High expression and potential synergy of human epididymis protein 4 and Annexin A8 promote progression and predict poor prognosis in epithelial ovarian cancer

Abstract

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-related deaths. Aberrant expression of human epididymis protein 4 (HE4) and Annexin A8 (ANXA8) plays crucial roles in some malignancies; however, their functions in EOC remain unclear. In this study, we utilized immunohistochemistry, real-time PCR, western blotting, immunofluorescence labeling, and gene interaction and enrichment pathway analyses to explore the roles of HE4 and ANXA8 in EOC. They were highly expressed in EOC tissues, which significantly correlated with higher tumor burden, advanced FIGO stages, poor differentiation, presence of > 1 cm residual tumor, and tumor recurrence. The expression patterns of HE4 and ANXA8 were similar, and Spearman's correlation analysis showed that they were positively correlated (r=0.671, P < 0.001). Large sample database analyses also showed significant positive correlation between their mRNA expression (R=0.304, 0.321, and 0.304 in TCGA, CCLE and GTEx, respectively, all P < 0.001). Kaplan-Meier survival analysis demonstrated that advanced FIGO stages, lymph node metastasis, residual tumor size > 1 cm, and high HE4 and ANXA8 expression were significantly associated with poor overall survival (all P < 0.05). Moreover, multivariate Cox analysis showed that advanced FIGO stages and HE4 expression were independent factors for poor survival (P < 0.001, 0.012, respectively). Interaction network analysis of genes associated with ANXA8, expressed in response to HE4, revealed that these genes participated in TP53 expression, autophagy regulation, and the PID FOXO pathway. In conclusion, the potential synergy between HE4 and ANXA8 may exacerbate the disease condition. Thus, targeting HE4 and ANXA8 could be a novel therapeutic strategy for ovarian cancer.

Keywords: ANXA8; HE4; epithelial ovarian cancer; prognosis.

Figure 1

The expression of HE4 and ANXA8 in different groups of epithelial ovarian tissues and their correlation analysis. A. Immunopositivity for HE4 and ANXA8 are represented by brown staining in epithelial ovarian cancer, epithelial borderline ovarian tumor, epithelial benign ovarian tumor, and normal ovarian tissues. Scale bars: lower, 100 μm; upper, 50 μm. B. The correlation between HE4 and ANXA8 expression, as detected by IHC staining in EOC patient samples and pooled ovarian samples, by Spearman correlation analysis. C. Correlation of WFDC2 with ANXA8 gene in expression by Pearson correlation analysis of database on cancer samples (TCGA), cancer cell lines (CCLE), and normal tissues (GTEx). Note that every dot in the TCGA and GTEx dataplot represents one cancer type or one tissue type.

Figure 2

Correlation of clinicopathological factors and expression of HE4 and ANXA8 with the prognosis of patients with epithelial ovarian cancer. A. Overall survival curve for all the ovarian cancer patients, the 5-year survival rate and median survival time are shown; Kaplan-Meier survival analysis showing that late FIGO stages, lymphatic node metastasis, residual tumor size > 1 cm and high expression of HE4 and ANXA8 are correlated with poor OS (LogRank: all P < 0.01). B. Cox-proportional hazard model analysis of factors affecting patient’s overall survival for clinicopathological variables and the IHC expression in 130 patients of EOC. Variables with P < 0.05 in the univariate analysis were included in the multivariate analysis. Abbreviation: HE4, Human epididymis protein 4; ANXA8, Annexin A8.

Figure 3

The correlation between HE4 and ANXA8 expression in epithelial ovarian cancer cells and gene network analysis of ANXA8-associated genes. A. Real-time PCR results showing the expression of HE4 and ANXA8 after HE4 gene transfection in ovarian cancer cell line ES-2. B. Western blot results showing the expression of HE4 and ANXA8 after HE4 gene transfection in ovarian cancer cell line ES-2. C. Double-labeling immunofluorescence showing the colocalization of HE4 and ANXA8 in ovarian cancer ES-2 cells. HE4 (green, a), ANXA8 (red, b), the nucleus (blue, c), and merged image (d) are shown. Scale bar=50 μm. D. Gene network was generated by the IPA^® platform, the genes marked with red and green represent the upregulated and downregulated genes, respectively, dotted lines indicate indirect interactions and solid lines indicate direct interaction between the genes. E. The enriched pathway and biological process analysis was conducted by Metascape analysis.