Overexpression of brachyury contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma

Abstract

Aims: Brachyury overexpression has been reported in various human malignant neoplasms, but its expression and function in hepatocellular carcinoma progression and metastasis remains unknown. The present study aimed to evaluate the critical role of Brachyury in HCC metastasis.

Methods: The expression of Brachyury in human HCC (SMMC7721, HepG2, FHCC98, and Hep3B) and control cell lines was analyzed using quantitative reverse-transcriptase polymerase chain reaction and immunoflourence methods. Cancerous tissues collected from patients with HCC (n = 112) were analyzed using immunohistochemical method; a microarray analysis of HCC tissues was performed to explore the clinicopathological variables of HCC. The migratory and invasive capacities of Brachyury-SMMC7721 and Brachyury-HepG2 transfected cells were evaluated using in vitro scratch wound healing and Matrigel invasion assays, respectively. Further, six-week-old male BALB/c nude mice (n = 10) model was used in vivo assay.

Results: Elevated expression of Brachyury was detected in HCCs (62.5%) compared with that in adjacent nontumorous tissues. Clinicopathological analysis revealed a close correlation of Brachyury expression with distant metastasis and poor prognosis of HCC. Overexpression of Brachyury promoted epithelial-mesenchymal transition (EMT) and metastasis of HCC cells in vitro and in vivo. Brachyury overexpression enhanced Akt activation by inhibiting phosphatase and tensin homolog (PTEN), which led to subsequent stabilization of Snail, a critical EMT mediator.

Conclusion: The study findings suggest that elevated Brachyury facilitates HCC metastasis by promoting EMT via PTEN/Akt/Snail-dependent pathway. Brachyury plays a pivotal role in HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target.

Figure 1

Expression of Brachyury is up-regulated in human HCCs. (A) Expression of Brachyury in one human normal liver cell lines (QZG) and four hepatoma cell lines was examined by western blot assay. (B) Brachyury expression in 40 pairs of HCC and the surrounding tissues were detected by real-time PCR. (C) Representative western blot showing the expression of Brachyury protein in tumor tissue (T) and paired peri-tumor tissue (N) from eight HCC patients. (D) Relative immunohistochemical staining of Brachyury expression in paired HCC tissue samples (n = 112), and the representative view (E).

Figure 2

Brachyury overexpression predicts poor prognosis of HCC. (A) HCC patients were divided into Brachyury-positive expression group (n = 70) and Brachyury-negative expression group (n = 40). The 5-year overall of 112 HCC patients were compared between the low and high Brachyury groups. (B) The 3-year overall of 112 HCC patients were compared between the low and high Brachyury groups.

Figure 3

Brachyury enhances metastatic potential of HCC cells. (A) The invasive properties of the cells were analyzed in by an invasion assay using a Matrigelcoated Boyden chamber. Migrated cells were plotted as the average number of cells per field of view from 3 different experiments, as described in Materials and Methods. (B) The cell migration rate between SMMC-7721/Brachyury and SMMC-7721/GFP cells was compared by wound-healing assay. Microscopic observation was recorded at 0 and 24 hours after scratching the cell layer. (C) Representative view of lung tissue sections from each group are shown (H&E stain; magnification × 100). The number of lung metastatic foci in each group (n = 10) of HepG2/Brachyury or HepG2/GFP xenografted mice was calculated microscopically 6 weeks after tail vein injection. *p < 0.05.

Figure 4

Brachyury promotes EMT in HCC cells. (A) HepG2/ Brachyury and control cells were cultured on Poly-L-Lysine coated glass coverslips for 24 hours followed by immunofluorescence assay. (B) Relative expression of E-cadherin, γ-catenin, and Vimentin in HepG2/ Brachyury cells compared to control cells. (C) Representative view of Western blot assay of E-cadherin and Vimentin in tumor tissue (T) and paired peri-tumor tissue (N) from HCC patients with up-regulated Brachyury in tumor. (D) Scatter plot with fitted values intervals for T/NT (ratio of tumor tissue and paired peri-tumor tissue) expression of Bachyury and Vimentin. (E) Scatter plot with fitted values intervals for T/NT expression of Bachyury and E-cadherin.

Figure 5

Bachyury regulates EMT via Akt/Snail pathway. (A) Western blot assay demonstrated overexpression Bachyury repressed PTEN and enhanced p-Akt, Snail, but not t-Akt expression in HepG2 cells. (B) AKT inhibitor prevented the Brachyury-mediated increase in p-AKT and Snail expression and abolished Bachyury-induced HCC EMT phenotype. (C) SiRNA against snail abolished Bachyury-induced Snail expression and HCC EMT phenotype.