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Loss of Beta-Catenin Expression in Metastatic Gastric Cancer

Matthias P.A. Ebert, Jun Yu, Juliane Hoffmann, Alba Rocco, Christoph Röcken, Sabine Kahmann, Oliver Müller, Murray Korc, Joseph J. Sung, Peter Malfertheiner

From the Department of Gastroenterology, Hepatology, and Infectious Diseases and Institute of Pathology, Otto-von-Guericke University, Magdeburg; Max-Planck-Institute for Molecular Physiology, Dortmund, Germany; Department of Medicine and Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong; and Division of Endocrinology, Diabetes and Metabolism, University of California, Irvine, CA.

Address reprint requests to Matthias Ebert, MD, Otto-von-Guericke University, Department of Gastroenterology, Hepatology, and Infectious Diseases, Leipzigerstr 44, D-39120 Magdeburg, Germany; email: Matthias.Ebert{at}medizin.uni-magdeburg.de.

	ABSTRACT

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Purpose: Beta-catenin (ß-catenin) participates in intercellular adhesion and is an integral part of the Wnt signaling pathway. The role of ß-catenin in the pathogenesis of gastric cancer and its metastasis is largely unknown.

Patients and Methods: Immunohistochemistry and Western blot analysis were used to analyze the expression of ß-catenin in 87 human gastric cancers, in metastasis and cancer cell lines. The ß-catenin and the adenomatous polyposis coli (APC) genes were analyzed for gene mutations. Furthermore, methylation of the ß-catenin promoter in cell lines was assessed by treatment with 5'-azadeoxycytidine and sodium bisulfite genomic sequencing.

Results: ß-Catenin expression was present at either the cell membrane or the cytoplasm in 34 of 75 primary gastric cancers. Expression of ß-catenin was significantly more frequent in intestinal-type (P = .0049) and well-differentiated gastric cancers (P < .001). There were no quantitative differences between gastric cancers and the nonmalignant gastric tissues, as determined by Western blot analysis. One of 18 metastatic cancer lesions and four of five gastric cancer cell lines expressed ß-catenin protein. N87 cells, derived from the liver metastasis of a gastric cancer, did not express ß-catenin. Treatment with 5'-azadeoxycytidine restored ß-catenin protein levels in this cell line, which exhibited significantly more 5-methylcytosines in the ß-catenin promoter compared with the other cell lines.

Conclusion: ß-Catenin expression is lost in a subgroup of primary gastric cancers, is frequently absent in metastases, and exhibits nuclear localization in cancers with either ß-catenin or APC gene mutations. Interestingly, the loss of ß-catenin expression in metastatic gastric cancers may result from hypermethylation of the ß-catenin promoter.

	INTRODUCTION

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DESPITE THE declining incidence of gastric cancer in some parts of the world, it is one of the leading causes of cancer-related deaths worldwide.^1,2 It is believed to develop in a multistep process that includes the activation and overexpression of oncogenes, such as K-sam and c-met,^3,4 as well as the inactivation of tumor suppressor genes such as adenomatous polyposis coli (APC) and TP53.⁵ In addition, microsatellite instability is present in approximately 30% of gastric cancers.⁶ Interestingly, the alteration of the expression of adhesion molecules is a frequent event in gastric cancer.^7,8 Thus diffuse-type gastric cancers frequently harbor E-cadherin gene mutations.⁹ In addition, we and others have reported the frequent downregulation of E-cadherin and alpha-catenin (-catenin) expression in gastric cancer.^10,11 In addition to -catenin, beta-catenin (ß-catenin) is also part of this adhesion complex; however, it also participates in the Wnt signaling pathway, involving APC, ß-catenin, and the Tcf-Lef transcription factor.^12,13 APC and glycogen synthetase kinase 3-beta regulate the levels of ß-catenin, leading to the phosphorylation of its serine and threonine residues at the amino-terminal region of the ß-catenin protein.¹⁴ ß-catenin expression is frequently altered in several malignancies, including gastric, colon, and hepatocellular cancer.^15–18 Recent reports indicate that ß-catenin expression may be increased in gastric cancers, and ß-catenin mutations have been identified primarily in gastric cancers of the intestinal type.^18–20 However, the expression and mutation of the ß-catenin and APC genes have not been studied in relation to the changes underlying the progression and formation of metastasis in gastric cancer. The purpose of this study was to assess the expression of ß-catenin in primary tumors and distant metastasis of patients with gastric cancer and to elucidate the molecular mechanisms that may underlie the accumulation or loss of ß-catenin expression in gastric cancer and its metastasis, respectively.

	PATIENTS AND METHODS

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Patients
Tissue specimens were obtained by upper gastrointestinal endoscopy from six patients without gastric disease and by surgical resection from 87 patients (63 male and 24 female) with gastric cancer, with a mean age of 60 years (range, 26 to 79 years). Tissues were taken from the tumor and a tumor-free location that was at least 6 cm from the tumor and that was confirmed to be without any tumor cell infiltration by histologic assessment. Immediately after removal, all tissues for molecular analysis were put in liquid nitrogen and stored at -80°C until use. This study was approved by the ethics committee of the University of Magdeburg (Magdeburg, Germany).

Histology
Formalin-fixed tissues were processed as previously described, and sections were stained with hematoxylin and eosin for histologic evaluation. Gastric cancer was classified as intestinal type (n = 63) or diffuse type (n = 24) according to the Lauren system, and tumor stages were assessed using the tumor-node-metastasis system.^21,22

Cell Lines
The human gastric cancer cell lines KATO III, MKN45, MKN28, AGS, and N87 were obtained from Riken Cell Bank (Tsukuba, Japan) and American Type Culture Collection (Rockville, MD). Various cell lines from both primary gastric cancer and metastasis of gastric cancers with different grades of differentiation, ß-catenin, and APC gene status were chosen for this study. All cell lines, except AGS, were maintained in RPMI medium with 10% fetal bovine serum. AGS cell line was kept in F-12K medium with 10% fetal bovine serum. For immunocytochemical analysis, the cells were immersed in agar, fixed in 10% buffered formalin, and embedded in paraffin.

Treatment of Cells With 5-Aza-2'-Deoxycytidine (5-aza-dC)
Cells were seeded at a density of 1 x 10⁶ cells/60-mm dish. Twenty-four hours later, cells were treated with 1, 5, and 10 µmol/L of 5-aza-dC (Sigma Chemical Co, Deisenhofen, Germany). The same concentration of dimethyl sulfoxide was also used as a control for nonspecific solvent effect on cells. Total cellular protein was isolated 3 and 5 days after addition of 5-aza-dC, as previously described.²³

Sodium Bisulfite Genomic Sequencing
Genomic DNA from the cell lines was extracted using the High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN). One microgram of genomic DNA was treated with sodium bisulfite using the CpGenome DNA Modification Kit (Intergen, Purchase, NY) according to the manufacturer’s instructions.²⁴ Polymerase chain reaction (PCR) primers were designed to amplify a CpG-rich region of the promoter spanning 38 CpG sites. Primer sequences were as follows: 5'-GGTTTGGGATAGGGGAGGA-3', 5'-CACAAAAAACTCTTATAAAT-3'.²⁵ The PCR product was cloned into pCR4-TOPO (Invitrogen, Leek, the Netherlands). Multiple clones from each PCR product were sequenced using an ABI Prism 310 DNA Sequencer (Perkin-Elmer, Wellesley, MA).²⁶

Preparation of Nuclear Proteins
Approximately 1 to 2 x 10⁷ cells were collected by low-speed centrifugation in a buffer containing 10 mmol/L of HEPES (pH 7.9), 10 mmol/L of KCl, 1.5 mmol/L of MgCl₂, 0.5 mmol/L of dithiothreitol (DTT), and 0.5 mmol/L of phenylmethyl sulfonyl fluoride with inhibitors (10 µg/mL of aprotinin, 10 µg/mL of leupeptin, 10 µg/mL of antipain, and 10 µg/mL of pepstatin a). The pellet was resuspended in 100 µL of nuclear extract buffer (20 mmol/L of HEPES [pH 7.9], 25% glycerol, 0.4 mol/L of NaCl, 1 mmol/L of EDTA, 0.5 mmol/L of DTT, and 0.5 mmol/L of phenylmethyl sulfonyl fluoride) with inhibitors. The cells were sonicated and collected by centrifugation. Protein concentration was measured using bichinchoninic acid protein assay reagent kit (Pierce, Rockford, IL), and the nuclear protein pellets were stored at -70°C until further use.

Western Blot Analysis
Gastric cancer cell lines and tissues were homogenized in lysis buffer-containing Tris-HCl (pH 7.4), 0.5% Triton X-100, and protease inhibitor cocktail (Roche, Indianapolis, IN). Protein concentrations were measured by the method of Bradford.²⁷ Before separation, samples containing 50 µg/mL of total protein were resuspended in loading buffer (0.1 mol/L of Tris-HCl [pH 6.8], 0.2 mol/L of DTT, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, and 20% glycerol), denatured by boiling for 8 minutes and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to Trans-Blot polyvinylidene fluoride membrane (Applied Biosystems, Foster City, CA), and membranes were probed with primary goat polyclonal antibody against human ß-catenin (SC-1496, Santa Cruz, CA) at a dilution of 1:1,000 for 3 hours. Blots were washed in TBST (150 mmol/L/L of NaCl, 50 mmol/L of Tris [pH 7.5], and 0.1% Tween-20) and incubated with secondary antigoat horseradish peroxidase–conjugated antibody. Enhanced chemiluminescence (Pierce) was determined by exposure to x-ray film.

Immunohistochemistry
Paraffin-embedded sections were deparaffinized and rehydrated by xylene and ethanol. Endogenous peroxidase activity was blocked by incubation for 30 minutes in 0.3% H₂O₂. After the sections were washed twice with phosphate-buffered saline, they were blocked with normal rabbit serum for 20 minutes and were incubated with the ß-catenin goat antihuman antibody (1:400) for 2 hours. The anti–ß-catenin antibody (c-18) is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of human ß-catenin. The anitbody reacts with ß-catenin of mouse and human origin. The specificity of the antibody was confirmed by Western blot analysis. Immunohistochemical staining was performed according to the manufacturer’s instructions using the Streptavidin-HRP Systems kit (KPL, Gaithersburg, MD), followed by counterstaining with hematoxylin.²⁸

Detection of ß-Catenin Gene Mutation
Detection of mutations in exon 3 of the ß-catenin gene in human gastric cancers and cancer cell lines was carried out by single-strand conformation polymorphism (SSCP) analysis as previously described.¹⁹ Briefly, PCR primers were designed to amplify a 298–base pair DNA fragment encompassing exon 3 of the ß-catenin gene (sense: 5'-ACAAACTGTTTTGAAAATCCA-3, antisense: 5'-CGAGTCATTGCATACTGTCC-3').²⁵ The PCR mixture contained 50 ng of DNA as template in 10 µL of reaction containing 4 mmol/L of MgCl₂, 0.2 mmol/L each deoxynucleotide triphosphate, 5 pmol of each primer, and 0.08 U of Taq polymerase (Eppendorf Netheler-Hinz GmbH, Hamburg, Germany). Thereafter, the PCR fragment encoding exon 3 of the ß-catenin gene was analyzed by SSCP. Briefly, 5 µL of the PCR product was diluted in 3 µL of denaturation buffer, and 4 µL of the mixture was loaded on a 0.5 x mutation detection enhancement gel (FMC Bioproducts Rockland, ME). The fragments were separated at 4°C by horizontal electrophoresis and the DNA was visualized using modified silver staining.^28,29

Detection of APC Gene Mutations
The mutation cluster region from nucleotides 3570 through 4800 of the APC gene was screened for sequence alterations by PCR of two overlapping fragments and direct sequencing. The first fragment was amplified using the primers 5'-TCCTTCATCACAGAAACAGT-3' and 5'-GCTGGATTTGGTTCTAGGG-3', and the second fragment was amplified using the primers 5'-GGTCAGCTGAAGATCCTGTG-3' and 5'-GATGACTTTGTTGGCATGGCA-3'. PCR was carried out for 30 cycles: strand separation for 60 seconds at 92°C, annealing for 60 seconds at 62°C, and elongation for 60 seconds at 72°C. PCR products were purified and reaction products were analyzed on an ABI Prism 310 DNA automated sequencer. PCR and sequence analysis of mutated samples were repeated twice to exclude PCR errors.²⁷

Statistical Analysis
The Fisher’s exact test and ² test were used to determine statistical difference. A P value of less than .05 was considered statistically significant.³⁰

	RESULTS

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ß-Catenin Expression in the Normal and Cancerous Stomach
The presence of ß-catenin protein in the normal gastric mucosa and gastric cancers was first assessed using Western blot analysis. In Western blot analysis, ß-catenin expression was observed in all biopsies taken from patients without clinically evident gastric disease (Fig 1A), which was located at the cell membrane of gastric epithelial cells (not shown). In patients with gastric cancer, ß-catenin protein levels were assessed in both the cancer and the matched nonmalignant gastric tissue. Although the 85-kd band corresponding to the ß-catenin protein was detected in all cases, no significant difference in the protein levels was found in cancerous and nonmalignant gastric tissue by Western blot analysis (Fig 1B).

View larger version (35K): [in this window] [in a new window]   Fig 1. Western blot analysis. (A) Normal gastric mucosa (N) expressing the beta-catenin protein (85 kd). (B) No apparent difference in the levels of beta-catenin protein was identified in the nonmalignant (N) and cancerous (T) gastric tissue.

View larger version (144K): [in this window] [in a new window]   Fig 2. Immunohistochemistry. Beta-catenin expression was observed at the cell membrane (A; arrowheads) or in the cytoplasm (B) of gastric cancer cells. Beta-catenin expression was detected only in one gastric cancer metastasis (C; arrowheads); other metastatic cancer cells did not exhibit beta-catenin immunoreactivity (D; arrowheads). Magnifications: A–C, x400; D, x120.

Expression of ß-Catenin in Human Gastric Cancer Cell Lines
The ß-catenin expression was also analyzed in gastric cancer cell lines using Western blot analysis and immunocytochemistry. Although KATOIII, AGS, MKN45, and MKN28 cells exhibited ß-catenin protein, the N87 cells did not express ß-catenin as determined by Western blot analysis (Fig 3). An additional incubation with an anti–beta-actin antibody was performed to demonstrate equal loading of lanes and to confirm the integrity of the protein homogenates used for Western blot analysis. Immunocytochemical analysis of AGS, N87, and MKN28 cells revealed cytoplasmic and nuclear staining in AGS and MKN28 cells, whereas in N87 cells, no specific immunoreactivity was observed (not shown). After preparation of nuclear proteins, an additional Western blot analysis was performed. Only AGS and MKN28 cells exhibited a weak band, representing nuclear expression of ß-catenin in these cells (Table 2).

View larger version (52K): [in this window] [in a new window]   Fig 3. Western blot analysis. Upper panel, four of the five cancer cell lines expressed beta-catenin. Lower panel, incubation with 5'-azadeoxycytidine led to the restoration of beta-catenin protein levels in N87 cells after 3 days of treatment; after 5 days the protein levels were increased in all three cell lines.

Treatment of Cells With 5-Aza-dC Restores ß-Catenin Expression in Metastatic Gastric Cancer
In contrast to KATOIII, AGS, MKN45, and MKN28 cells, N87 cells did not express ß-catenin, as determined by Western blot analysis and immunocytochemistry. Three cell lines (KATOIII, AGS, and N87) were treated with 5-aza-dC. Expression of ß-catenin was restored in N87 cells after 3 days of treatment with this demethylating agent. After 5 days of treatment, ß-catenin protein levels were increased in all tested cell lines (Fig 3).

Analysis of the Promoter Region of the ß-Catenin Gene in Gastric Cancer Cell Lines
Because demethylation with 5-aza-dC leads to the restoration of ß-catenin expression in N87 cells, we next analyzed the 5-methylcytosine residues in the 5'-promoter region of the ß-catenin gene in N87 and the other cell lines. Using the sodium bisulfite genomic sequencing method, we analyzed 38 CpG islands in a 338–base pair region containing part of the promoter region of the ß-catenin gene (Fig 4A). Multiple methylation sites were identified in N87 cells (Fig 4B), whereas only one 5-methylcytosine was observed in several sequenced clones derived from KATO III and AGS cells.

View larger version (24K): [in this window] [in a new window]   Fig 4. (A) Promoter region. Primer sequences and TATA box are underlined. N1, nucleotides according to Genbank; N2, nucleotides relative to transcription start site (arrow). (B) Sequencing results from five clones for each cell line. () Unmethylated and (•) methylated CpG sites.

	DISCUSSION

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Several studies reported a high frequency of mutations of the ß-catenin gene in gastric cancers. More recently, however, other studies reported that there is a low frequency of ß-catenin gene mutations in gastric cancer.^18–20 Our own data from the present study and a previous study¹⁹ revealed only one mutation in the ß-catenin gene in 40 gastric cancer samples.¹⁹ These results are in agreement with reports by Candidus et al²⁰ and Tong et al,³² who also reported a very low frequency of ß-catenin gene mutations in their series. However, in the analysis of the expression of the ß-catenin protein, several other studies reported a high frequency of abnormal ß-catenin immunoreactivity in gastric cancer.^7,32 Thus loss of membranous ß-catenin expression was reported in the study by Jawhari et al⁷ in 14 of 24 diffuse-type gastric cancers and in 24 of 63 intestinal-type gastric cancers. Our analysis revealed a similar frequency of loss of ß-catenin expression in gastric cancers. However, in our study, the loss of ß-catenin immunoreactivity was also more frequent in diffuse-type gastric cancers. These findings are in line with a previous report by Woo et al,³³ who found abnormal ß-catenin expression more frequently in diffuse-type gastric cancers as well. In addition, we observed a strong and significant association of ß-catenin expression with the degree of differentiation, indicating that poor differentiation is associated with reduced ß-catenin expression. Although nuclear immunoreactivity was present in 16 of 139 cancers in the study by Miyazawa et al,³⁴ we could not confirm this observation in our analysis. However, in the cell lines that we analyzed, we found nuclear expression of ß-catenin in AGS and MKN28 cells by immunocytochemistry and Western blot analysis of nuclear proteins. Because nuclear localization of ß-catenin seems to be associated with mutations of the ß-catenin or APC gene, we also screened these cell lines for mutations of the ß-catenin and APC gene and found mutations in both cell lines. Thus, although in human gastric cancers ß-catenin gene mutations seem to be infrequent, the accumulation and nuclear localization of ß-catenin in gastric cancer cell lines is associated with mutations of either the ß-catenin or APC gene, confirming previous reports in other human cancers.^17,35

Interestingly, the quantitative analysis of ß-catenin expression in human gastric cancers and their matched normal gastric mucosa by Western blot analysis did not reveal any significant difference. Although ß-catenin accumulation is a frequent event in colon or hepatocellular cancers,^16,17 a direct comparison of ß-catenin protein levels in cancers versus normal tissues has not been previously reported. Our data did not reveal any quantitative difference in ß-catenin protein levels in gastric cancers versus normal mucosa. In agreement with a previous report by Jawhari et al,⁷ we also identified membranous expression of ß-catenin in gastric epithelial cells.¹⁹ Furthermore, ß-catenin expression has been identified in endothelial cells and neurons in the adjacent normal gastric mucosa by Jawhari et al.⁷ Although ß-catenin is expressed by other cells apart from the gastric cancer cells that all contribute to the overall ß-catenin expression detected by Western blot analysis in the cancerous and noncancerous stomach, immunohistochemical analysis identified abnormal immunoreactivity in the cancer cells in this and other studies, indicating a selective loss of ß-catenin expression in the cancer cells.^7,19

The role of catenins in the progression of gastric cancer and the formation of metastasis is not well understood. Reduced expression of -catenin was reported in gastric cancer and in cancers with infiltrative growth and lymph-node metastasis.⁸ However, to date, only few reports have analyzed the role of ß-catenin in this context. In a study by Miyazawa et al,³³ nuclear ß-catenin expression was found in infiltrating cancers, whereas Jawhari et al⁷ reported loss of membranous staining in 63% of lymph nodes analyzed for ß-catenin expression by immunohistochemistry. In our analysis, ß-catenin expression was only detected in one of the 18 lymph node and peritoneal metastases by immunohistochemistry. Moreover, the analysis of gastric cancer cell lines by Western blot analysis revealed ß-catenin expression in four of the five gastric cancer cell lines analyzed. Interestingly, only one cancer cell line (ie, N87 cells) did not exhibit ß-catenin protein expression. This cell line was derived from a liver metastasis of a gastric cancer patient, whereas all of the other cell lines were derived from primary gastric cancers.³⁶ Thus the loss of ß-catenin expression seems to be a uniform event in distant metastasis of gastric cancers. To elucidate the potential molecular mechanism underlying this phenomenon, we studied exon 3 of the ß-catenin gene and the APC gene for mutational inactivation. Although we found mutational changes in AGS and MNK28 cells, which were associated with nuclear ß-catenin accumulation, N87 cells harbored neither ß-catenin nor APC gene mutations.

Epigenetic changes of tumor-related genes through hypermethylation of CpG sites in the 5'-promoter regions of various genes has been reported in different cancers.³⁷ Thus hypermethylation within a promoter of a tumor-suppressing gene may lead to inhibition of gene transcription and loss of its function. Aberrant CpG-island methylation has been reported in various cancers, including gastric cancers.^38,39 Although the downregulation of E-cadherin expression through hypermethylation of CpG-islands in its promoter has been reported in several studies, the methylation status of the catenins has not been investigated to date. We found a loss of ß-catenin expression in metastasis of gastric cancers. Fresh human metastatic tissue was not available for treatment with 5'-azadeoxycytidine, a demethylating agent. Interestingly, the N87 cells, derived from a liver metastasis of a gastric cancer patient, also did not exhibit ß-catenin expression as determined by Western blot analysis and immunocytochemistry. Therefore, we treated these cells, along with AGS and KATOIII cells, with 5-aza-DC and observed the restoration of ß-catenin protein levels in N87 cells. The methylation in N87 cells was confirmed by bisulfite genomic sequencing of the promoter region of the ß-catenin gene.

In summary, our data indicate that ß-catenin expression is frequently lost in diffuse-type gastric cancers and that the loss of ß-catenin expression in distant metastasis may be due to hypermethylation of the ß-catenin promoter. Inasmuch as the formation of metastasis is dependent on the loss of the expression of adhesion molecules, the reduced expression of ß-catenin in metastasis of gastric cancer and its possible inactivation through hypermethylation of its promoter indicate a novel mechanism for epigenetic changes underlying the formation of metastasis and the progression of gastric cancer. Our findings therefore raise the possibility that therapeutic reversal of this epigenetic alteration may ultimately have a role in suppressing gastric cancer metastasis.

	NOTES

 
M.P.A.E. is supported by the Heisenberg-Programm and a grant from the Deutsche Forschungsgemeinschaft (Eb 187/4-1; Eb 187/5-1).

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Submitted October 2, 2002; accepted January 24, 2003.

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