Cytokeratin 18 Affects Metastasis And Stemness Of Breast Cancer Through Regulation Of EpCAM Via Wnt/β-catenin Signaling Pathway

Background and Object:Breast cancer is one of the most common malignant tumors in the clinic,causing approximately 500,000 deaths worldwide each year,which is a serious threat to women’s health.Recurrence and metastasis are the leading causes of death in breast cancer patients.The latest research showed that a small number of cancer stem cells(CSCs)in tumors are one of the causes of tumor recurrence and metastasis.Epithelial mesenchymal transition(EMT),a process known to promote metastasis in cancer,has been shown to promote tumor stemness in recent years.Down-regulation of cytokeratin 18(CK18)is one of the hallmarks of EMT.This study aims to investigate the role and mechanism of CK18 in metastasis and stemness of breast cancer.Epithelial cell adhesion molecule(EpCAM)is a newly established tumor-associated protein that is abnormally expressed in many malignant epithelial tumors and can be activated by Wnt/β-catenin signaling pathway.It is confirmed to be one of CSCs markers.Wnt/β-catenin signaling pathway is a classical signaling pathway involved in tumorigenesis,metastasis,drug resistance and stemness,and EpCAM is one of the targets of this pathway.This study aims to investigate whether CK18 affects the metastasis and prognosis of breast cancer through regulation on EpCAM via Wnt/β-catenin signaling pathway.Methods:1.The expression of CK18 in breast cancer patients and adjacent non-tumor tissues was determined by immunohistochemistry.2.Western blot was used to determine the difference of CK18 in MCF-7 and MDA-MB-231 and MCF-7/MX cells.3.The expression of CK18 in MCF-7 cells was interfered using shRNA,and the knockdown of CK18 expression was detected by Western blot.Two monoclonal cell lines MCF-7/siCK18-7D and-3C with different knockdown levels were screened.Subsequent experiments were performed with MCF-7,negative control MCF-7/siNC,and two monoclonal cell lines.4.Western blot and plate clone formation method were used to detect the difference of stemness in all above cell lines.5.The expressions of β-catenin,EpCAM and Claudin-1 in breast cancer patients and adjacent non-tumor tissues were determined by immunofluorescence.6.The expression of Wnt pathway-related proteins in all above cells lines was determined by Western blot and immunofluorescence.7.Western blot and immunofluorescence were used to determine the expression of EpCAM and Claudin-1 protein in all above cells.Results:1.Low expression of CK18 predicts poor prognosis in breast cancer.In 60 breast cancer patients,CK18 expression was associated with lymph node metastasis(P < 0.01),tumor stage(P < 0.05),and E-cadherin(P < 0.01).There was no significant correlation between CK18 expression with age,family history,tumor size,histological grade,estrogen receptor(ER),progesterone receptor(PR),human epidermal growth factor 2(Her-2)and Ki-67(P > 0.05).There was significant correlation between CK18 expression and disease free survival(DFS)(P < 0.05).2.Down-regulation of CK18 promoted metastasis and stemness of breast cancer cells.Compared with MCF-7 cells,the expression of CK18 protein in highly invasive human breast cancer cell lines MDA-MB-231 was decreased(P < 0.05),and the expression of CK18 protein in MCF-7/MX cells with more stemness was also significantly decreased(P < 0.001).3.Knockdown of CK18 expression promoted the stemness of MCF-7 cells.Compared with MCF-7 cells,the expression of PCNA,stem cell markers and clone formation in MCF-7/siNC cells were almost no changed(P > 0.05),and significantly increased in CK18 down-regulation MCF-7/siCK18-7D and-3C cells(P < 0.05).4.Knockdown of CK18 in MCF-7 cells up-regulated the expression of EpCAM and down-regulated Claudin-1.Compared with MCF-7,the expression of EpCAM and Claudin-1 in MCF-7/siNC cells was not significantly different(P > 0.05),and the expression of EpCAM was increased in CK18 down-regulation cells(P < 0.01),while Claudin-1 expression was decreased remarkably(P < 0.001).5.Knockdown of CK18 in MCF-7 cells activated the Wnt/β-catenin pathway,inhibited Wnt pathway in MCF-7/siCK18-3C and reversed up-regulation of EpCAM and down-regulation of Claudin-1.Compared with MCF-7,the Wnt pathway proteins level in the negative control MCF-7/siNC cells were almost no changed(P > 0.05).The expression of p-GSK3β and β-catenin was increased in the CK18down-regulation cells(P < 0.05),GSK3β expression decreased.ICG-001,the inhibitior of Wnt signaling pathway,significantly inhibited the expression ofβ-catenin in MCF-7/siCK18-3C cells(P < 0.05),while EpCAM and CSCs markers expressions decreased(P < 0.01),and Claudin-1 expression increased(P < 0.01).Conclusions:1.CK18 is associated with breast cancer prognosis.2.CK18 inhibits the metastasis and stemness of breast cancer by down-regulating EpCAM expression and up-regulating the Claudin-1 levels through Wnt/β-catenin pathway.