TES functions as a Mena‐dependent tumor suppressor in gastric cancer carcinogenesis and metastasis

Figures

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  Fig. 1. Overexpression of testin LIM domain protein (TES) suppresses gastric cancer (GC) cell viability in vitro. a Proliferation rate of Ad-TES transfected SGC7901 and MKN45 cells were compared with Ad-Control cells by proliferation assay. The results are expressed as mean ± standard deviation (SD) of at least three independent experiments. Student’s t-test, *P < 0.05. b Representative pictures of colony formation assay in Ad-TES transfected SGC7901 and MKN45 cells. The cells transfected with Ad-Control were used as control. c Summary of cell cycle distributions detected by flow cytometry shows that the proportion of cells at S phase was much lower in Ad-TES-transfected SGC7901 and MKN45 cells than that in control cells. Values are expressed as mean ± SD of at least three independent experiments. Student’s t test, *P < 0.05

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  Fig. 2. Overexpression of TES suppresses the tumorigenicity of SGC7901 and MKN45 cells in vivo. a Tumor growth curves of Ad-TES-transfected SGC7901 and MKN45 cells in nude mice were compared with Ad-Control-transfected cells in tumorigenicity assay. The data were analyzed by ANOVA. The average tumor volume is expressed as mean ± SD in seven inoculated nude mice for each group. b Representative pictures of dissected tumors from nude mice 6 weeks following injection of Ad-TES or Ad-Control-transfected SGC7901 and MKN45 cells and control cells (n = 7 mice per group), respectively. The tumor volumes are smaller in the Ad-TES group than in the Ad-Control group. c Tumor weights were compared between the Ad-TES group and the Ad-Control group by ANOVA. The results are expressed as mean ± SD

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  Fig. 3. Overexpression of TES suppresses migration and invasion of GC cells both in vitro and in vivo. a Representative pictures show that the Ad-TES- and Ad-Control-transfected SGC7901 and MKN45 cells invaded through the matrigel. Magnification: ×100. b Invaded, Ad-TES-transfected SGC7901 and MKN45 cells were quantified. Ad-Control-transfected SGC7901 and MKN45 cells were used as controls. The results are expressed as mean ± SD of at least three independent experiments. Student’s t-test, *P < 0.05. c Representative pictures show that the Ad-TES- and Ad-Control-transfected SGC7901 and MKN45 cells migrated through the uncoated transwell membrane. Magnification: ×100. d Migrated Ad-TES transfected SGC7901 and MKN45 cells were quantified. Ad-Control-transfected SGC7901 and MKN45 cells were used as controls. The results are expressed as mean ± SD of at least three independent experiments. Student’s t-test, *P < 0.05. e Representative lungs derived from nude mice inoculated with Ad-TES- or Ad-Control-transfected MKN45 cells are shown. The formation of metastatic nodules at the surface of lungs could be significantly suppressed by TES overexpression. f Metastatic nodules in the lung were…

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  Fig. 4. TES inhibits the interaction between Mena and Lpd. a Western blotting of immunoprecipitates prepared using antibodies against TES revealed that TES interacted with Mena. b Western blotting of immunoprecipitates prepared using antibodies against Mena revealed that Mena interacted with TES. c Mena protein expression in GC cell lines detected by Western blotting. d The association of Lpd and TES expression in Ad-TES- or Ad-Control-transfected MKN45 cells detected by Western blotting. Overexpression of TES had no effect on the Lpd expression in MKN45 cells. e Western blotting of immunoprecipitates from Ad-TES-transfected MKN45 cells show that overexpression of TES reduced the level of Lpd in the immunoprecipitates prepared using antibodies against Mena. The immunoprecipitates from Ad-Control-transfected MKN45 cells were served as control. The loading volume of immunoprecipitates was 10 μL for each sample. Three independent experiments were performed. f The level of Lpd (band intensity) binding to Mena in Ad-TES-transfected cells and Ad-Control-transfected cells were compared by Student’s t-test. The results are expressed as mean ± SD of at least three independent experiments

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  Fig. 5. TES inhibits migration and invasion of GC cells in a Mena-dependent fashion. a Expression of TES and Mena in siMena-transfected MKN45 cells was detected by Western blotting. Overexpression of TES significantly suppressed the migration (b) and invasion (c) of siNC-transfected MKN45 cells compared with those of siMena-transfected MKN45 cells. Control: MKN45 cells transfected with Ad-Control and siNC. siMena: MKN45 cells transfected with Ad-Control and siMena. Ad-TES: MKN45 cells transfected with Ad-TES and siNC. Ad-TES + siMena: MKN45 cells transfected with Ad-TES and siMena. The results are expressed as the mean ± SD of three independent experiments. Magnification: ×200. *P < 0.05

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  Fig. 6. Mena protein expression in GC specimens was detected by immunohistochemistry. Weak Mena staining was observed in noncancerous gastric mucosa (A) and in well-differentiated gastric adenocarcinoma (B). Strong Mena staining was observed in moderately differentiated (C) and poorly differentiated gastric adenocarcinoma (D). Magnification: ×200

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  Fig. 7. Kaplan–Meier overall survival curves of 172 GC patients after gastrectomy. a The survival rate of patients with high Mena expression was significantly lower than that of patients with low Mena expression (log-rank test, P = 0.017). b Among the 99 patients with high Mena expression, the survival rate of patients with high TES expression was significantly higher than that of patients with low TES expression (log-rank test, P = 0.010). c Among the 73 patients with low Mena expression, there was no significant association between TES expression and patient survival (log-rank test, P = 0.158)