50% of sporadic breast cancers, suggesting a role in the biology of these tumors. LMO4 was recently found to interact with the breast/ovarian tumor suppressor BRCA1, with LMO4 repressing BRCA1 transcriptional activity (33). Combined disruption of both mitogen-activated protein kinase kinase/extracellular signal-activated kinase and interleukin-6/signal transducers and activators of transcription 3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells (34), whereas cathepsin E inhibits vessel growth and induces apoptosis (35). Vav3 modulates tyrosine kinase signaling and changes in cell morphology and induces neoplastic transformation (36). Whereas the function of the most of the kallikreins remains to be elucidated, two members are useful biomarkers for prostate cancer and several others are potentially informative markers for breast cancer, Alzheimer's disease, and Parkinson's disease (37). IRF and S100C/A11 are involved in growth inhibition by TGF-ß in human hepatocellular carcinomas (38, 39). Interestingly, IRF and S100C are up-regulated or down-regulated after RUNX3 transfection, which is an important mediator of TGF-ß signaling. This coincides with restoration of responsiveness to TGF-ß in RUNX3-stable transfectants.

Among genes related to immunity, CD26/dipeptidyl peptidase IV (DPPIV) is a 1 10-kDa glycoprotein that is expressed on surfaces of numerous cell types and having multiple biological functions. A key factor of CD26/DPPIV biology is its enzymatic activity and its physical and functional interaction with other molecules. DPPIV takes part in immunoregulation, signal transduction, and apoptosis (40). Furthermore, CD26 seems involved in tumor progression. TOLL-like receptor 10 (TLR10) is the most recently identified human homologue of the Drosophila TOLL protein. In humans, TOLL-like receptors recognize pathogen-associated molecular patterns as part of innate immune host defenses (41). The complement regulatory protein decay–accelerating factor (CD55) inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B (42).

Several genes related to apoptotic process were down-regulated or up-regulated in our cell lines derived from stable transfectants of RUNX3. Caspase 9 is essential for the process of the apoptosis, whereas activation of mitomycin C–induced apoptosis process (43) seems related to the potency to develop metastases. Several genes involved in TGF-ß signaling are involved in TGF-ß-dependent apoptosis. IRFs and S100A11 are well known in the apoptotic signaling (38). Although the function of N-methyltransferase is still unclear, some methyltransferases are known to be involved in cancer metastases (44).

In conclusion, our results indicated that silencing of RUNX3 in gastric cancer affects the expression of important genes involved in metastases including cell adhesion, proliferation, and apoptosis; such silencing would promote peritoneal metastasis. Assessing RUNX3 methylation may be well suited to diagnosis of peritoneal metastases of gastric cancers. Furthermore, induction of RUNX3 may have value in treating such dissemination. Whether the identified genes are actually regulated by RUNX3 and are involved in peritoneal dissemination is a matter to be further investigated. Because RUNX3 regulates different target genes in each type of cells, identification of these genes could suggest a wide range of therapeutic modalities and targets.


   Footnotes
 
Grant support: Ministry of Health and Welfare and Ministry of Education, Science, and Culture grant-in-aid for Cancer Research and grants from the Uehara Memorial Foundation and Inamori Foundation, Japan.

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 4/ 8/05; revised 5/26/05; accepted 6/16/05.


   References
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 Abstract
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 References
 

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