TGIF1 plays a carcinogenic role in esophageal squamous cell carcinoma through the Wnt/β‑catenin and Akt/mTOR signaling pathways

Abstract

TGFB induced factor homeobox 1 (TGIF1), a transcriptional corepressor, has been reported to be involved in tumorigenesis and cancer development. However, the role of TGIF1 in the growth and metastasis of esophageal cancer is poorly studied. In the present study, it was found that TGIF1 was highly expressed in esophageal cancer tissues and cell lines. The silencing of TGIF1 by siRNA interference significantly inhibited the proliferation, migration, invasion and epithelial‑mesenchymal transition (EMT) process of KYSE‑150 esophageal cancer cells, and promoted cell apoptosis. Correspondingly, the upregulation of TGIF1 significantly promoted the proliferation and metastatic potential of Eca‑109 cells, and reduced apoptosis. Furthermore, the data indicated that the Wnt/β‑catenin and Akt/mammalian target of rapamycin (mTOR) signaling pathways were inhibited by TGIF1 knockdown, and were promoted by the overexpression of TGIF1. It was also confirmed that TGIF1 knockdown reduced tumor growth, inhibited Wnt/β‑catenin and Akt/mTOR pathway activation, and reversed the TGF‑β1‑mediated EMT process in a tumor xenograft model. Taken together, the data of the present study suggest that TGIF1 plays an oncogenic role in the progression of esophageal cancer. It may carry out this role by regulating the Wnt/β‑catenin and Akt/mTOR signaling pathways.

Keywords: esophageal cancer; TGFB induced factor homeobox 1; proliferation; invasion; Wnt/β‑catenin; Akt/mammalian target of rapamycin.

Figure 1

TGIF1 is upregulated in esophageal cancer and promotes cell proliferation. (A) mRNA expression of TGIF1 in esophageal cancer tissues (red box) and normal tissues (black box) analyzed using the GEPIA database. (B) Expression of TGIF1 mRNA in esophageal cancer cell lines (TE-10, KYSE-150, TE-1, kyse410 and Eca-109) and the human esophageal epithelial cell line, Het1A. (C) mRNA expression of TGIF1 in KYSE-150 cells transfected with siRNA-TGIF1-1#/2#/3# or siRNA control. (D) Western blot analysis was used to detect TGIF1 protein expression in KYSE-150 cells transfected with siRNAs. (E) mRNA expression of TGIF1 in Eca-109 cells transfected with pcDNA3.1-TGIF1 or pcDNA3.1 vector. (F) Western blot analysis was used to detect TGIF1 protein expression in Eca-109 cells. (G and H) CCK-8 assay was used to measure the viability of (G) KYSE-150 and (H) Eca-109 cells transfected with siRNAs or pcDNA3.1-TGIF1. (I and J) Colony formation assay was performed to examine colony-forming abilities of the (I) KYSE-150 and (J) Eca-109 cells. ^*P<0.05, **P<0.01 vs. respective control. TGIF1, TGFB induced factor homeobox 1.

Figure 2

TGIF1 enhances the migration and invasion abilities of esophageal cancer cells. (A and B) Following transfection with siRNAs or pcDNA3.1 vector, (A) KYSE-150 and (B) Eca-109 cell migration was measured by Transwell assay (magnification, ×100). (C and D) Transwell assay was performed to examine the invasion of (C) KYSE-150 and (D) Eca-109 cells (magnification, ×100). (E) Expression of EMT-related proteins (E-cadherin, N-cadherin and Snail) was detected by western blot analysis in KYSE-150 and Eca-109 cells. ^*P<0.05, ^**P<0.01, vs. respective control. TGIF1, TGFB induced factor homeobox 1.

Figure 3

TGIF1 decreases the apoptosis of esophageal cancer cells. (A and B) Flow cytometry was used to determine the rate of apoptosis of (A) KYSE-150 and (B) Eca-109 cells following transfection. (C) Expression of apoptosis-related proteins (Bcl-2, Bax and cleaved caspase-3) was detected by western blot analysis in the KYSE-150 and Eca-109 cells. ^*P<0.05, vs. respective control. TGIF1, TGFB induced factor homeobox 1.

Figure 4

TGIF1 promotes the activation of the Wnt/β-catenin and Akt/mTOR signaling pathways. (A) Expression of Wnt/β-catenin-related and Akt/mTOR-related proteins were detected by western blot analysis in KYSE-150 and Eca-109 cells. (B) Quantitative analysis of the results of western blot analysis in KYSE-150 cells. (C) Quantitative analysis of the results of western blot analysis in Eca-109 cells. ^*P<0.05, vs. respective control. TGIF1, TGFB induced factor homeobox 1; mTOR, mammalian target of rapamycin.

Figure 5

TGIF1 enhances the growth of esophageal cancer and increases the expression of EMT-related proteins in vivo. (A) Relative tumor volume growth cure of KYSE-150-derived tumors. (B) Relative tumor volume growth cure of Eca-109-derived tumors. ^*P<0.05, ^**P<0.01, vs. respective control. (C) Expression of EMT-related proteins (E-cadherin, N-cadherin and Snail) was detected by immunohistochemistry in the KYSE-150and Eca-109-derived tumors. TGIF1, TGFB induced factor homeobox 1; RTV, relative tumor volume; N-cad, N-cadherin; E-cad, E-cadherin.

Figure 6

TGIF1 promotes the activation of the Wnt/β-catenin and Akt/mTOR signaling pathways in vivo. Expression levels of Wnt/β-catenin-related and Akt/mTOR-related proteins were detected by immunohistochemistry in the KYSE-150 and Eca-109 tumors. TGIF1, TGFB induced factor homeobox 1; mTOR, mammalian target of rapamycin.