ZEB1 Stimulates Breast Cancer Growth By Up-regulating HTERT Expression

Background:Breast cancer accounted for 30% of the new cancer diagnoses in women in 2017 and was the leading cause of cancer death among young Chinese women in 2015.As early as 50 years ago,scientists found that immortality was an essential hal mark of cancer and the cornerstone of many malignant characteristics of cancer,such as rapid growth and distant metastasis.Zinc finger E-box binding homeobox 1(ZEB1)is a transcription factor belonging to the ZEB protein family that binds to specific DNA sequences known as E-boxes(5’-CACGTG-3’)to control target gene expression.Because one of its best-known targets is the epithelial-mesenchymal transition(EMT)-related gene E-cadherin,many researchers have concentrated on its functions in migration and invasion of various cancer types.However,accumulating evidence has shown that ZEB1 is also involved in cancer cell proliferation and apoptosis.Objective: To discover relationship between hTERT and ZEB1 expression in breast cancer patients and breast cancer cell lines.To exam the influence of ZEB1 on hTERT expression and telomerase function in the breast cancer cell lines MDA-MB-231 and MCF-7 and revealed the involvement of ZEB1 in hTERT-mediated cell proliferation and apoptosis.To investigate the mechanism by which ZEB1 regulates hTERT.Research methods: qRT-PCR analysis of ZEB1 and hTERT expression level of 133 breast cancer tissue samples,IHC of ZEB1 and hTERT in tissue microarrays.qRT-PCR and Western blot analysis of ZEB1 and hTERT expression in the breast cancer cell lines MDA-MB-231 and MCF-7 transfected with ZEB1 overexpression plasmids and ZEB1 si RNA.Telomerase activity assay and Telomere lengths test of transfected cel s.qRT-PCR and Western blot analysis of ZEB1 and hTERT expression in cel s with hTERT knockdown alone or simultaneous ZEB1 overexpression and hTERT knockdown.MTT assay and apoptosis assay of cel s co-transfected with ZEB1 overexpression plasmid negative control and hTERT si RNA negative control,ZEB1 overexpression plasmid negative control and hTERT si RNA,or ZEB1 overexpression and hTERT si RNA.Ch IP assays and dual-luciferase reporter assays of interactions between ZEB1 and hTERT promoter region.DNA sequencing of cell and tissue samples to exam mutations located in hTERT promoter region.Co-IP assays of ZEB1 and YAP interaction.Ch IP assays and dual-luciferase reporter assays of interactions between YAP and hTERT promoter region.Dual-luciferase reporter assays of the protein complex of ZEB1 and YAP interaction with part region of hTERT promoter.qRT-PCR and Western blot analysis of YAP’ influence in hTERT.qRT-PCR and IHC of xenograft tumors to explore the relationship of ZEB1 and hTERT in vivo.Results: hTERT expression is positively correlated with ZEB1 expression in breast cancer.ZEB1 promotes hTERT expression and telomerase activity in breast cancer cell lines.ZEB1 cooperated with co-activator YAP to form a protein complex to increases the transcriptional activity of the hTERT promoter in order to control hTERT expression.ZEB1 overexpression promotes tumor growth and hTERT expression in vivo.Conclusion: In the present study,we il uminated a new ZEB1-hTERT signaling pathway that regulates breast cancer cell proliferation and apoptosis.In summary,this study identified ZEB1 as a tumor promoter in breast cancer.ZEB1 not only promoted the invasiveness of breast tumors,as previously reported49 but also controlled cell proliferation and death by regulating hTERT expression in vitro and in vivo.Thus,ZEB1,a multifunctional cancer stimulatory factor,should be further investigated to elucidate its potential role,which may help in the treatment of breast cancer.