Molecular Mechanisms Of HTERT/ZEB1 Pathway Promote Epithelial-to-mesenchymal Transition(EMT) In Colorectal Cancer

BackgroundsIn human cancers, high expression of telomerase 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 esophageal cancer, gastric cancer and liver cancer. Epithelial-to-mesenchymal transition (EMT) which is a requirement for tumor invasion and metastasis, and 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 this study, we found that hTERT and Zinc-finger E-box binding homeobox 1(ZEB1) form a complex, and this complex directly binds to the E-cadherin promoter to inhibit E-cadherin expression and promotes EMT in colorectal cancer cells. Overexpression of hTERT in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even when we overexpression hTERT. Taken together, our finding suggests that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting hTERT and ZEB1 may prevent cancer progression.ResultsPart 11. hTERT promote sepithelial-to-mesenchymal transition (EMT) in colorectal cancer cell lines(SW480 and HCT116 cells).2. Western blot and immunofluorescence experiment showed hTERT reduced E-cadherin expression, and increased N-cadherin and Vimentin expression.3. qPCR analysised mesenchyme related markers and found that Snail high expressed in SW480 cells when we overexpression hTERT in SW480 and HCT116 cells.4. Dual luciferase experiments showed hTERT can reduce E-cadherin promoter activity in SW480 and HCT116 cells.5. Top-Fop showed Wnt signal was activated by hTERT in SW480 cells but not in HCT116 cells.6. We detected the Wnt activity when we used Wnt inhibitor(XAV) in SW480 and HCT116 cells. The Wnt activity down-regulated and E-cadherin restored in SW480 cells but not had any chang in HCT116 cells.7. We interferenced β-catenin in SW480 and HCT116 cells, The Wnt activity down-regulated and E-cadherin restored in SW480 cells but had not any change in HCT116 cells.8. Western blot examine nucleus β-catenin expression in SW480 and HCT116 cells. The nucleus β-catenin increased in SW480 cells but had not changed in HCT116 cells.9. Dual luciferase experiments showed when we repressed Wnt activity in SW480 and HCT116 cells, the E-cadherin prmoter activity recovered in SW480 cells(p<0.01) but not in HCT116 cells(p>0.05).Part 21. Co-immunoprecipitation was used to identify if hTERT interactive ZEB1 in HCT116 cells. The result showed that hTERT could pull out ZEB1 and ZEB1 also pull out hTERT in HCT116 cells. The confocal laser scanning microscope also observed hTERT and ZEB1 colocalization in HCT116 cells.2. We designed three primers that target the ZEB1 binding sites within E-cadherin E-box elements and chromatin immunoprecipitation experiment showed hTERT interacted with the E-cadherin promoter within the-1959bp to-1954bp region.3. Western blot and immunofluorescence experiment showed interference ZEB1 can reverse EMT in HCT116 cells when overexpressing hTERT. After overexpressd hTERT the E-cadherin down-regulated on the contrary the N-cadherin and vimentin increased. But when we intereferencing ZEB1 the E-cadherin recovered and the N-cadherin and vimentin down-regulated.4. Dual luciferase report system to detect E-cadherin promoter activity after ZEB1 knockdown, and found that E-cadherin promoter activity was increased even overexpression hTERT in HCT116 cells.5. Chromatin immunoprecipitation assays showed that hTERT overexpression increased its association with the E-cadherin promoter, and this association was decreased after ZEB1 knockdown.6. Western blot found that when overexpressing hTERT the ZEB1 was up-regulated in SW480 cells but not in HCT116 cells.7. Transwell and wounding exprements assays showed when we overexpressing hTERT the migration is increased(p<0.01), but after interferencing ZEB1 the migration is reduced in HCT116 cells(p<0.01). The tail vein injection of nude mice also indicated that hTERT could promote lung metastasis(p<0.01) and distrubed ZEB1 could prevent metastasis(p<0.01).Conclusion1. Here provide the first evidence for the hTERT promote metastasis by epithelial-to-mesenchymal transition(EMT) of colorectal cancer(CRC). And we also find a new way for the hTERT promote EMT of tumor. In this study we found that hTERT could downregulate E-cadherin expression and increased N-cadherin and Vimentin expression. Further study found hTERT could promote EMT by restrain the E-cadherin promoter activity, and this way independent Wnt signal pathway in HCT116 cells which is a classical pathway for hTERT to promote EMT in other tumors. This result will supplement the thoery of hTERT promote tumor metastasis.2. On the basis of this study, we conclude that hTERT could activate the EMT in colorectal cancer cells to prmote cancer metastasis. hTERT and ZEB1 can combine into a protein complex, which binds to E-cadherin promoter region together to inhibit the expression of E-cadherin, and finally contributes to the occurrence of EMT. In summary, we demonstrate for the first time that E-cadherin is a direct hTERT/ZEB1 target in CRC cells and acts as an effector of this signaling pathway to regulate genes associated with tumor invasiveness. Our findings provide novel insights into hTERT promoted the occurrence of EMT in CRC. The establishment of such a link establishes hTERT as an important diagnostic predictor and a potential therapeutic target in CRC.