CtBP2 overexpression promotes tumor cell proliferation and invasion in gastric cancer and is associated with poor prognosis

Abstract

C-terminal binding protein-2 (CtBP2), a transcriptional corepressor, has been reported to correlate with tumorigenesis and progression and predict a poor prognosis in several human cancers. However, few studies on CtBP2 in gastric cancer (GC) have been performed. In this research, we evaluated the correlations between CtBP2 expression and the clinicopathological characteristics, as well as prognosis of GC patients. The effects of silencing CtBP2 expression on GC cells biology activity were also assessed. The results showed that CtBP2 was overexpressed in GC tissues and closely correlated with poor differentiation, advanced tumor stage and poor prognosis in GC patients. CtBP2 induced epithelial-to-mesenchymal transition (EMT) and repressed PTEN to increase proliferation rate, migration, and invasion in GC cells. Silencing CtBP2 inhibited GC growth in nude mice model. In conclusion, CtBP2 is overexpressed in GC and may accelerate GC tumorigenesis and metastasis, which could represent an independent prognostic marker and promising therapeutic target for GC.

Keywords: CtBP2; gastric cancer; prognosis.

Figure 1. Immunohistochemical (IHC) analysis of CtBP2 expression in 352 gastric cancer (GC) and matched normal tissues

Representative examples of IHC results. A. CtBP2 showed low expression in normal gastric tissues but high expression in GC tissues (magnification, ×200 and 400). B. CtBP2 expression in well-, moderately, and poorly differentiated GC tissues; the staining results were weak, moderate and strong, respectively (magnification, ×200 and 400).

Figure 2

A. Western blotting analysis showed that the protein levels of CtBP2 were higher in six representative GC tissues than in matched adjacent normal gastric tissues. B. The expression of CtBP2 was higher in the GC cell lines (HGC-27 and SGC-7901) than in the normal gastric cell line GES1. C. GC cell lines (HGC-27 and SGC-7901) were transiently transfected with CtBP2-shRNA#2 for 48 h. Western blotting analysis demonstrated the silencing of CtBP2. Representative WB images showed the expression of PCNA, E-cadherin, N-cadherin, PTEN in untreated, Control-shRNA and CtBP2-shRNA#2 treated cells, respectively.

Figure 3. Analysis of GC patient survival prognosis using the Kaplan-Meier method

A. The CtBP2 high-expression group (blue line) had significantly worse prognoses than the CtBP2 low-expression group (red line) (p < 0.001). B. The overall survival of stage I and II GC patients was significantly higher than for stage III and IV GC patients (p < 0.001). C, D. The CtBP2 high-expression group (blue line) had a significantly worse prognosis than the CtBP2 low-expression group (red line) at stage III or IV (p < 0.001 and p = 0.004, respectively).

Figure 4. Knockdown of CtBP2 suppresses cell proliferation in vitro

A, B. Colony formation assays showed that the downregulation of CtBP2 reduced the mean colony number in both HGC-27 and SGC-7901 cells. C, D. The downregulation of CtBP2 suppressed the growth rate of HGC-27 and SGC-7901 cells in CCK-8 cell proliferation assays. E, F. Cell cycle analysis of the role of CtBP2. Knockdown of CtBP2 resulted in an increase in the G1/S ratio in both HGC-27 and SGC-7901 cells (* p < 0.05, **p < 0.01, *** p < 0.001).

Figure 5

Transwell migration A, B. and invasion C, D. assays showed that knockdown of CtBP2 expression significantly reduced migration and invasion in both HGC-27 and SGC-7901 GC cells (* p < 0.05).

Figure 6. Knockdown of CtBP2 suppresses GC tumourigenesis in vivo

A. Representative pictures of GC tumors excised from BALB/c-nude injected with HGC-27-CtBP2-shRNA#2 (above) and HGC-27-control-shRNA (bellow) after 2,3,4,5 week-tumor-formation respectively. B. The growth rate of tumourigenesis in vivo after GC cells injection was shown in the growth curve (mean ± SD, p < 0.05).