Abstract
CUL4A regulate the termination of autophagy in a physical process. However, the relationship between CUL4A and autophagy in cancer is unclear. We retrospectively investigated 99 intrahepatic cholangiocarcinoma (iCCA) cases. Whole sections were used for immunohistochemical analysis for p62, and LC3B expression. Q-score was defined as the sum of the labeling intensity and proportion. The cut-off point for immunoreactivity was set. CUL4A was overexpressed in cell lines and autophagy reflux was compared after manipulation. The iCCA cases with CUL4A overexpression had significantly higher prevalence of intact activated autophagy (42.4 vs. 15.2%; p = 0.003), which was significantly associated with advance tumor stage (34.1% vs. 15.4%; p = 0.032), less extensive necrosis (8.3 vs. 49.3%; p < 0.001), and shortened disease-free survival (mean, 19.6 vs. 65.5 months, p = 0.015). In vitro, iCCA cells with CUL4A overexpression significantly increased LC3II level as compared to the cells under basal condition. Although both cell types showed intact autophagy with increased LC3II expression after bafilomycin A1 treatment, the accumulation of LC3II was higher in CUL4A-overexpressing cells. CUL4A overexpression increased the proliferation of cells as compared with control cells. After treatment with bafilomycin A1, proliferation was inhibited in both cell types, but the effects were more prominent in the cells overexpressing CUL4A. CUL4A promotes autophagy, and exhibits significantly higher autophagic flux which affects the proliferation of iCCA cells; these effects correlated with advance tumor stage and poor prognosis. Thus, targeting autophagy may be potentially therapeutic in iCCA.
Highlights
CUL4A is a member of the cullin family of proteins and comprises a multifunctional ubiquitinprotein ligase E3 complex
The phenotypes were defined according to the expression of light chain 3 beta (LC3B) and p62 as follows [17, 18]: (1) Intact activated type: LC3B+/p62− and (2) non-intact type: LC3B+/p62+ indicative of autophagy activation, which was impaired at later steps during the process; LC3B−/p62+ representing a basal level of autophagy which was impaired at later steps of process; and LC3B−/p62− mimicked the basal level of autophagy
We elucidated the clinical effects of CUL4A in intrahepatic cholangiocarcinoma (iCCA) that are probably mediated through intact activated autophagy, which affects the proliferation of iCCA cells; these effects correlated with advance tumor stage and poor prognosis
Summary
CUL4A is a member of the cullin family of proteins and comprises a multifunctional ubiquitinprotein ligase E3 complex. This cullin-RING ubiquitin ligase (CRL) mediates the process of ubiquitylation or ubiquitination through a cascade of enzymatic reactions involving E1, E2, and Pathology & Oncology Research. GRAPHICAL ABSTRACT | Overexpression of CUL4A in iCCA cells. The expression of the reporter gene in the lentivirus was observed by immunofluorescence staining for green fluorescent protein (GFP) along with DAPI staining. Percentage of cells expressing CUL4A-mGFP was normalized to DAPI-stained nuclei (blue).
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