Western blot analysis revealed Par-3 protein variants of 180, 150 and 100 kDa with differential expression in all HCC cell lines (Huh-7, HepG2, Hep3B, PLC-5 and SK-Hep-1) (Figure 1). Interestingly, the poorly-differentiated HCC cells, SK-Hep-1, expressed more multiple forms of Par-3 protein variants than other well-differentiated HCC cell lines.

We examined the expression of Par-3 in paraffin-embedded primary HCC tumors with surrounding non-cancerous parenchyma from 111 patients and 31 matched extrahepatic metastatic tumors by immunohistochemical staining. Negative control slides were negatively unstained with Par-3 (Figure 2A). The expression of Par-3 was increased in 47 (42.3%) of 111 primary HCC tumors and not in non-cancerous cells adjacent to tumors (Figure 2B and Table 1). Moreover, Par-3 was overexpressed in 31 matched metastatic HCC specimens, as illustrated in brain (Figure 2C) and rectum (Figure 2D). Expression of Par-3 was not significantly related to most clinicopathological characteristics, but was associated with tumor multiplicity (p = 0.002), Alpha-fetoprotein level (p = 0.046) and subsequent extrahepatic metastasis (p = 0.037) (Table 1).

Multivariate analysis confirmed Par-3 expression as a predictor of distant HCC metastasis (p = 0.037) (Table 2). The cumulative rate of developing extrahepatic metastasis within five years with primary HCC was significantly higher with positive rather than negative Par-3 expression (40.2% ± 8.0% vs. 23.4% ± 6.0%, p = 0.047) (Figure 3). Furthermore, the expression of Par-3 was significantly increased in metastatic HCC samples than in their primary tumors (21 with increased Q-score > 2, and 10 with no difference in Q-score, p < 0.001). These observations suggest a strong association of Par-3 expression and extrahepatic metastasis of HCC.

After a mean follow-up of 52.0 ± 28.4 months after surgery, 27 patients (24.3%) remained free of HCC, 54 patients (48.6%) had died because of their disease and 30 patients (27.0%) were still alive with disease recurrence and/or distant metastasis. Survival analysis revealed a significantly better overall five-year survival with negative rather than positive Par-3 expression in primary HCC tumors (59.6% ± 6.3% vs. 41.7% ± 7.3%, p = 0.047) (Figure 4). The increased expression of Par-3 in primary tumors had no significant effect on progression-free survival in these patients (data not shown). In addition, Cox proportional-hazard regression models revealed that Par-3 overexpression was significantly associated with poor overall survival (hazard ratio 2.049, 95% confidence interval 1.082–3.884, p = 0.028), but not associated with progression-free survival (Table 3). Thus, overexpression of Par-3 in primary tumors is an important predictor of poor overall survival with HCC.

To examine whether Par-3 expression is associated with 14-3-3ɛ (Par-5), we determined the 14-3-3ɛ expression by IHC analysis. Negative control slides were unstained with 14-3-3ɛ (Figure 5A). 14-3-3ɛ was significantly overexpressed in primary (Figure 5B) and metastatic HCC tumors, as representatively illustrated in brain and rectum (Figure 5C,D). Furthermore, overexpression of 14-3-3ɛ is significantly correlated with Par-3 in primary HCC tumors (p = 0.014) (Table 1).

We retrospectively enrolled (from January 1999 to December 2001) and obtained tissue from 111 HCC patients who underwent surgery for tumor resection in Taichung Veterans General Hospital. The mean follow-up was 52.0 ± 28.4 months. In total, tissue from 31 patients (27.9%) showed metastasis 5 to 88 months after the surgery for primary HCC. The metastasis sites included bone, abdominal and chest wall, brain, mesentery, peritoneum, adrenal gland and retroperitoneum. The paraffin-embedded surgical specimens composed of the primary tumors with surrounding non-cancerous liver parenchyma and metastatic tumors underwent pathology examination. We examined pathological features, including Barcelona-Clinic Liver Cancer (BCLC) [31] staging and clinical outcomes. This study was approved by the Institutional Review Board of Taichung Veterans General Hospital.

For immunohistochemistry analysis of Par-3 expression in paraffin-embedded tissues, we used an automatic immunostaining device and ultraView detection kit (Ventana XT Medical System, Tucson, AZ, USA) with a primary rabbit polyclonal antibody for Par-3 (1:100, Millipore, Clone 07–330, Temecula, CA, USA). Detection of 14-3-3ɛ expression was performed according to [28], described previously. A negative control was incubation without the primary antibody. The intensity of Par-3 and 14-3-3ɛ proteins staining were semiquantitatively scored by a Quick-score (Q-score) method based on intensity and heterogeneity [28–30,32–35]. Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). For heterogeneity, the proportion of tumor cells positively stained with Par-3 was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%) and 4 (76%–100%). The Q-score of a given tissue sample was the sum of intensity and heterogeneity scores and ranged from 0 to 7. The scoring of each sample was evaluated independently and blindly by 2 pathologists. A Q-score ≥2 was considered to be an overexpressed or positive expression of Par-3/14-3-3ɛ, and a Q-score <2 was considered a normal or negative expression of Par-3/14-3-3ɛ. Some rare cases with <5% weakly stained specimens were considered to be a negative expression.

Huh-7, HepG2, Hep3B, PLC-5 and SK-Hep-1 human hepatoma cells were maintained in DMEM (Gibco, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (FBS; Hyclone Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin and 100 units/mL streptomycin in a humidified incubator with 5% CO₂ at 37 °C. Expression of Par-3 protein was determined by Western blot analysis. In brief, HCC cells were cultured to 90% confluence, harvested and lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA; Millipore, Temecula, CA, USA) containing cocktail protease inhibitors (Roche, Indianapolis, IN, USA). Cell lysates were clarified by centrifugation at 15,000 rpm for 20 min at 4 °C. Protein concentration was determined by use of a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). In total, 20 μg protein from each sample was applied to a gradient SDS-PAGE gel and immunoblotted onto PVDF membranes, which were blocked for 1 h in PBST (0.1% Tween 20, 2.67 mM KCl, 1.47 mM KH₂PO₄, 137.93 mM NaCl, 8.1 mM Na₂HPO₄, pH 7.4) containing 5% nonfat dry milk, then incubated with primary antibody against Par-3 (Sigma-Aldrich, St. Louis, MO, USA) overnight, washed 3 times with PBST for 5 min, then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Protein levels were determined by use of enhanced chemiluminescence reagents.

One-Way ANOVA was used to analyze differences for clinicopathological variables. Multivariate logistic regression was used to determine factors predicting extrahepatic metastasis. The Wilcoxon signed-rank test was used to analyze the differences between primary tumors and matched metastatic tissues by Par-3 staining density. Kaplan-Meier curves were plotted, and the log rank test was used to analyze time-related probabilities of metastasis, overall survival and progression-free survival. Cox proportional hazards regression models were used to evaluate the impact of prognostic factors on survival. p < 0.05 was considered statistically significant and p = 0.05 to 0.10 marginally significant.