Peritumoral Small EphrinA5 Isoform Level Predicts the Postoperative Survival in Hepatocellular Carcinoma

<div><h3>Background</h3><p>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).</p> <h3>Methodology/Principal Findings</h3><p>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 (<em>p = </em>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 (<em>p = </em>0.002) and overall survival (<em>p = </em>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 (<em>p</em><0.05) and migration (<em>p</em><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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

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

<p>(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 (<i>p = </i>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 <i>p-</i>values of 0.019 and 0.045, respectively.</p

Association between ephrinA5 isoforms, clinical parameters and disease-free survival/overall survival.

a<p>Kaplan-Meier analysis with log rank test.</p>b<p>Relative expression of ephrinA5 mRNA assessed by real-time RT-PCR using peritumoral liver tissues.</p><p>CI: Confidence Interval,</p>*<p>P<0.05.</p

Stepwise multivariate Cox proportional hazard model for independent predictors for postoperative survival.

<p>HR: Hazard Ratio, CI: Confidence Interval.</p

Clinical parameters of HCC patients analyzed.

<p>Clinical parameters of HCC patients analyzed.</p

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

<p>(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 <i>p</i><0.05 (*), <i>p</i><0.01 (**), or <i>p</i><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.</p