Abstract
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. 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. 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. 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.
Highlights
Mothers against decapentaplegic homologue 4 (SMAD4) is a tumor suppressor gene associated with gastrointestinal carcinogenesis
The level of SMAD4 was lower in gastric adenocarcinoma (Fig. 1A)
SMAD4 functions as a tumor suppressor, in that clinicopathologically, inactivation of SMAD4 is associated with pathogenesis and a prognosis of gastric carcinoma [12, 27]
Summary
Mothers against decapentaplegic homologue 4 (SMAD4) is a tumor suppressor gene associated with gastrointestinal carcinogenesis. 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]; the role of hypermethylation as a mechanism for SMAD4 gene inactivation remains unknown They are rare in gastric cancer [7], SMAD4 mutations can result in changes in the tertiary structure and stability of the protein (20 – 22). 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
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