Inactivation of SMAD4 tumor suppressor gene during gastric carcinoma progression.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Wang
Li-Hui
LH
Research Institute of Pharmaceutical Science, Department of Pharmacy, Seoul National University College of Pharmacy, Seoul, Korea.
Kim
Seok-Hyung
SH
Lee
Jung Hyun
JH
Choi
Yoon-La
YL
Kim
Young Chul
YC
Park
Tae Sung
TS
Hong
Yun-Chul
YC
Wu
Chun-Fu
CF
Shin
Young Kee
YK
D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019175:DNA Methylation; D018450:Disease Progression; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051901:Smad4 Protein / Q000096:biosynthesis* / Q000235:genetics*; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*
C497796:SMAD4 protein, human; D051901:Smad4 Protein; D046988:Proteasome Endopeptidase Complex