p53 and ANXA4/NF‑κB p50 complexes regulate cell proliferation, apoptosis and tumor progression in ovarian clear cell carcinoma

Abstract

Annexin IV (ANXA4) is highly expressed in ovarian clear cell carcinoma (OCCC); however, its underlying molecular mechanism in OCCC remains unknown. The present study aimed to identify the molecule that ANXA4 may act on and to determine its underlying molecular mechanism. Immunohistochemistry, co‑immunoprecipitation and western blotting were performed to detect the expression and interaction of ANXA4, and its associated proteins. Furthermore, MTT assay, flow cytometry, western blotting and gene expression profile enrichment analysis were performed to identify the potential role and molecular mechanism of ANXA4 in OCCC. The results demonstrated that ANXA4 and nuclear factor‑κ‑light‑chain‑enhancer of activated B cells (NF‑κB) p50 nuclear expression levels were significantly higher in OCCC tissues compared with other subtypes of ovarian cancer, such as serous and mucinous. In addition, a significantly positive correlation was observed between ANXA4 and NF‑κB p50 expression in OCCC; however, the expression levels of mutant p53 and ANXA4 were negatively correlated in a linear manner. These results suggest that ANXA4 and NF‑κB p50 may be potential independent risk factors for poor prognosis. ANXA4 and NF‑κB p50 were demonstrated to interact and their expression was co‑localized. The cBioPortal database was used to construct a protein‑protein interaction network between ANXA4, NF‑κB p50 and p53, and functional pathway analysis indicated that the genes were predominantly enriched in the cell cycle and during apoptosis. Transfection of the ANXA4 gene increased the expression of NF‑κB p50, as well as its downstream targets, Cyclin D1 and B‑cell lymphoma‑2 (Bcl‑2). Furthermore, transfection of the ANXA4 gene increased proliferation and decreased apoptosis of OCCC cells. Treatment with the NF‑κB inhibitor, BAY 11‑7082, decreased Cyclin D1 and Bcl‑2 expression levels. Collectively, the results of the present study suggest that wild p53 activates ANXA4 transcription, promotes its expression and enhances NF‑κB p50 and ANXA4 interaction. This in turn activates the NF‑κB signaling pathway, promotes cell cycle progression and inhibits apoptosis, thus contributing to the malignant progression of OCCC. Thus, ANXA4 and NF‑κB p50 may be used as prognostic biomarkers, and may be molecular therapeutic targets in OCCC.

Figure 1

Nuclear expression of ANXA4 and NF-κB p50 in different ovarian tissues. Immunohistochemistry was performed to detect ANXA4 and NF-κB p50 expression in normal ovarian tissues and tissues from different subtypes of ovarian cancer (magnification ×400). OCCC, ovarian clear cell carcinoma; ANXA4, Annexin IV; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells.

Figure 2

The association between ANXA4 and NF-κB p50 expression on the survival time of patients with ovarian clear cell carcinoma. Kaplan-Meier survival analysis demonstrated that high expression levels of ANXA4 and NF-κB p50 were independent risk factors for overall survival. ANXA4, Annexin IV; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells.

Figure 3

Expression levels of ANXA4, NF-κB p50 and mutant p53 in ovarian clear cell carcinoma tissues (magnification ×400). ANXA4, Annexin IV; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells.

Figure 4

Interaction between ANXA4 and NF-κB p50 in OCCC cell lines. (A) Co-immunoprecipitation analysis was performed to determine the association between ANXA4 and NF-κB p50. (B) Double-labeling immunofluorescence analysis demonstrated co-localization of ANXA4 and NF-κB p50 in OCCC cells. (C) Western blot analysis was performed to detect ANXA4 and NF-κB p50 expression following transfection of the ANXA4 gene. (D) Immunocytochemistry analysis was performed to detect ANXA4 and NF-κB p50 expression following transfection of the ANXA4 gene. ^*P<0.05. ANXA4, Annexin IV; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells; OCCC, ovarian clear cell carcinoma; IB, immunoblotting; IP, immunoprecipitation; NOT, anti-IgG antibody (negative control).

Figure 5

Protein-protein interaction network on ANXA4, NF-κB p50 and TP53 was constructed using Cbioportal database. Each node represents a protein, while the edges between the nodes represent the interaction between the proteins. The thickness of the line correlates to the strength of the association. The stronger the interaction between proteins, the more connectivity, the bigger the nodes and the darker the color.

Figure 6

GO and signaling pathway enrichment analyses. (A) Signaling pathway bubble map. (B) Functional bubble map of gene enrichment in protein-protein interaction networks. The size of the bubble represents the number of genes in the signaling pathway or the number of genes involved in the function. Color represents P-value, the darker the color the more significant the result. GO, gene ontology.

Figure 7

Effects of ANXA4 and NF-κB p50 on cell proliferation and apoptosis. (A) Expression changes of Cyclin D1 and Bcl-2 in ES-2 cells before and after transfection of the ANXA4 gene, and addition of the NF-κB inhibitor. (B) Proliferation capacity of ES-2 cells before and after addition of the NF-κB inhibitor, detected via the MTT assay. (C) Apoptosis of ES-2 cells before and after addition of the NF-κB inhibitor, detected via Annexin V-APC/PI double-staining. ^*P<0.05. ANXA4, Annexin IV; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells; Bcl-2, B-cell lymphoma 2. ^*ES-2 compared with ES-2-A4-O; #ES-2-A4-O compared with ES-2-A4-O + BAY 11-2078.