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Clinical Cancer Research 13, 102, January 1, 2007. doi: 10.1158/1078-0432.CCR-06-1467
© 2007 American Association for Cancer Research
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Human Cancer Biology

Inactivation of SMAD4 Tumor Suppressor Gene During Gastric Carcinoma Progression

Li-Hui Wang1,2, Seok-Hyung Kim3, Jung Hyun Lee1, Yoon-La Choi4, Young Chul Kim5, Tae Sung Park3, Yun-Chul Hong6, Chun-Fu Wu2 and Young Kee Shin1

Authors' Affiliations: 1 Research Institute of Pharmaceutical Science, Department of Pharmacy, Seoul National University College of Pharmacy, Seoul, Korea; 2 Department of Pharmacology, Shenyang Pharmaceutical University College of Pharmacy, Shenyang, China; 3 Department of Pathology, Chungbuk National University, College of Medicine, Cheongju, Korea; 4 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine; 5 Department of Statistics, Seoul National University College of Nature Science; and 6 Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Korea

Requests for reprints: Young Kee Shin, Molecular Pathology Laboratory, College of Pharmacy, Department of Pharmacy, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea. Phone: 82-2880-9126; Fax: 82-2872-1795; E-mail: ykeeshin{at}snu.ac.kr.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Mothers against decapentaplegic homologue 4 (SMAD4) is a tumor suppressor gene associated with gastrointestinal carcinogenesis. The aim of the present study is to more precisely characterize its role in the development and progression of human gastric carcinoma.

Experimental Design: The expression of SMAD4 was investigated in 283 gastric adenocarcinomas and related lesions, as well as in 9 gastric carcinoma cell lines. We also analyzed the methylation status of SMAD4 gene by using methylation-specific PCR, examined loss of heterozygosity (LOH) of this gene locus by using a vicinal marker, and detected exon mutation of SMAD4 through exon-by-exon amplification. Moreover, we assessed whether MG132, a proteasome inhibitor, affected the SMAD4 protein level.

Results: We found loss of SMAD4 protein expression in the cytoplasm (36 of 114, 32%) and in the nucleus (46 of 114, 40%) of gastric cancer cells. The loss of nuclear SMAD4 expression in primary tumors correlated significantly with poor survival, and was an independent prognostic marker in multivariate analysis. We also found a substantial decrease in SMAD4 expression at both the RNA and protein level in several human gastric carcinoma cell lines. In addition, we found that LOH (20 of 70, 29%) and promoter hypermethylation (4 of 73, 5%) were associated with the loss of SMAD4 expression. SMAD4 protein levels were also affected in certain gastric carcinoma cell lines following incubation with MG132.

Conclusion: Taken together, our results indicate that the loss of SMAD4, especially loss of nuclear SMAD4 expression, is involved in gastric cancer progression. The loss of SMAD4 in gastric carcinomas was due to several mechanisms, including LOH, hypermethylation, and proteasome degradation.

Mothers against decapentaplegic homologue 4 (SMAD4) was initially isolated as a tumor suppressor gene on chromosome 18q21.1 in pancreatic ductal adenocarcinomas (1). In pancreatic and colorectal cancer, inactivation of the SMAD4 gene through mutation occurs frequently in association with malignant progression (2, 3). However, SMAD4 mutations are less frequently associated with breast (4), head and neck (5), prostatic (6), esophageal, and gastric cancers (7). Nevertheless, SMAD4 inactivation at the gene or protein level has been shown to be essential for the progression of several tumors (8–13).

The mechanisms of SMAD4 inactivation can be divided into at least four groups, depending on where the mutations are and how they disrupt SMAD4 function. First, homozygous deletion or loss of heterozygosity (LOH) are two of the major mechanisms for the inactivation of SMAD4 (2, 3, 14). Homozygous disruption of SMAD4 in mice caused early embryonic lethality, making it difficult to study the function of SMAD4 during later stages of development and tumorigenesis in Smad4-knockout mice (15). However, it has been shown that haploid loss of Smad4 initiated gastric carcinogenesis in mice (15, 16), indicating that LOH of the SMAD4 locus is important in the development of gastric cancer. Second, hypermethylation as a mechanism of inactivation of tumor suppressor genes is well characterized (17). However, in two previous studies regarding hypermethylation of SMAD4, either the first exon of SMAD4 was omitted from the hypermethylation assay, or an irrelevant promoter region of SMAD4 was used (18, 19); thus, the role of hypermethylation as a mechanism for SMAD4 gene inactivation remains unknown. Third, although they are rare in gastric cancer (7), SMAD4 mutations can result in changes in the tertiary structure and stability of the protein (20–22). Fourth, SMAD4 shuttles continuously between the cytoplasm and the nucleus (23). SMADs associate with transcription activators and repressors in the nucleus, and microtubules in the cytoplasm (24), which potentially act as nuclear or cytoplasmic retention factors. In addition, it was shown that embryonic liver fodrin, a ß-spectrin, is an important adaptor protein in the transforming growth factor ß signaling pathway, and is required for SMAD3 and SMAD4 localization and signaling (25, 26). Thus, several factors can potentially affect the localization and function of SMAD4 in gastric carcinoma. Recently, loss of SMAD4 expression was observed in gastric carcinomas, and was associated with poor prognosis in patients with advanced gastric cancer (12, 27). Based on these findings, we initiated a comprehensive study of the role of SMAD4 in gastric carcinoma.

In the present study, we examined the subcellular localization and expression of SMAD4 in gastric carcinomas and other gastric lesions using immunohistochemistry-based tissue microarray analysis, and analyzed the clinical significance of SMAD4 down-regulation in gastric adenocarcinoma. We investigated potential mechanisms of SMAD4 inactivation using allelic deletion marker analysis, hypermethylation analysis of specific regions of the SMAD4 promoter (dense CpG islands, TATAA and CCAAT boxes), mutation analysis in patients that displayed single allelic loss of SMAD4 and negative SMAD4 staining, and analysis of the proteasome inhibitor MG132 in gastric carcinoma cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tissue specimens. Tissues from patients with gastric carcinoma and other gastric lesions (114 carcinoma, 20 high-grade dysplasia, 17 low-grade dysplasia, 48 intestinal metaplasia, 30 chronic atrophic gastritis, and 54 normal gastric epithelium) were retrospectively identified from the surgical pathology files of Chungbuk National University Hospital (Cheongju, Korea). Pathology slides were analyzed according to tumor size, histologic grading, depth of invasion, and presence of nodal metastasis. Ethical oversight and approval was obtained from the Institutional Review Board of Chungbuk National University Hospital. The 114 gastric carcinomas (age, 49-85 years; average age, 66.1 years; 40 females and 74 males) comprised 32 early cases and 82 advanced cases. Of these, 26 (22.8%) were classified as well differentiated, 41 (36.0%) as moderately differentiated, and 31 cases (27.2%) as poorly differentiated adenocarcinomas. Tumor-node-metastasis staging according to the American Joint Committee on Cancer was used. All archival materials were routinely fixed in 10% neutral-buffered formalin, and embedded in paraffin. Then, these samples were used to generate tissue microarray slides (for details, see Supplementary Materials and Methods).

Immunohistochemistry and evaluation of results. Immunostaining of SMAD4 and SMAD2/3 was done on consecutive sections obtained from paraffin-embedded material, using mouse monoclonal anti-SMAD4 antibody (B-8; Santa Cruz Biotechnology, Santa Cruz, CA), and goat polyclonal anti-SMAD2/3 antibody (N-19; Santa Cruz Biotechnology). The experimental procedures were done as previously described (28).

The evaluation of both the intensity of immunohistochemical staining and the proportion of positively stained epithelial cells was previously described (29). Cytoplasmic and nuclear staining were independently analyzed. A detailed description of the analysis is provided in the Supplementary Materials and Methods.

Nucleic acid extraction. Cancer and normal tissues on slides were collected by scraping with a sterile razor using a microdissection technique (30). A G-spin Genomic DNA Extraction Kit (Intron, Sungnam, Korea) was used to extract DNA from tumor tissues, normal lymphocytes, and gastric cancer cells. Total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA was reverse-transcribed using Superscript II Transcriptase (Invitrogen) and oligo-dT and random primers. The experimental procedure was done as described in the manufacturer's instructions.

Bisulfite treatment and methylation-specific PCR. Bisulfite treatment of DNA and methylation-specific PCR (MSPCR) were done as previously described (31). DNA from normal lymphocytes was used as the negative control in the MSPCR assay; normal human lymphocyte DNA methylated in vitro with SssI methylase (New England Biolab, Beverly, MA) according to the manufacturer's instructions was used as the positive control. Water blanks and PCR mixtures without template were also used as negative controls in each assay. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. All methylated and representative unmethylated PCR fragments were sequenced as described in Supplementary Materials and Methods. The primers and PCR conditions used were also provided in Supplementary Table S1.

LOH assay. Matched normal and tumor genomic DNA samples were analyzed for LOH using a highly polymorphic CA repeat microsatellite marker, D18S1110, on the long arm of chromosome 18. The marker has been mapped to 4 kb downstream of SMAD4. Primer sequences for this marker (Supplementary Table S1) were obtained from the Sanger Institute (http://www.ensembl.org). PCR amplification and analysis were done as previously described (ref. 32; Supplementary Materials and Methods).

Mutation of SMAD4. Exon-by-exon amplification of the 11 exons of SMAD4 (encompassing the coding sequence and all intron/exon boundaries) was done on DNA from patients with a single allelic loss of SMAD4 and negative SMAD4 expression, as previously described (33). The untranslated regions of SMAD4 in nine gastric carcinoma cell lines were also amplified (see Supplementary Table S1 for primers and PCR conditions). All PCR products were purified and sequenced (Cosmogenetech, Seoul, Korea).

Cell lines, Western blotting, drug treatment, and MTT assay. Nine human stomach cancer cell lines, i.e., SNU5, SNU16, SNU216, SNU484, SNU638, MKN28, MKN74, KATOIII, and AGS, were maintained. Western blotting was done according to standard procedures, using 100 µg of whole-cell extracts. The antibodies used were as follows: anti-SMAD4 mouse monoclonal antibody (B-8; Santa Cruz Biotechnology), anti-p53 mouse monoclonal antibody (DO-7; Vector Laboratories, Burlingame, CA), and anti-ß-actin goat polyclonal antibody (Pierce, Rockford, IL). A ratio of protein band intensities of SMAD4 and ß-actin were calculated. SMAD4 protein was classed as "absent", "weak", "moderate", or "strong", compared with ß-actin.

MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; Sigma, St. Louis, MO) was freshly prepared in DMSO before use. A vehicle control consisting of DMSO alone (<0.1%) was included in the analysis. Five tumor cell lines were cultured for 24 h, then treated with 10 µmol/L of MG132 for 48 h. Cells were collected by centrifugation, then proteins were extracted and analyzed by Western blot. Cell growth was measured using the MTT assay, as previously described (34).

Reverse transcription PCR and real-time PCR analysis of SMAD4. Reverse transcription-PCR and nucleotide sequencing were carried out on SMAD4 cDNAs from gastric carcinoma cells lines. Real-time quantitative reverse transcription-PCR was also carried out using the Light Cycler Real-time PCR Detection system (Roche, Mannheim, Germany). Real-time PCR primers for amplifying SMAD4 and the control gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) are shown in Supplementary Table S1. Universal ProbeLibrary probe no. 78 was purchased from Roche and used as the TaqMan probe. Universal ProbeLibrary was detected at 530 nm using the FAM reporter dye. HPRT primers and the TaqMan probe were obtained from TIP Molbiol (Berlin, Germany). The HPRT TaqMan probe was labeled with DYXL and BHQ2 to create DYXL-TTCGTTTCCTTGGTCAGGCAGTATAATC-BHQ2. SMAD4 and HPRT were detected in the same tube at 530 and 705 nm, respectively. The conditions for PCR were as follows: 95°C for 10 min (1 cycle), 95°C for 7 s, 55°C for 40 s (single; 45-cycle quantification), 40°C for 30 s (1 cycle). To confirm the fidelity of the reported SMAD4 cDNA, SMAD4 cDNAs encompassing the 5'-flanking non–coding exons of SMAD4 were amplified and sequenced. The primers and PCR conditions are provided in Supplementary Table S1.

Data analysis. Statistical analysis of group differences were done by Pearson's {chi}2 test and ANOVA. With regard to survival analysis, we analyzed 114 patients with gastric carcinoma using Kaplan-Meier analyses. We used log-rank tests in order to compare the survival curves between groups. Univariate and multivariate survival analyses were then conducted using the Cox regression model. P < 0.05 was regarded as statistically significant. All statistical analyses were done using SPSS software (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression profile of SMAD4 in gastric lesions and positive correlation with prognostic factors. Cytoplasmic and nuclear SMAD4 expression in normal gastric epithelium and various gastric lesions, including chronic atrophic gastritis, intestinal metaplasia, low-grade dysplasias, high-grade dysplasias, and carcinomas were semiquantitated. SMAD4 was markedly enhanced in both low-grade and high-grade dysplasias. However, the level of SMAD4 was lower in gastric adenocarcinoma (Fig. 1A ). Overall, cytoplasmic SMAD4 was absent in 36 of 114 carcinomas (32%), and SMAD4 nuclear staining was absent in 46 carcinomas (40%). In 21 carcinomas (18%), both cytoplasmic and nuclear SMAD4 were completely absent. The intensity of staining and representative tissues from different tumor-node-metastasis stages are presented in Fig. 1B and C. The loss of SMAD4 expression was more prevalent in advanced stage cancers compared with early stage cancers. Specifically, the rate of loss of cytoplasmic SMAD4 expression increased from 23% in early stage (stage I + II) to 39% in advanced stage (stage III + IV) carcinomas. The loss of nuclear staining was also more frequent in advanced stage (55%) compared with early stage carcinomas (23%). Interestingly, 21 of 46 nuclear SMAD4-negative tumors (45.7%) had moderate to high levels of cytoplasmic SMAD4 (Supplementary Materials and Methods). This abnormal distribution of SMAD4 correlated with abnormal SMAD2/3 staining (negative nuclear staining with positive cytoplasmic staining) (P < 0.001, Supplementary Table S2).


Figure 1
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  		Fig. 1. Immunohistochemical analysis of SMAD4 in gastric carcinomas and related lesions. A, gastric carcinoma (CA, n = 114), high-grade dysplasia (HD, n = 20), low-grade dysplasia (LD, n = 17), intestinal metaplasia (IM, n = 48), chronic atrophic gastritis (CAG, n = 30), and normal gastric epithelium (NL, n = 54) samples were subjected to immunohistochemistry for SMAD4 expression. Left, average immunoreactive score (IS) of cytoplasmic staining for each type of lesion. Right, average immunoreactive score (IS) of nuclear staining for type of lesion. B, relationship between the immunoreactive scores of individual gastric carcinoma samples and the gastric cancer stage. Patients were divided according to their gastric cancer stage (I-IV) and the immunoreactive scores of their samples are shown in color. C, SMAD4 expression in representative gastric carcinoma tissue according to tumor-node-metastasis staging as follows: stage I, strong cytoplasmic and nuclear staining; stage II, strong nuclear staining with weak cytoplasmic staining; stage III, negative nuclear staining with weak cytoplasmic staining; stage IV, negative nuclear and cytoplasmic staining (original magnification, x400).

 
There was a significant correlation between reduced nuclear expression of SMAD4 and unfavorable variables, including large tumor size, nodal metastasis, advanced gastric cancer, poor differentiation (high grade), deep tumor invasion, and advanced clinical staging (P < 0.05). There was also a significant correlation between down-regulation of cytoplasmic SMAD4 and large tumor size, nodal metastasis, poor differentiation, and deep tumor invasion (P < 0.05). These findings were observed in terms of both expression rate (number of positive cells) and average staining intensity (data not shown).

Correlation of SMAD4 expression with patient survival. The relationship between SMAD4 expression and prognosis in patients with gastric carcinoma was analyzed using survival curves calculated according to the Kaplan-Meier method (Fig. 2A and B ). This revealed that the absence of nuclear SMAD4 expression correlated strongly with decreased 1-year and 3-year survival levels (P = 0.015), and there was a marginal but significant correlation between overall survival and SMAD4 cytoplasmic expression (P = 0.047; Table 1 ). Univariate and multivariate analyses using the Cox proportional hazards model were carried out to assess the independent predictive value of SMAD4 nuclear expression. As classic prognostic variables, patient age at diagnosis, sex, and chemotherapy were included in the model. We found that SMAD4 nuclear expression served as an independent prognostic variable after both univariate and multivariate analysis (univariate P = 0.008; multivariate P = 0.014; Table 2 ).


Figure 2
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  		Fig. 2. Relationship between SMAD4 expression by gastric carcinoma samples and the overall survival of the patients. A, Kaplan-Meier survival curve of patients with the indicated level of SMAD4 nuclear staining. B, Kaplan-Meier survival curve according to the indicated levels of SMAD4 cytoplasmic staining.

 

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  		Table 1. Univariate analysis of the overall survival of gastric cancer patients using Kaplan-Meier survival analysis

 

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  		Table 2. Results of univariate and multivariate analyses of the overall survival of patients with gastric carcinoma using Cox-proportional hazards regression

 
Promoter methylation status in gastric carcinomas. To elucidate the mechanism of SMAD4 down-regulation in gastric carcinoma, the methylation state of the SMAD4 promoter was examined. Using gastric carcinoma cell lines, we first confirmed the location of the transcription start site through reverse transcription-PCR and nucleotide sequencing. Our data on the identity of the transcription start site was consistent with the European Molecular Biology Laboratory database (http://www.ensembl.org), despite the presence of several alternative transcripts encoding exon 1 and exon 2 of SMAD4 (Supplementary Fig. S1). We then designed primers to amplify sequences up to 200 bp upstream of the SMAD4 transcription start site, which included the dense CpG islands, and examined the methylation status of the promoter region using MSPCR (see Fig. 3A ). We found that in 4 of the 73 (5%) gastric carcinomas analyzed, the SMAD4 promoter was hypermethylated (Fig. 3B). Of the 16 samples that had a complete loss of SMAD4 expression in both the nucleus and cytoplasm, 3 also displayed promoter hypermethylation (Supplementary Table S3). Although hypermethylation was rather rare, there was a significant correlation between promoter methylation and loss of SMAD4 expression (P < 0.05; Table 3 . Bisulfite DNA sequencing of the gastric carcinoma samples confirmed the promoter methylation status (Fig. 3C).


Figure 3
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  		Fig. 3. SMAD4 promoter methylation and LOH in gastric carcinomas. A, schematic representation of the promoter region of SMAD4. Red boxes, nonprotein coding exons; blue box, coding exons; black circles, CpG islands; solid black arrows, MSP primers. B, representative results of MSPCR analysis of SMAD4 promoter methylation in gastric carcinomas. U, unmethylated; M, methylated; NC, negative control; PC, positive control; DW, distilled water. C, methylation status of the SMAD4 promoter analyzed by bisulfite sequencing in gastric carcinomas. Case 1, sites –111, –118, –123, and –130 are unmethylated; case 2, the CpG sites at –111, –118, –123, and –130 were completely methylated. CpG sites are underlined. The nucleotide number for the location of the CpG sites is based on a transcription start site at +1. D, schematic representation of the SMAD4 locus showing the location of the microsatellite marker D18S1110, located at only 4 kb from SMAD4. E, representative results of LOH analysis from normal lymphocytes (N) and tumor specimens (T) from three patients with gastric cancer using the marker D18S1110. RE, retained heterozygosity; MSI, microsatellite instability.

 

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  		Table 3. Correlation between LOH and hypermethylation and SMAD4C, SMAD4N, and cellular SMAD4 expression using {chi}2 test

 
LOH in gastric carcinomas. The 76 DNA samples isolated from matched normal and tumor tissues were analyzed for possible LOH at the SMAD4 gene locus. Typically, three pairs of primers specific for the polymorphic markers D18S474, D18S363, and D18S46 had been used to analyze the SMAD4 locus (Fig. 3D). We chose to analyze marker D18S1110 because it was much closer to the SMAD4 locus, located only 4 kb from SMAD4 (Fig. 3D; Supplementary Table S5). Normal and tumor samples were analyzed using the dinucleotide repeat assay, and we found that 70 of the 76 samples (92%) were informative for this marker. The tumor samples corresponding to the normal samples that were informative for this marker were similarly analyzed, and the relative intensities of the alleles were compared. The comparative ratios for most of the informative cases centered near 1.0, indicating no significant allelic imbalance (Fig. 3E, left). In 20 of 70 informative cases (29%), significant allelic imbalance was observed in the tumor sample, with comparative ratios >1.35 (Table 1). These cases were scored as positive for allelic loss (Fig. 3E, middle). Of these 20 LOH-positive cases, 10 exhibited neither nuclear nor cytoplasmic SMAD4 expression, indicating an association between LOH at the SMAD4 locus and loss of SMAD4 expression in gastric carcinomas (P < 0.05; Table 3). LOH was observed in the 53% cases of complete loss of both cytoplasmic and nuclear SMAD4 expression (Supplementary Table S4). Interestingly, microsatellite instability was noted in one case that presented abnormal-sized alleles in the tumor DNA compared with the normal counterpart (see Fig. 3E, right). However, there was no significant correlation between LOH and either clinicopathologic variables, or the patients' survival (Table 1).

SMAD4 mutation in gastric carcinomas. To investigate whether additional genetic events contributed to the loss of SMAD4 expression, we directly sequenced the coding exons of SMAD4 from nine cases having both LOH and loss of SMAD4. We found no mutations in any of these cases (data not shown).

SMAD4 expression pattern in gastric carcinoma cell lines. Based on the frequency of LOH on chromosome 18q in gastric carcinomas, we analyzed SMAD4 expression in a panel of nine individual human gastric carcinoma cell lines. SMAD4 was analyzed by quantitative reverse transcription-PCR and Western blot (Fig. 4A and B ). A substantial decrease in SMAD4 expression at the RNA and/or protein level was found in SNU216, MKN28, MKN74, and AGS cells, whereas SNU484 and SNU638 had relatively high levels of SMAD4 protein expression. Interestingly, LOH analysis revealed that only in SNU484 and SNU638 cell lines, was the heterozygosity of the SMAD4 locus preserved (Fig. 4C). This result strongly suggested that allelic loss is an important mechanism of down-regulation SMAD4 in gastric cancer. We also examined SMAD4 promoter methylation status in nine gastric cancer cell lines using MSPCR. As seen in Fig. 4D, we found no evidence for hypermethylation of the SMAD4 promoter region. We did mutation analysis in nine gastric cancer cell lines, and found that in one, SNU216, there was an insertion mutation of 37 nucleotides in exon 8 (Supplementary Fig. S2), which is consistent with previous results (35). No mutations were found in the coding or noncoding regions of SMAD4 in the eight cell lines. A summary of the data is shown in Table 4 .


Figure 4
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  		Fig. 4. Genetic alterations in SMAD4 in gastric cancer cell lines. A, SMAD4 mRNA was measured by real-time PCR. The quantity of mRNA was normalized to that of HPRT. B, SMAD4 protein expression profiles in nine gastric cancer cell lines. Protein bands were analyzed by scanning densitometry. Actin was used to normalize for protein levels. C, LOH status of the SMAD4 locus. Except for SNU484 and SNU638, all cell lines showed LOH. D, methylation status of the SMAD4 promoter. MSPCR-U bands were observed in all cell lines, whereas none of the cell lines displayed MSPCR-M bands. U, unmethylated; M, methylated.

 

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  		Table 4. Summary of SMAD4 expression and genetic alterations in gastric cancer cell lines

 
Posttranslational regulation of sMAD4 in gastric cancer cell lines. To examine whether posttranslational modification of SMAD4 affected its expression in gastric cancer cell lines, we cultured SNU16, SNU484, MKN28, MKN74, and AGS cells in the presence of MG132, a proteasome inhibitor. The extent and rate of cell death varied with each cell line after MG132 treatment (Fig. 5A ). Of the four adherent cell lines (SNU484, MKN28, MKN74, and AGS), AGS cells exhibited the sharpest decline in viability in the presence of MG132 treatment. This result is consistent with the previous data showing that MG132 induced apoptosis in AGS cells (34). The level of SMAD4 increased in SNU16, SNU484, and AGS cells, but not MKN28 and MKN74 cells, following treatment with MG132. As a control, the expression level of p53 after treatment with MG132 was also examined. We found enhanced expression of p53 in AGS cells, and reduced expression was detected in SNU484, MKN28, and MKN74 cells, in the presence of MG132 (Fig. 5B).


Figure 5
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  		Fig. 5. Cell viability and protein expression in gastric cancer cells treated with MG132. A, effect of MG132 on cell viability. Open columns, 1 µmol/L of MG132 treatment; closed columns, 10 µmol/L of MG132 treatment. B, protein levels of SMAD4 and p53 were determined following 10 µmol/L of MG132 treatment.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
SMAD4 functions as a tumor suppressor, in that clinicopathologically, inactivation of SMAD4 is associated with pathogenesis and a prognosis of gastric carcinoma (12, 27). However, the mechanism of SMAD4 inactivation in carcinogenesis remains largely unknown. Promoter CpG methylation is a frequent event in gastric carcinoma (36, 37), suggesting that promoter methylation is an important pathway leading to the inactivation of tumor suppressor genes. In this study, we examined the SMAD4 sequences between nucleotides –4 and –278 (transcriptional start site being +1), as the candidate promoter region (see also Fig. 3A, GenBank accession no. DQ660374). MSPCR analysis showed that hypermethylation in gastric carcinomas was relatively infrequent (5%), but hypermethylation correlated significantly with loss of nuclear and cytoplasmic SMAD4, suggesting that DNA hypermethylation is involved in the suppression of SMAD4 in gastric cancer.

The SMAD4 gene is located on chromosome region 18q21, and LOH in this region has been reported in 33% to 42% of gastric carcinomas (27, 38). A summary of SMAD4 LOH and a comparison of chromosome 18q21 microsatellite markers are presented in Supplementary Table S5. Compared with previous reports, our result of 29% LOH is relatively low. One reason for this may be that in previous studies, markers that were more distal to the SMAD4 locus were used. As a result, LOH in these markers could reflect the presence of other tumor suppressor genes, such as SMAD2 and DCC. In the present study, LOH of the SMAD4 locus was correlated significantly with loss of nuclear and cytoplasmic expression of SMAD4. Consistent with patient data, in several gastric cancer cell lines, there was obvious correlation between LOH and expression of SMAD4, both at the RNA and protein levels. These results suggested that LOH plays an important role in the inactivation of SMAD4.

It is generally believed that LOH alone does not result in the suppression of SMAD4 expression because many genes are expressed monoallelically (39). Furthermore, hypermethylation of SMAD4 is relatively rare in gastric cancers. In the current model of cancer progression, two "hits" are required to inactivate a tumor suppressor, such as LOH and a mutational hit in the remaining allele. We directly sequenced the open reading frame of SMAD4 in samples from patients that carried a single allelic loss of SMAD4, and were negative for protein expression. However, we found no mutations in SMAD4 in any of the tissue samples of gastric carcinoma. This is consistent with previous results (7), which showed that mutations were infrequent in gastric cancers. Furthermore, a mutation assay of the SMAD4 coding and uncoding regions in nine gastric carcinoma cell lines revealed the presence of only one mutation, an insertion mutation, in SNU216. These results suggested that mutation of SMAD4 plays a minor, if any, role in the inactivation of this gene in gastric carcinoma.

To explore the mechanisms for the reduced SMAD4 expression in gastric carcinoma in detail, we investigated the effect of posttranslational modification of SMAD4 using the proteasome inhibitor, MG132. The protein levels of SMAD4 increased significantly in SNU484 and AGS cells after treatment with MG132, indicating that the proteasome degradation pathway plays a crucial role in the down-regulation SMAD4 protein in gastric carcinomas. We also observed that the level of SMAD4 protein decreased in MKN28 and MKN74 cells after MG132 treatment. However, the level of p53, a well known proteosome degradation pathway–dependent protein, also decreased in these cells after MG132 treatment, indicating that these cells were insensitive to MG132. Our results support the concept that the mechanism of SMAD4 down-regulation in gastric carcinoma is different from other cancers, such as pancreatic and colorectal cancers, which are caused by homozygous deletions or intragenic mutations (2).

The finding that loss of nuclear expression of SMAD4, and not cytoplasmic expression, strongly correlated with prognosis in gastric cancer is subject to several interpretations. First, the function of SMAD4 is not the simple carriage of R-SMADs into the nucleus, but rather must be at the level of cooperating with R-SMADs in the transcription factor complex within the nucleus (40). This implies that nuclear SMAD4 plays a more critical role in cellular homeostasis compared with cytoplasmic SMAD4. Second, SMAD4 plays a crucial role as a tumor suppressor protein in the nucleus, through its involvement in the regulation of p15 and p21, which are important modulators of cell proliferation and apoptosis, and CDH1 (E-cadherin), which is involved in tumor-infiltrative growth and lymph node metastasis (41, 42).

Nuclear SMAD4–negative tumors (45.7%) had moderate to high levels of cytoplasmic SMAD4. This pattern of SMAD4 accumulation correlated with cytoplasmic accumulation of SMAD2/3. One reason for this could be that dysregulation of microtubules, a cytoplasmic retention factor for SMADs (24), and embryonic liver fodrin, a mediator of SMADs nuclear import (25, 26), are involved not only in cytoplasmic accumulation of SMAD4, but also of SMAD2/3 (see Supplementary Materials and Methods for details). Therefore, dysregulation of nucleocytoplasmic shuttling may be another mechanism leading to functional loss of nuclear SMAD4.

Based on our data, the "two-hit" model of tumor suppressor, involving both a genetic and epigenetic event does not fully explain the loss of SMAD4 expression in gastric carcinoma because mutation, LOH, and hypermethylation were insufficient to explain the loss of SMAD4. Therefore, we propose a modified two-hit model for gastric cancer, which includes a non–genetic/epigenetic event, such as transcriptional regulation, proteasome degradation, or abnormal nucleocytoplasmic shuttling. This modified two-hit model could also incorporate downstream components of other major signaling pathways, when genetic and epigenetic events are not dominant.

In conclusion, we investigated the significance of loss of SMAD4 in gastric cancer, and explored the possible underlying mechanisms. Our findings suggest that SMAD4 is a critical prognostic factor, and that various mechanisms, including dysregulation of nucleocytoplasmic shuttling, and down-regulation by LOH, hypermethylation, or proteasome degradation, are involved in the loss of SMAD4 in gastric carcinoma.


   Acknowledgments
 
We are very grateful to Jung Sun Lee and Mee Young Sim for their technical assistance including slide cutting and immunohistochemistry.


   Footnotes
 
Grant support: In part by a Seoul R&BD Program and a grant from the Korea Health 21 R&D Project of the Ministry of Health and Welfare (A050742), Korea. National Research Laboratory Program of Korea Science and Engineering Foundation (M10500000126, to T.S. Park).

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.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

The sequence data described in this paper have been submitted to the NCBI GenBank under accession no. DQ660374.

Received 6/16/06; revised 9/ 9/06; accepted 10/ 6/06.


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