An hTERT/ZEB1 complex directly regulates E-cadherin to promote epithelial-to-mesenchymal transition (EMT) in colorectal cancer

Abstract

In human cancer, high telomerase expression is correlated with tumor aggressiveness and metastatic potential. Telomerase activation occurs through telomerase reverse transcriptase (hTERT) induction, which contributes to malignant transformation by stabilizing telomeres. Previous studies have shown that hTERT can promote tumor invasion and metastasis of gastric cancer, liver cancer and esophageal cancer. Epithelial-to-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis, plays a key role in cancer progression. Although hTERT promotes EMT through Wnt signaling in several cancers, it is unknown if other signaling pathways are involved. In the present study, we found that hTERT and ZEB1 form a complex, which directly binds to the E-cadherin promoter, and then inhibits E-cadherin expression and promots EMT in colorectal cancer cells. hTERT overexpression in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even during hTERT overexpression. Taken together, our findings suggest that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting them may prevent cancer progression.

Keywords: CRC; EMT; ZEB1; hTERT.

Figure 1. hTERT promotes EMT in colorectal cancer cell lines

A. Morphological changes in SW480 and HCT116 cells after hTERT overexpression. B. hTERTexpression in SW480 and HCT116 cells as detected by Western blot. C. Western blot of E-cadherin, N-cadherin and vimentin in SW480 and HCT116 cells.

Figure 2. hTERTpromotes EMT independent of the Wnt signaling pathway

A. Colorectal cancer cells were co-transfected with 100 nM hTERT plasmid and a TOP-FLASH reporter (0.1 ng/well in each 96-well plate), and Wnt signal agonists(LiCl) and inhibitor(XAV) were added TOP-FLASH reporter activation was tested 48 h later. A scrambled construct was used as a negative control (NC). *P < 0.05 compared to control. B. Western blot of E-cadherin in HCT116 and SW480 cells(Lane1, No treatment; Lane2, hTERT overexpression; Lane3, Wnt inhibition; Lane4, hTERT overexpression plus Wnt inhibition). C–D. Colorectal cancer cells were co-transfected with 100 nM hTERT plasmid and a luciferase reporter containing the E-cadherin promoter (0.1 ng/well in each 96-well plate), and luciferase activity was tested 48 h later. A scrambled construct was used as a negative control (NC). *P < 0.05 compared to control. (C) The relative luciferase activity of the E-cadherin promoter in HCT116 cells. (D) The relative luciferase activity of the E-cadherin promoter in SW480 cells.

Figure 3. Interfere with β-catenin in HCT116 and SW480 cells

A. β-catenin knocks down in HCT116 and SW480 cells. B. HCT116 and SW480 cells were co-transfected with 100 nM hTERT plasmid and a TOP-FLASH reporter (0.1 ng/well in each 96-well plate), and 100 nM β-catenin interfere plasmid were added TOP-FLASH reporter activation was tested 48 h later. A scrambled construct was used as a negative control (NC). *P < 0.05 compared to control. C. The relative luciferase activity of the E-cadherin promoter in HCT116 and SW480 cells. D. Western blot of E-cadherin in HCT116 and SW480 cells(Lane1, No treatment; Lane2, hTERT overexpression; Lane3, hTERT overexpression plus β-catenin inhibition). E. Western blot to detect the β-catenin expression in nucleu and the total protein of HCT116 and SW480 cells.

Figure 4. hTERT and ZEB1 form a complex and bind the E-cadherin promoter

A. HCT116 cells were collected andlysates were subjected to IP using anti-hTERT, anti-ZEB1and rabbit IgG antibodies. Co-precipitating proteins were detected by western blot. B. Immunofluorescence staining for hTERT (green) and ZEB1 (red) in HCT116 cells. A representative mergedimage is shown (yellow denotes colocalization). C. Three binding sites for the E-cadherin promoter were designed. D. PCR quantification of immunoprecipitated E-cadherin promoterin ChIP assays from HCT116 cells with Abs against hTERT and the respective control IgG. Amplified E-cadherin promoter regions contained ZEB1 binding sites at −1959 and −1954. Values represent relative binding to input.

Figure 5. hTERT promotes EMT through the ZEB1 pathway

A. ZEB1 knocks down in HCT116 cells. B. Western blot of E-cadherin, N-cadherin and vimentin after hTERT overexpression and ZEB1 knock down in HCT116 cells. C. The relative luciferase activity of the E-cadherin promoter in NC-HCT116, hT-HCT116 and shZ-hT-HCT116 cells. D. PCR quantification of the E-cadherin promoter at −1959and −1954 immunoprecipitated in ChIP assays from HCT116, hT-HCT116 and shZ-hT-HCT116 cells with Abs against hTERT andthe respective control IgG. Values represent relative binding to input.

Figure 6. The hTERT/ZEB1 complex promotes the metastatic potential of HCT116 colorectal cancer cells in vivo and in vitro

A–B. Cell invasion was measured by Transwell assays 48 h after incubation. The number of cells from three random areas of the membrane was counted using light microscopy; *P < 0.05. C–D. The migratory properties of cells were tested in wound healing assays 24 h after incubation. The distance of cells from three random areas of the wound was counted using light microscopy; *P < 0.05. E. Metastasis observed after intravenous injection of cells. Metastases were observed by H&E staining at different magnifications.

Figure 7. Model of hTERT/ZEB1 complex regulation of E-cadherin expression