Extensive analysis of D7S486 in primary gastric cancer supports TESTIN as a candidate tumor suppressor gene.
BACKGROUND
High frequency of loss of heterozygosity (LOH) was found at D7S486 in primary gastric cancer (GC). And we found a high frequency of LOH region on 7q31 in primary GC from China, and identified D7S486 to be the most frequent LOH locus. This study was aimed to determine what genes were affected by the LOH and served as tumor suppressor genes (TSGs) in this region. Here, a high-throughput single nucleotide polymorphisms (SNPs) microarray fabricated in-house was used to analyze the LOH status around D7S486 on 7q31 in 75 patients with primary GC. Western blot, immunohistochemistry, and RT-PCR were used to assess the protein and mRNA expression of TESTIN (TES) in 50 and 140 primary GC samples, respectively. MTS assay was used to investigate the effect of TES overexpression on the proliferation of GC cell lines. Mutation and methylation analysis were performed to explore possible mechanisms of TES inactivation in GC.


RESULTS
LOH analysis discovered five candidate genes (ST7, FOXP2, MDFIC, TES and CAV1) whose frequencies of LOH were higher than 30%. However, only TES showed the potential to be a TSG associated with GC. Among 140 pairs of GC samples, decreased TES mRNA level was found in 96 (68.6%) tumor tissues when compared with matched non-tumor tissues (p < 0.001). Also, reduced TES protein level was detected in 36 (72.0%) of all 50 tumor tissues by Western blot (p = 0.001). In addition, immunohistochemical staining result was in agreement with that of RT-PCR and Western blot. Down regulation of TES was shown to be correlated with tumor differentiation (p = 0.035) and prognosis (p = 0.035, log-rank test). Its overexpression inhibited the growth of three GC cell lines. Hypermethylation of TES promoter was a frequent event in primary GC and GC cell lines. However, no specific gene mutation was observed in the coding region of the TES gene.


CONCLUSIONS
Collectively, all results support the role of TES as a TSG in gastric carcinogenesis and that TES is inactivated primarily by LOH and CpG island methylation.
Ma
Haiqing
H
State Key Laboratory of Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou 510060, PR China.
Weng
Desheng
D
Chen
Yibing
Y
Huang
Wei
W
Pan
Ke
K
Wang
Hui
H
Sun
Jiancong
J
Wang
Qijing
Q
Zhou
Zhiwei
Z
Wang
Huiyun
H
Xia
Jianchuan
J
D015153:Blotting, Western; D002897:Chromosomes, Human, Pair 7; D003598:Cytoskeletal Proteins; D005260:Female; D016147:Genes, Tumor Suppressor; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D060588:LIM Domain Proteins; D019656:Loss of Heterozygosity; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D016601:RNA-Binding Proteins; C429688:TES protein, human; D025521:Tumor Suppressor Proteins