Peritumoral small ephrinA5 isoform level predicts the postoperative survival in hepatocellular carcinoma

Abstract

Background: EphrinA5, a member of Eph/Ephrin family, possesses two alternative isoforms, large ephrinA5 isoform (ephrinA5L) and small ephrinA5 isoform (ephrinA5S). EphrinA5L is a putative tumor suppressor in several types of human cancers. However, the role of ephrinA5S in hepato-carcinogenesis remains unclear. In this study, we evaluate the role of ephrinA5 isoforms in human hepatocellular carcinomas (HCC).

Methodology/principal findings: A total of 142 paired HCCs and peritumoral liver tissue was examined for relative expression of ephrinA5L and ephrinA5S by using quantitative real-time polymerase chain reaction. We analyzed their expression in relation to clinical parameters, disease-free survival and overall survival. Functional assays were performed to dissect the possible underlying mechanisms. Both ephrinA5L and ephrinA5S were significantly downregulated in HCCs, as compared to those in peritumoral tissue (p = 0.013 and 0.001). Univariate analysis demonstrated that ephrinA5S was positively correlated with old age and histological grade. In multivariate analysis, high ephrinA5S expression in peritumoral tissue had better disease-free survival (p = 0.002) and overall survival (p = 0.045) in patients with HCC after surgical resection. Functional analysis in HCC cell lines revealed that ephrinA5S had a more potent suppressive effect than ephrinA5L on cell proliferation (p<0.05) and migration (p<0.01). Furthermore, forced expression of both ephrinA5 isoforms in HCC cell lines significantly down-regulated epidermal growth factor receptor (EGFR) expression by promoting c-Cbl-mediated EGFR degradation.

Conclusions/significance: EphrinA5S might be a useful prognostic biomarker for HCCs after surgical resection. EphrinA5, especially ephrinA5S, acts as a tumor suppressor in hepatocarcinogenesis. Peritumoral small ephrinA5 isoform level could determine the postoperative survival in hepatocellular carcinoma.

Figure 1. Relative expression of ephrinA5L and ephrinA5S and its relation to disease-free survival and overall survival.

(A) RNA of 142 paired human HCC tissues was extracted and subjected into primer-specific real-time PCR to detect the expression of ephrinA5L and ephrinA5S. Both ephrinA5L and ephrinA5S were significantly downregulated in tumor as compared with normal tissues (p = 0.013 and 0.001). (B) Kaplan-Meier curve for the disease-free survival and overall survival of HCC patients with high and low ephrinA5S expression. The disease-free survival and overall survival were significantly different in log-rank test with p-values of 0.019 and 0.045, respectively.

Figure 2. EphrinA5 isoforms suppress cell proliferation and migration.

(A) Cells were transfected with 1 µg of pIRESneo-ephrinA5 isoforms or pIRESneo vector, and analyzed at the indicated times by ACP assay. Ectopic expression of ephrinA5 significantly inhibited cell growth in both HepG2 and Hep3B cells as compared to the vector control at 72 hrs. The level of significance was set at p<0.05 (*), p<0.01 (**), or p<0.001 (***). EphrinA5S exerted a stronger suppressive effect than ephrinA5 on both cell lines HepG2 and Hep3B. (B) Cells treated with 3 concentrations of ephrinA5-Fc were analyzed at the indicated time points by MTT assay. EphrinA5-Fc significantly reduced cell proliferation of HepG2 and Hep3B (p<0.05). (C) Expression patterns of Eph receptors in hepatoma cell lines. Primary HCCs and paratumoral tissues were analyzed by RT-PCR. EphB2, A2 and A3 expressed in both cell lines and all human HCC tissues analyzed. (D) Cell migration was compared between HepG2 and Hep3B cells transfected with pIRESneo-ephrinA5 isoforms and vector control. 5 × 10⁴ cells were plated in Transwell inserts and cultured for 24 hr in triplicates. Data were analyzed with Student’s t-tests. Ectopic expression of ephrinA5 significantly decreased the cell migratory ability of both cell types. The level of significance was set at p<0.05 (*), p<0.01 (**), or p<0.001 (***). EphrinA5S also had a stronger inhibotory effect on cell migration.

Figure 3. EphrinA5 isoforms suppressed EGFR expression by enhancing c-Cbl-mediated EGFR degradation.

(A) Both ephrinA5L and ephrinA5S reduced EGFR protein expression level in Hep3B cells. Ectopic ephrinA5 reduced endogenous EGFR protein expression (left panel) but had no transcriptional modification of EGFR in RT-PCR (right panel). The differences were statistically significant between the treated group and untreated group. Experiments in each group were performed in triplicate. The level of significance was set at p<0.05 (*), p<0.01 (**), or p<0.001 (***). (B) Ectopic expresison of ephrinA5L and ephrinA5S reduced endogenous EGFR protein expression in Hep3B cells, which was rescued after c-Cbl knockdown by siRNA.