Cullin 4A is associated with epithelial to mesenchymal transition and poor prognosis in perihilar cholangiocarcinoma

AIM To explore the functional role of cullin 4A(CUL4A), a core subunit of E3 ubiquitin ligase, in perihilar cholangiocarcinoma(PHCC).METHODS The expression of CUL4 A in PHCC cell lines was evaluated by Western blot and quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry(IHC) was adopted to investigate the relationship between CUL4 A expression and clinicopathological characteristics of PHCC. Univariate analysis and multivariate regression analysis were performed to analyze the risk factors related to overall survival(OS) and progression-free survival(PFS) of PHCC patients. Wound healing, Transwell and Matrigel assays were utilized to explore the function of CUL4 A in PHCC metastasis. Furthermore, expression of epithelial to mesenchymal transition(EMT) markers was verified in cells with CUL4 A knockdown or overexpression. The relationship between CUL4 A expression and E-cadherin expression was also analyzed by IHC assay. Finally, the role of ZEB1 in regulating CUL4 A mediated PHCC was detected by IHC, Western blot, Transwell and Matrigel assays.RESULTS CUL4 A overexpression was detected in PHCC cell lines and clinical specimens. Clinicopathological analysis revealed a close correlation between CUL4 A overexpression and tumour differentiation, T, N and TNM stages in PHCC. Kaplan-Meier analysis revealed that high CUL4 A expression was correlated with poor OS and PFS of PHCC patients. Univariate analysis identified the following four parameters as risk factors related to OS rate of PHCC: T, N, TNM stages and high CUL4 A expression; as well as three related to PFS: N stage, TNM stage and high CUL4 A expression. Further multivariate logistic regression analysis identified high CUL4 A expression as the only independent prognostic factor for PHCC. Moreover, CUL4 A silencing in PHCC cell lines dramatically inhibited metastasis and the EMT. Conversely, CUL4 A overexpression promoted these processes. Mechanistically, ZEB1 was discovered to regulate the function of CUL4 A in promoting the EMT and metastasis.CONCLUSION CUL4 A is an independent prognostic factor for PHCC, and it can promote the EMT by regulating ZEB1 expression. CUL4 A may be a potential therapeutic target for PHCC.