Wang Li-Hui, Kim Seok-Hyung, Lee Jung Hyun, Choi Yoon-La, Kim Young Chul, Park Tae Sung, Hong Yun-Chul, Wu Chun-Fu, Shin Young Kee
Research Institute of Pharmaceutical Science, Department of Pharmacy, Seoul National University College of Pharmacy, Seoul, Korea.
Clin Cancer Res. 2007 Jan 1;13(1):102-10. doi: 10.1158/1078-0432.CCR-06-1467.
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.
母亲对脱尾蛋白同源物4（SMAD4）是一种与胃肠道癌变相关的肿瘤抑制基因。本研究的目的是更精确地描述其在人类胃癌发生发展中的作用。
在283例胃腺癌及相关病变以及9种胃癌细胞系中研究SMAD4的表达。我们还使用甲基化特异性PCR分析了SMAD4基因的甲基化状态，使用邻近标记检测该基因位点的杂合性缺失（LOH），并通过逐个外显子扩增检测SMAD4的外显子突变。此外，我们评估了蛋白酶体抑制剂MG132是否影响SMAD4蛋白水平。
我们发现胃癌细胞的细胞质中（114例中有36例，32%）和细胞核中（114例中有46例，40%）存在SMAD4蛋白表达缺失。原发性肿瘤中细胞核SMAD4表达缺失与生存率低显著相关，并且在多变量分析中是一个独立的预后标志物。我们还发现几种人类胃癌细胞系中SMAD4在RNA和蛋白水平均有显著下降。此外，我们发现LOH（70例中有20例，29%）和启动子高甲基化（73例中有4例，5%）与SMAD4表达缺失有关。用MG132处理某些胃癌细胞系后，SMAD4蛋白水平也受到影响。
综上所述，我们的结果表明SMAD4的缺失，尤其是细胞核SMAD4表达的缺失，与胃癌进展有关。胃癌中SMAD4的缺失是由多种机制导致的，包括LOH、高甲基化和蛋白酶体降解。
