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

Abstract

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 CUL4A 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 CUL4A 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 CUL4A in PHCC metastasis. Furthermore, expression of epithelial to mesenchymal transition (EMT) markers was verified in cells with CUL4A knockdown or overexpression. The relationship between CUL4A expression and E-cadherin expression was also analyzed by IHC assay. Finally, the role of ZEB1 in regulating CUL4A mediated PHCC was detected by IHC, Western blot, Transwell and Matrigel assays.

Results: CUL4A overexpression was detected in PHCC cell lines and clinical specimens. Clinicopathological analysis revealed a close correlation between CUL4A overexpression and tumour differentiation, T, N and TNM stages in PHCC. Kaplan-Meier analysis revealed that high CUL4A 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 CUL4A expression; as well as three related to PFS: N stage, TNM stage and high CUL4A expression. Further multivariate logistic regression analysis identified high CUL4A expression as the only independent prognostic factor for PHCC. Moreover, CUL4A silencing in PHCC cell lines dramatically inhibited metastasis and the EMT. Conversely, CUL4A overexpression promoted these processes. Mechanistically, ZEB1 was discovered to regulate the function of CUL4A in promoting the EMT and metastasis.

Conclusion: CUL4A is an independent prognostic factor for PHCC, and it can promote the EMT by regulating ZEB1 expression. CUL4A may be a potential therapeutic target for PHCC.

Keywords: Cullin 4A; Epithelial to mesenchymal transition; Metastasis; Perihilar cholangiocarcinoma; Prognosis; ZEB1.

Figure 1

Cullin 4A is overexpressed in perihilar cholangiocarcinoma. A: Expression of CUL4A protein was detected in normal biliary epithelial cells (HIBEpic) and PHCC cell lines by Western blot assays; B: Expression of CUL4A mRNA was detected in normal biliary epithelial cells (HIBEpiC) and PHCC cell lines by qRT-PCR assay; C: Representative images of CUL4A IHC staining in PHCC tumour tissues and normal adjacent tissues. Corresponding semiquantification of CUL4A expression is shown. Numbers in (B) indicate the fold changes of band densities based on at least three independent experiments. ^bP < 0.01 based on the Student’s t-test. Data are represented as mean ± SD. CUL4A: Cullin 4A; PHCC: Perihilar cholangiocarcinoma.

Figure 2

Cullin 4A correlates with a poor prognosis in perihilar cholangiocarcinoma. A and B: The cut-off points of CUL4A expression for the overall survival (A) and progression-free survival (B) were analysed by the ROC curve analysis; C: Representative images of low and high CUL4A expression; D and E: Kaplan-Meier analysis and the log-rank test were adopted to investigate the overall survival (D) and progression-free survival (E) differences between the low and high CUL4A expression groups. P < 0.01 in D and E based on the log-rank test. CUL4A: Cullin 4A; PHCC: Perihilar cholangiocarcinoma.

Figure 3

Cullin 4A promotes the migration and invasion of perihilar cholangiocarcinoma cell lines. QBC939-shCUL4A and FRH0201-CUL4A cells or control cells were subjected to wound healing (A and C), Transwell migration (B and D, top), and Matrigel invasion (B and D, bottom) assays. A: Quantification was performed by measuring the uncovered areas compared with the controls; B: Quantification of migrated cells through the membrane and invaded cells through the Matrigel of each cell line is shown as proportions to their controls; C: Quantification was carried out by measuring the uncovered areas compared with the controls; D: Quantification of migrated cells through the membrane and invaded cells through Matrigel for each cell line is shown as proportions to their controls. ^bP < 0.01 based on the Student’s t-test. All results are from at least three independent experiments. Data are represented as mean ± SD. CUL4A: Cullin 4A.

Figure 4

Cullin 4A induces the epithelial to mesenchymal transition in perihilar cholangiocarcinoma. A: Expression levels of an epithelial marker (E-cadherin) and mesenchymal marker (vimentin) were analyzed by Western blot; B: Representative IHC images of E-cadherin expression in PHCC tissues and adjacent normal tissues; C: Statistical analysis of the semiquantification of E-cadherin expression in PHCC tissues and adjacent normal tissues; D: Linear regression analyses of IHC scores between CUL4A and E-cadherin expression in PHCC. ^bP < 0.01 based on the Student’s t-test. CUL4A: Cullin 4A; PHCC: Perihilar cholangiocarcinoma.

Figure 5

ZEB1 mediates the metastasis regulated by cullin 4A in perihilar cholangiocarcinoma. A: Representative IHC images of ZEB1 expression in PHCC tissues and adjacent normal tissues. Corresponding semiquantification of ZEB1 expression is shown; B: Linear regression analyses of IHC scores between CUL4A and E-cadherin expression in PHCC; C: Linear regression analyses of IHC scores between CUL4A and ZEB1 expression in PHCC; D: Western blot assay was performed to investigate the E-cadherin and vimentin expression after ZEB1 interference in CUL4A overexpressing FRH0201 cells; E and F: The Transwell and Migration assays were performed to analyse the migration and invasion ability changes in CUL4A overexpressing FRH0201 cells with ZEB1 depletion. ^bP < 0.01 based on the Student’s t-test. All results are from at least three independent experiments. Data are represented as mean ± SD. CUL4A: Cullin 4A; PHCC: Perihilar cholangiocarcinoma.