EphrinA5L and S are Downregulated in Human Hepatocellular Carcinomas

To elucidate the biological significance of ephrinA5 alternative isoforms in HCCs, we first examined the expression of ephrinA5L and ephrinA5S. Because no specific antibodies against ephrinA5L and ephrinA5S were available, primer-specific real-time PCR was used to study the mRNA expression of ephrinA5L and ephrinA5S in 142 paired HCCs and peritumoral liver tissue. The relative expression of ephrinA5L and ephrinA5S to reference samples are shown in Figure 1A. As compared to peritumoral liver tissue, ephrinA5L and ephrinA5S were simultaneously downregulated in 96 HCCs. Four HCCs showed ephrinA5L downregulation alone. Another 3 HCCs only had downregulated ephrinA5S. Both ephrinA5L and ephrinA5S were significantly downregulated in tumor tissue as analyzed by Wilcoxon matched pair test with p-values of 0.013 and 0.001, respectively. The downregulation of ephrinA5 mRNA in HCCs indicated its potential role as a tumor suppressor.

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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.

https://doi.org/10.1371/journal.pone.0041749.g002

EphrinA5S is an Independent Prognostic Predictor for Postoperative Survival

To evaluate the clinical significance of ephrinA5 isoforms, we performed linear regression analysis in the 142 HCC samples. Clinical parameters, including patients’ gender, age, HBV or HCV carrier, cirrhosis, alcoholic liver disease, degree of vascular invasion, capsule invasion, acites formation, histology grading, tumor size, alpha-fetoprotein, albumin, bilirubin, prothrombim time, creatinine, and AST/ALT for the recruited HCC cohort were summarized in table 1. In the univariate analysis, the expression of ephrinA5S positively correlated with old age (over 55 years) and histological grade. No significant association with other clinical or pathological parameters was determined (Table 2). EphrinA5L expression showed a positive correlation with serum creatinine level, and no significant difference was found in relation to other clinical parameters (Table S1). Of interest, the Kaplan-Meier survival curve with log-rank test showed that a higher ephrinA5S expression (relative expression level >0.3) in peritumoral liver tissues had a better disease-free survival and overall survival among this HCC cohort and the p-values were 0.019 and 0.045, respectively (Fig. 1B, Table 3). In addition, HBV surface antigen (HBsAg) was also significantly associated with better disease-free survival and overall survival, and serum albumin (Alb) was positively associated with disease-free survival (Table 3). On the contrary, elder age, microvascular invasion, larger tumor size, higher serological alpha-fetoprotein (AFP), and higher AST/ALT were associated with poor disease-free survival. To determine potential independent predictors for postoperative survival, a stepwise multivariate Cox proportional hazard model was performed. Higher ephrinA5S expression in peritumoral liver tissue, positive HBsAg and higher Alb showed a reduced lethal risk to 0.42×, 0.39× and 0.40×, respectively, whereas, elder age, larger tumor size and higher AFP exhibited 1.44×, 1.75× and 2.37× poor disease-free survival risk, respectively. For overall survival, only a high ephrinA5S expression in peritumoral liver and HBsAg reduced the lethal risk to 0.34× and 0.28×, respectively (Table 4).

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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.

https://doi.org/10.1371/journal.pone.0041749.g003

Both ephrina5l and ephrina5s Suppress Proliferation and Migration in HCC Cell Lines

To determine the biological functions of two ephrinA5 isoforms during tumor progression, we expressed the ephrinA5 alternative variants in Hep3B and HepG2 cell lines. In an ACP assay, the overexpression of ephrinA5L and ephrinA5S significantly suppressed cell proliferation. The proliferation of both HepG2 and Hep3B was significantly inhibited up to approximately 32% by ephrinA5S at 72 hr as compared to those with pIRESneo vectors (p = 0.015 and p = 0.00016, Fig. 2A). EphrinA5L only marginally suppressed Hep3B growth by 12% at 72 hr (p = 0.003). EphrinA5S exhibited a stronger suppressive effect than ephrinA5L on cell proliferation (p<0.05).

Next, we investigated the integrity of the eph receptor and ephrinA5 interaction in HCCs by analyzing the expression of EphB2, A2, A3 and A5 the preferred receptors for ephrinA5, in Hep3B and HepG2 cell lines. Except for EphA5, the expression of EphB2, A2 and A3 were detected in both cell lines. A similar eph receptor expression pattern was also found in both tumor and peritumoral liver tissues (Fig. 2C). However, EphA5 was detected in only one tumoral tissue. This result suggests that ephrinA5 is able to exhibit its tumor suppressor effect through its eph receptors in HCCs.

Next, we studied the in vitro effect of ephrinA5-Fc on cell proliferation. Hep3B and HepG2 were treated with a series of concentrations of ephrinA5-Fc for 72 hrs. A significant inhibitory effect on cell proliferation was observed in both Hep3B and HepG2 cells treated with a series of concentrations of ephrinA5-Fc (p<0.05; Fig. 2B). Although there was no statistical significance between different concentrations of ephrinA5-Fc, its suppressive effect had a dose-dependent trend.

Furthermore, we also examined the effect of ephrinA5 isoforms on cell migration with Transwell assays. The migratory activity of both cell lines was reduced to 35–40% and 60–65%, by ephrinA5L (p<0.001) and ephrinA5S transfectants (p<0.001), respectively, as compared to the pIRESneo vector control (Fig. 2D). These results indicate that both L and S isoforms of ephrinA5 were involved in regulating cell proliferation and migration. EphrinA5S had a more potent suppressive effect on both cell proliferation and migration.

EphrinA5L and ephrina5s Downregulate EGFR Expression

EphrinA5L acts as a tumor suppressor by negatively regulating EGFR expression in glioma [35]. To elucidate if there was a similar regulatory mechanism for both ephrinA5 isoforms in HCC, we further examined the potential ephrinA5L and ephrinA5S suppressive effects on EGFR expression. As in Fig. 3A, overexpression of both ephrinA5 isoforms significantly reduced EGFR protein expression as compared to cells transfected with control vector pIRESneo (left panel). However, the EGFR mRNA level was not affected by any ephrinA5 variant (Fig. 3A, right panel). To address the possibility that ephrinA5 enhanced c-Cbl, the EGFR E3-ligase, to associate with EGFR and thus promote EGFR degradation, ephrinA5L and ephrinA5S was overexpressed in Hep3B cells with and without c-Cbl siRNA treatment. Hep3B cells normally expressed EGFR without detectable ephrinA5 isoforms, whereas ectopic expression of both ephrinA5L and ephrinA5S inhibited the expression of EGFR in Hep3B cells. c-Cbl siRNA treatment partially rescued the expression of EGFR in cells with ectopic expression of ephrinA5L and ephrinA5S (Fig. 3B).

Patients

The study cohort consisted of 142 patients with HCCs, who underwent surgical resection at Lin-Kou Chang Gung Memorial Hospital between 2000 and 2008. Clinical and pathological characteristics were obtained from patient charts. Tumors were staged according to the seventh edition of the American Joint Committee on Cancer, and histological grade was scored according to the World Health Organization classification criteria. This study was approved by the Ethics Committee of Chang Gung Memorial hospital and written informed consent was obtained from each patient.

Detection of ephrina5 Isoforms by Quantitative Real Time Polymerase Chain Reaction (PCR)

Total RNA was isolated by TRIzol (Invitrogen, Carlsbad, CA). After calculating the concentration of each RNA sample using a Nanodrop detector (Thermo Scientific, Wilmington, DE), RNA samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI). Two micrograms of treated RNA samples was subjected to reverse transcription with SuperScript III (Invitrogen, Carlsbad, CA). Quantitative real time PCR was processed by ABI-3700 machine using a Quantifast syber green PCR kit (Qiagen, Valencia, CA), and GAPDH mRNA was used as an internal control. Real-time PCR products were also analyzed by gel electrophoresis to confirm a single PCR product. Primer sets are listed as follows.

ephrinA5L-forward: 5′-ACCAACAAATAGCTGTATGA -3′,

ephrinA5L-reverse: 5′-TCGGCTGACTCATGTACGGT -3′,

ephrinA5S -forward: 5′-ACCAACAAATGACACCGTA -3′,

ephrinA5S -reverse: 5′-CATCGCCAGGAGGAACAGTA -3′,

GAPDH-forward: 5′-AGCCTCAAGATCATCAGCAA -3′,

GAPDH-reverse: 5′-GGCATGGACTGTGGTCATGAG -3′.

Cell Lines, siRNA and Plasmids

HepG2 and Hep3B cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere. All siRNAs targeting ephrinA5 and c-Cbl were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pIRESneo-ephrinA5, CMV-based expression, and neomycin-selective plasmids containing ephrinA5 variant cDNA, were constructed by processing with an ephrinA5 cloning primer set: Forward, 5′-CATAAGCTTCCACCATGTTGCACGTGGAGATGTT-3′; reverse 5′-ATCGGATCCTGACTCATGTACGGTGTC-3′.

Transient Transfection of pIESneo-ephrinA5 Plasmids and siRNAs

HepG2 and Hep3B cell lines were seeded in a 6-well plate at a density of 3×10⁵ cells/well overnight. The pIRESneo-ephrinA5 plasmid (0.25 µg each) or 3 µg siRNAs including si-c-Cbl or si-scrambled were added to DMEM medium with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) for transfection. Forty-eight hours after transfection, cells were harvested and subjected to RT-PCR and western blotting to detect RNA and protein levels of ephrinA5, c-Cbl, and EGFR.

Detection of ephrina5, Eph Receptors, c-Cbl and EGFR mRNA Levels by Conventional RT-PCR

Total RNA was extracted with TRIzol. Two micrograms of treated RNA samples was subjected to RT-PCR, and GAPDH mRNA was used as an internal control. PCR products were analyzed by gel electrophoresis. The primer sets are listed as followed.

EphA2-forward: 5′-TCAGCAGCAGCGACTTCGAGGCA-3′,

EphA2-reverse: 5′-CAGTGGCCAGGGAAGGTGCA-3′,

EphA3-forward: 5′-ATGTTTCCAGACACGGTACC-3′,

EphA3-reverse: 5′-CCATCTTCCTGAGTAGAACTGTGAGG-3′,

EphA5-forward: 5′-CCTTCTGTGGTACGACACTTG-3′,

EphA5-reverse: 5′-GGTCTGCACACTTGACAGGTG-3′,

EphB2-forward: 5′-ATGGCGCCCCTCTCCTCTGGCATCA-3′,

EphB2-reverse: 5′-ACCGCTTGGTTCTTCCCGTG-3′,

ephrinA5-forward: 5′-GCAATCCCAGATAATGGAAGAA-3′,

ephrinA5-reverse: 5′-TCGGCTGACTCATGTACGGT-3′,

c-Cbl-forward: 5′-CGCTAAAGAATAGCCCACCTTAT-3′,

c-Cbl-reverse: 5′-ATGGCCTCCAGCCCAGAACTGAT-3′,

EGFR-forward: 5′-CGGGACATAGTCAGCAGTG-3′,

EGFR-reverse: 5′-GCTGGGCACAGATGATTTTG-3′,

GAPDH-forward: 5′-TGCACCACCAACTGCTTAGC-3′,

GAPDH-reverse: 5′-GGCATGGACTGTGGTCATGAG-3′.

Detection of ephrina5, c-Cbl and EGFR Protein Levels by Western Blot Analysis

Transfected cells were washed twice with PBS, then lysed in 200 µl of RIPA lysis buffer with protease inhibitors, and protein concentrations were determined using the Bradford Reagent (Bio-Rad, Hercules, CA). One hundred micrograms of protein from the supernatant was loaded onto an SDS-polyacrylamide gel, followed by western blot analysis to detect protein level by ephrinA5 (Abcam, Cambridge, UK), c-Cbl (Santa Cruz Biotechnology), β-actin (Novus Biologicals, Littleton, CO) and EGFR (Santa Cruz Biotechnology) antibodies. The intensity of each band was quantified by ImageQuant 5.2 (GE Healthcare, Piscataway, NJ).

Cell Growth Determined by ACP Assay and MTT Assay

Cells transfected with ephrinA5 plasmid or shRNA were washed twice with PBS and subjected to an acid phosphatase assay [50], to detect the proliferation rate. In the MTT assay, cells were treated with 1 µg/ml, 2 µg/ml and 5 µg/ml ephrinA5-Fc (Sigma-Aldrich Inc., St Louis, MO), respectively, and analyzed at the indicated time points.

Cell Migration Assay

The migratory ability of HepG2 and Hep3B cells was assessed by ThinCert™ Tissue Cell Culture Inserts (Greiner bio-one, Monroe, NC) with an 8 µm pore size membrane. Cells were suspended in a final concentration of 5×10⁵ cells/ml. The lower chambers were filled with 500 µl complete medium (DMEM supplemented with 10% FBS), and 100 µl of the cell suspension were loaded into each upper chamber substantially. The cultures were incubated in a humidified 5% CO₂ incubator at 37°C for 24 hours. The cells were fixed with 500 µl methanol for 15 minutes, and then the inner surface of the upper chambers was wiped with cotton swabs to remove the nonmigrating cells. The membranes were washed with 500 µl PBS and stained with 500 µl hematoxylin for 20 minutes at room temperature. The membranes were then washed again with 500 µl PBS. The stained cells were imaged by ImagePro 6.2 software and five random fields were counted at 100× magnification.

Statistical Analyses

A Wilcoxon matched pair test was used to analyze the significance of ephrinA5L and ephrinA5S expression in the paired HCC tissues and suppression of cell proliferation in the MTT assay. Univariate analysis was used to analyze the expression of ephrinA5 isoforms in relation to clinical parameters. Kaplan-Meier survival curves were used to see the differences between disease-free survival and overall survival, and the significance differences between the survival curves were calculated by using log-rank test. Multivariate survival analysis was carried out by using the Cox hazard regression model. Correlation coefficients between all findings were calculated using Pearson correlation. Student’s t-test was used to analyze continuous variables in the western blot, ACP and migration assays. All tests were two-sided and a p-value <0.05 was considered statistically significant. All analyses were performed by using SPSS 16.0 or Excel 2007.