Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25

Abstract

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Keywords: AZGP1; Cholangiocarcinoma; E3 ligase; TRIM25; apoptosis.

FIGURE 1

AZGP1 expression is associated with poor prognosis in cholangiocarcinoma (CCA). (A) Alpha‐2‐Glycoprotein 1, Zinc‐Binding (AZGP1) expression was obtained from the Gene Expression Omnibus (GEO) database (GSE26566). (B) Analysis of the GEPIA database showed that AZGP1 expression is downregulated in CCA. (C) The mRNA levels of AZGP1 in various stages of CCA. (D) Kaplan–Meier curve of high and low AZGP1 expression in CCA patient groups (log‐rank p = 0.37, p(HR) = 0.39, compared with normal controls). **p < 0.01 indicates significant differences from the control group.

FIGURE 2

AZGP1 Induces apoptotic cell death in CCA. (A) KKU‐213 and SNU‐1079 cells were transfected with AZGP1, and cell death was determined by hemocytometry. (B) Analysis of apoptosis by Annexin‐V/PI staining and flow cytometry; bar charts were drawn to quantify three separate flow cytometry experiments. (C) TUNEL assay of apoptotic KKU‐213 and SNU‐1079 cells and quantification of three independent experiments using bar charts. Scale bar: 10 μm. (D) Western blot analysis of AZGP1, cleaved PARP, cleaved caspase‐3, cleaved caspase‐9 and β‐Actin in KKU‐213 and SNU‐1079 cells with or without AZGP1 expression. *p < 0.05, **p < 0.01, ***p < 0.005 indicate significant differences from the control group.

FIGURE 3

AZGP1 and TRIM25 are associated with immunoprecipitation (IP) and immunohistochemistry (IHC). (A) AZGP1 mRNA and protein expression in CCA cells. (B) Co‐IP assay of interaction between AZGP1 and TRIM25 in HEK293T cells. (C) Box plot of TRIM25 expression obtained from the GEPIA databases. (D) The GEPIA database showed a Kaplan–Meier curve for TRIM25 high and low expression group of CCA (log‐rank p = 0.17, compared to normal controls). (E) IHC analysis of AZGP1 and TRIM25 expression in adjacent normal tissue and CCA tissue. Bar charts show quantitative data from the average of IHC scoring (Normal, n = 4; CCA, n = 63). ***p < 0.005 indicates significant differences from the normal.

FIGURE 4

AZGP1 degradation is mediated by TRIM25 PRY/SPRY domain. (A) Western blot analysis of AZGP1, TRIM25 and β‐Actin in KKU‐213 and SNU‐1079 cells with and without TRIM25. (B) Apoptotic cell death analysis of CCA cells with and without TRIM25 knockdown using Annexin‐V/PI staining and flow cytometry, and quantification of three independent flow cytometry experiments by bar charts. (C) HEK293T cells were transfected with TRIM25 or TRIM25 mutants, followed by AZGP1 IP and western blots analysis of AZGP1 and TRIM25‐HA expression. (D) HEK293T cells were transfected with the indicated plasmids. Wild‐type and mutant AZPG1 proteins were analysed by western blotting with indicated antibodies. All experiments were performed 72 h after TRIM25 knockdown in CCA cells. Bar charts show quantitative data from the average of three independent experiments. **p < 0.01 indicates significant differences from the control group.

FIGURE 5

Overexpression of AZGP1 suppresses tumour growth in vivo. (A) Representative images of mice injected with doxycycline‐induced AZGP1 expression cells. (B) The tumour growth curve of the implanted subcutaneous tumours. Tumour growth was measured twice per week. (C) The body weight of mice was measured twice per week. (D) IHC staining for AZGP1, Ki67 and cleaved caspase‐3 in doxycycline‐induced AZGP1 expression cells. Three independent experiments were quantified, and data were expressed in bar charts. Scale bar: 50 μm. *p < 0.05, **p < 0.01 indicate significant differences from the control group.