Extensive analysis of D7S486 in primary gastric cancer supports TESTIN as a candidate tumor suppressor gene

Abstract

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,TESandCAV1) whose frequencies of LOH were higher than 30%. However, onlyTESshowed the potential to be a TSG associated with GC. Among 140 pairs of GC samples, decreasedTESmRNA 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 ofTESpromoter was a frequent event in primary GC and GC cell lines. However, no specific gene mutation was observed in the coding region of theTESgene.Conclusions: Collectively, all results support the role ofTESas a TSG in gastric carcinogenesis and thatTESis inactivated primarily by LOH and CpG island methylation.