CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma.

CtBP2 expression was upregulated in HCC tissues and associated with poor prognosis after liver resection(A) CtBP2 was expressed mainly in the cytoplasm of tumor cells and CtBP2 expression was notably higher in HCC tissues (a) when compared to matched normal adjacent liver tissues (b). (B) The Mann–Whitney U test confirmed that CtBP2 expression was significantly higher in HCC tissues than adjacent liver tissues from 100 HCC patients ( p < 0.001). (C) Kaplan Meier overall survival curves indicated that the High CtBP2 Group had an obviously shortened post-surgical survival time when compared to the Low CtBP2 Group (HR = 3.071; 95% CI: 1.357, 6.951; p = 0.007). (D) There was more GLI1 expression in HCC tissues (a) compared to matched normal adjacent liver tissues (b), and most of the GLI1-positive cells showed nuclear staining. (E) SNAI1 expression was notably increased in HCC tissues by IHC (a) when compared to matched adjacent healthy liver tissues (b). (F) The Spearman rank test indicated that GLI1 expression correlated positively with CtBP2 expression (r = 0.701, p < 0.001, left panel) and SNAI1 (r = 0.759, p < 0.001, right panel) in HCC tissues, respectively.

The expression of CtBP2 in HCC cell lines(A) CtBP2 mRNA levels in five different HCC cell lines was assessed by qRT-PCR. (B) CtBP2 protein expression in five different HCC cell lines was examined by Western blotting.

Elevated CtBP2 expression promoted Huh7 cell mobility and invasion by inducing EMT(A) Both qRT-PCR and Western blotting assays verified that CtBP2 expression was increased after stable transfection of CtBP2 expression plasmid. (B) The migration capacity of Huh7 cells, as assessed by wound healing assays, was enhanced by CtBP2 overexpression. (C) Transwell invasion assays demonstrated that elevating CtBP2 expression increased the invasive capability of Huh7 cells. (D) Overexpression of CtBP2 decreased E-cadherin expression and increased the expression of N-cadherin, Vimentin and Fibronectin in Huh7 cells, as measured by Western blotting. (E) Double immunofluorescence staining confirmed that CtBP2 overexpression resulted in the downregulation of E-cadherin and the upregulation of N-cadherin in Huh7 cells.

CtBP2 knockdown inhibited MHCC97H cell migration and invasion capacities by repressing EMT(A) Both qRT-PCR and Western blotting assays confirmed that siRNA transfection eliminated CtBP2 expression in MHCC97H cells. (B) Wound healing assays indicated that CtBP2 siRNA suppressed MHCC97H cell migration. (C) The invasive ability of MHCC97H cells was repressed by CtBP2 siRNA in Transwell invasion assays. (D) Western blotting assays demonstrated that CtBP2 suppression lead to E-cadherin upregulation and N-cadherin, Vimentin and Fibronectin downregulation in MHCC97H cells.

GLI1 increased CtBP2 expression directly by binding to its promoter(A) Both qRT-PCR and Western blotting assays demonstrated that the expression of GLI1 (left panel) and CtBP2 (right panel) were notably elevated after GLI1 overexpression in Huh7 cells. (B) A schematic model of the locations and sequences of the 14 potential GLI1 binding sites (BS). The consensus GLI1 binding sequence is listed here. (C) Luciferase reporter assays in Huh7 GLI1 cells indicate that the pGL3−1307 (−1307/−1 bp CtBP2 promoter fragment) has the strongest promoter activity. When the fragment between −1307 bp and −676 bp was deleted, the luciferase activity greatly reduced. The pGL3−1350 (−1350/−652 bp CtBP2 promoter fragment) displayed the second highest level of promoter activity. (D) Luciferase reporter assays demonstrated that the luciferase activity of pGL3−1350 was notably enhanced after GLI1 overexpression in Huh7 cells. This supports the ability of the GLI1 protein to bind the CtBP2 promoter in the −1350/−652 bp region. (E) DNA fragments capable of binding the GLI1 protein in the cell nucleus were analyzed by ChIP assay. As the upper panel shows, the PCR assay showed that the GLI1 protein was capable of directly binding to the −1350/−652 bp CtBP2 promoter fragment. After equalization using RNA Polymerase II (Input), more CtBP2 promoter fragment was bound to the GLI1 protein in Huh7 GLI1 cells. Similarly, a relative rtPCR assay demonstrated that elevated GLI1 expression increased the CtBP2 promoter occupancy of GLI1 in Huh7 cells, as presented in the lower panel.

CtBP2 interacted with SNAI1 and played a critical role in GLI1/SNAI1 induction of EMT(A) Both qRT-PCR and Western blotting assays demonstrated that SNAI1 siRNA abated SNAI1 expression (left panel) in Huh7 cells and that CtBP2 expression was enhanced by CtBP2 expression plasmid transfection in Huh7 cells without SNAI1. (B) Western blotting showed that CtBP2 overexpression lead to E-cadherin downregulation and N-cadherin, Vimentin and Fibronectin upregulation in Huh7 cells with normal SNAI1 expression. However, silencing SNAI1 counteracted the regulatory effect of CtBP2 on E-cadherin, N-cadherin, Vimentin and Fibronectin. (C) Co-IP assays demonstrated direct CtBP2 and SNAI1 binding in Huh7 GLI1 cells. (D) Both qRT-PCR and Western blotting verified that siRNA abolished CtBP2 expression in Huh7 GLI1 cells. (E) Western blotting assays demonstrated that CtBP2 knockdown in Huh7 GLI1 cells lead to E-cadherin upregulation and N-cadherin, Vimentin and Fibronectin downregulation, while SNAI1 expression was not significantly affected. However, silencing CtBP2 did not affect the expression of E-cadherin, N-cadherin and Fibronectin. Neither the expression of Vimentin nor the expression of SNAI1 could be detected in Huh7 Vector cells transfected with CtBP2 siRNAs or Scr siRNAs.

Elevated CtBP2 expression accelerated HCC xenograft growth and promoted EMT in HCC xenograft tissues(A) Representative pictures of HCC xenografts from both Huh7 Vector (left panel) and Huh7 CtBP2 cells (right panel). (B) Xenograft size in the Huh7 CtBP2 group was significantly larger than in the Huh7 Vector Group ( p = 0.001).

Compared to the Huh7 Vector group, there was more CtBP2 and N-cadherin protein expression in xenograft tissues from the Huh7 CtBP2 group, whereas E-cadherin expression was decreasedGLI1 and SNAI1 expression was undetected in xenograft tissues from either the Huh7 CtBP2 or the Huh7 Vector Group.

Working model of the role of CtBP2 in EMT induced by the GLI1/SNAI1 axis in HCCGLI1 upregulates both SNAI1 and CtBP2 simultaneously. SNAI1 binds the E-cadherin promoter after binding CtBP2, leading to the EMT phenotype of HCC cells.