Low expression of plakoglobin in intratumoral liver tissue is associated with poor survival after curative resection of Hepatocellular Carcinoma

Background: Plakoglobin also known as γ-catenin, is a member of the Armadillo family. It’s a structural and functional homologue of protein β-catenin with dual functions in cell-cell adhesion and cell signaling. However, the role of plakoglobin in hepatocellular carcinoma (HCC) is unclear to date. Our aim was to investigate the expression of plakoglobin in HCC and its correlation with prognosis and then to further study the underlying function. Methods: Western Blot and qRT-PCR were used to examine the expression of plakoglobin in HCC cell lines and normal hepatocyte cell line. Immunohistochemical method was used to analyze the expression and relationship of plakoglobin in tissue microarray with 191 HCC patients through Kaplan-Meier analysis. Results: Patients with high expression of plakoglobin had a better survival compared with those with low expression (OS, P=0.002; DFS, P=0.012). Plakoglobin is an independent prognostic factor for patients (OS, P=0.026, DFS, P=0.045). Plakoglobin was inversely consistent with the metastatic potential ability of HCC cell lines. Conclusions: Our founding indicates plakoglobin is an independent prognostic marker of HCC, and patients with low expression of that had a poor survival. Plakoglobin may serve as a potential target for anti-HCC.


Introduction
Plakoglobin (also known as γ-catenin), discovered more than 30 years ago as an adhesion protein associated with the cell membrane, is a member of the Armadillo family of proteins [1]. Plakoglobin is a structural and functional homologue of protein β-catenin, containing a central armadillo repeat domain [2]with dual functions in cell-cell adhesion and cell signaling [3,4]. Both plakoglobin and β-catenin could interact with the cytoplasmic domain of cadherins, such as E-cadherin, tethering the cadherincatenin complex to the cytoskeleton, though plakoglobin was found to be only loosely associated with Ecadherin [3]. In addition, several evidences suggest that plakoglobin has signaling ability similar to βcatenin when it's not sequestered at the plasma membrane as a part of the desmosomes, plakoglobin is capable to participate in cell signaling [5,6]. However, unlike β-catenin that has a well-de ned oncogenic potential as the terminal component involved in the Wnt signaling pathway [7,8], plakoglobin is generally considered as a tumor or metastasis suppressor [9,10], although its role in cancer is still controversial [3].
Recent some studies suggest that plakoglobin acts as a tumor-suppressor protein in some tumors, such as lung primary cancer, prostate cancer and esophageal cancer, its expression in these cancers is very low or completely absent [11,12]. Whereas other studies suggest that plakoglobin has a tumor-inducing effect or oncogenic activity [13][14][15]. It is possible that plakoglobin may have different roles in different malignancies. Recently, an immunohistochemical study revealed that 58% of human hepatocellular carcinoma (HCC) cases showed that reduced levels of E-cadherin associated with a declined expression of p120 catenin, α-and β-catenin and plakoglobin in a comparable percentage of HCC patients [16].
Additionally, our previous study revealed that β-catenin could be activated by hypoxia in HCC and contributed to enhance the metastatic potential and poor prognosis [17]. In this situation, to investigate exact role of plakoglobin involved in HCC is an outstanding issue.
HCC is one of the most common type of primary tumors in the liver with increasing incidence. It is the second most common cause of cancer death in men and sixth-greatest cause of cancer death in women worldwide [18]. Only about 20%-30% of HCC patients are curable by surgical resection with a reported 5year 40%-50% survival rate and a high incidence of postoperative recurrence rate [19,20], most patients diagnosed HCC are not candidate for curative treatments [21]. Postoperative recurrence may occur as a result of intrahepatic metastasis or de novo tumor occurrence [22]. Therefore, understanding the mechanism of tumor recurrence after curative resection is crucial for relieving this global health burden and improving the outcome of HCC patients. Metastasis and tumorigenesis is a multi-step process that the tumor cells acquire the ability to degrade the basement membrane and move from the primary site to distant sites forming the secondary tumors, which is the major cause of recurrence and cancer-related deaths. During this process, the intercellular adhesive complexes (mainly called adherens junctions and desmosomes) do limit the abnormal proliferation, migration and invasion and maintain the tissue integrity [23,24]. However, loss of the desmosomal adhesion is a precondition for the epithelialmesenchymal transition, indicated that the conversion of early stage primary tumors to invasive cancers.
In this study, to investigate the signi cance of plakoglobin in HCC patients' prognosis, we detected the expression of plakoglobin and analyzed the correlation between its expression and clinic pathological parameters in HCC patients. Moreover, we evaluated the expression of plakoglobin in several human HCC cell lines. Our results support the hypothesis that plakoglobin may act as a tumor suppressor in HCC.

Materials And Method
Patients and specimens In our study, a total of 191 HCC specimens were collected randomly from patients who underwent radical resection of pathologically con rmed tumors from 1999 to 2006 at the Liver Cancer Institute and Zhongshan Hospital (Fudan University, Shanghai, China). No patients received any preoperative anticancer treatment. The ethics committee of Zhongshan Hospital approved this research. All patients provided written informed consent to participate in this study.
Tissue microarray (TMA) and Immunohistochemistry (IHC) A tissue microarray (TMA) was constructed as described previously [25]. Brie y, two cores were taken from each representative tumor tissue and from peritumoral liver tissue (in collaboration with Shanghai Biochip Company Ltd, Shanghai, China). Duplicate cylinders from two different areas, intratumoral and peritumoral (a total of four punches for each patient), were obtained. The rabbit anti-plakoglobin (Cat. No. A303-718A, CST) was used in the immunochemistry to investigate the expression. The density of positive staining was measured with the use of a computerized image system composed of a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, United Kingdom).

Western Blot
Cells were lysed using cell lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100) containing protease and phosphatase inhibitors. Equivalent amounts of whole cell extracts were subjected to SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with respective primary antibody overnight at 4°C, followed by the incubation of the appropriate HRP-conjugated secondary antibody for 2 h at room temperature. Blots were visualized with an ECL detection kit (Pierce, IL) and analyzed using Quantity One 1-D Analysis Software (Bio-Rad, San Francisco, CA).

Statistical analysis
Data analysis was performed with SPSS 20.0 for Windows (SPSS Inc. Chicago, IL, USA). Unpaired Student's t-test or Pearson's χ² test was used to compare quantitative variables. Kaplan-Meier analysis was used to determine the survival and recurrence, the Cox regression model was used to perform multivariate analysis. P<0.05 was considered statistically signi cant.

Patient characteristics and intratumoral plakoglobin expression
We summarized the 191 HCC patient (160 males, 31 females) characteristics in Table1. In this study, basic clinicopathological features, including age, gender, hepatitis B surface antigen (HBs-Ag), liver cirrhosis, a-fetoprotein, tumor size, tumor encapsulation, microvascular invasion, tumor differentiation and TNM stage were collected (Table1). According to the expression of plakoglobin in intratumoral tissues, these patients were classi ed into the high and low group, we investigated the relationship between plakoglobin expression and clinicopathological features. The results revealed that plakoglobin was statistically signi cant association with age, gender, HBs-Ag (P=0.017), microvascular invasion (P=0.014) and TNM stage (P=0.040 and P=0.027). However, none of the other clinicopathological features such as alpha-fetoprotein (AFP), liver cirrhosis, tumor size, encapsulation or differentiation was correlated with plakoglobin expression in intratumoral tissues.

Low expression in HCC patients showed poor prognosis
We analyzed tissue from 191 patients with HCC using immunohistochemistry (IHC). Plakoglobin staining was mainly on the plasma membrane of tumor cells or hepatocytes ( Figure 1A), the results revealed that patients with high expression of plakoglobin had a better overall survival (OS, P=0.002) and a better disease free survival (DFS, P=0.012) ( Figure 1B). There were no statistically signi cant associations between the peritumoral plakoglobin expression and the OS (P=0.145) and DFS (P=0.377) rate after radical surgical resection of HCC ( Figure 1C). In addition to plakoglobin expression, univariate analysis revealed that AFP-1 (P=0.033), tumor differentiation (P=0.005), tumor size (P<0.001) and microvascular invasion (P<0.001) were associated with OS rate of patients with HCC. However, only the tumor size (P=0.022) and microvascular invasion (P=0.003) were associated the DFS of HCC patients (Table 2). Multivariate Cox's proportional Hazard model analysis suggested that high expression of plakoglobin as an independent prognostic factor for the OS (P=0.026) and DFS (P=0.045) rates of HCC patients (Table   3). Besides, microvascular invasion was also an independent prognostic factor for HCC. Moreover, we detected the expression of plakoglobin by western blot in four pairs tumor and matched peritumoral tissues randomly collected from four patients ( Figure 1D). The results showed that the expression of plakoglobin was lower in tumor tissues than that in peritumoral tissues.

Expression level of plakoglobin in HCC cell lines
We detected the expression of plakoglobin mRNA by qPCR in four HCC cell lines (Figure 2A), and found that the expression level of plakoglobin mRNA was higher in L02 cell, and lower in HCC cell lines; moreover, the expression level of plakoglobin was inversely consistent with the metastatic potential ability of HCC cell lines. Furthermore, we detected the expression level of plakoglobin by western blot analysis in the above-mentioned ve cell lines ( Figure 2B), which showed that the result was consistent with that detected by qPCR.

Discussion
Plakoglobin was found more than thirty years ago as an adhesion protein associated with the cell membrane, it's a homologue of protein β-catenin. Although the role of β-catenin, an important component of the canonical Wnt signaling pathway, is well understood in various aspects of liver pathology, there is little information on the role of plakoglobin. The most documented role of plakoglobin within the cell is in cell-cell adhesion as a desmosomal protein. Recent emerged evidence has demonstrated that plakoglobin has novel roles in intracellular signaling and the regulation of gene expression. Similar to β-catenin, plakoglobin has two major roles in the cell: the mediation of cell-cell adhesion and cellular signaling, sometimes replace β-catenin for its function in the Wnt signaling pathway [27]. Despite some reports revealed that plakoglobin expression induced the mesenchymal-epithelial transition (MET) and suppressed tumorigenesis and metastasis, the underlying mechanisms that could account for its function activities had remained unclear, though the induction of MET by plakoglobin may occur via a common pathway involved β-catenin [28]. Some studies have suggested a tumor promoting activity of plakoglobin in malignancies; while others suggested it acted as a tumor suppressor, plakoglobin loss correlates with tumor progression, metastasis and poor prognosis. Loss of plakoglobin is thought to enhance the β-catenin signaling due to reduced competition for Lef/Tcf binding [29]. In breast cancer, Holen et al. found that plakoglobin acted as a tumor suppressor, loss of plakoglobin promoted the decreasing of cell-cell contact, and increased the invasive behavior of breast cancer cells in vivo [30]; however, the results of Aceto et al. suggested that plakoglobin acted as a metastasis promoter in breast cancer circulating tumor cells [14]. The role of plakoglobin in solid tumors existed controversy. While the role of plakoglobin in hepatocellular carcinoma had remained unclear.
In our study, we preliminary explored the expression of plakoglobin and its relationship with HCC. Our results revealed that HCC patients with high expression of plakoglobin had a better prognosis than those with low expression, and the expression of plakoglobin in peritumoral tissues showed no signi cance with the prognosis. To some extent, our results were consistent with some reported studies, suggesting that plakoglobin acted as a suppressor in hepatocellular carcinoma. Although the precise mechanisms of plakoglobin function on hepatocellular carcinoma development need to be further investigated, our study con rms the value of plakoglobin in the prediction of prognosis and the development of new therapeutic interventions.

Conclusions
In conclusion, our founding indicates plakoglobin is an independent prognostic marker of HCC, and patients with low expression of that had a poor survival. Plakoglobin may serve as a potential target for anti-HCC. Abbreviations HCC, hepatocellular carcinoma; MET, mesenchymal-epithelial transition; TMA, tissue microarray; HBs-Ag, hepatitis B surface antigen Declarations Ethics approval and consent to participate Zhongshan Hospital Research Ethics Committee approved the present study. Written informed consent for the use of their tissue and clinicopathological data was obtained from all patients.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interest
The authors declare that they have no con icts of interest.   Abbreviations: AFP: a-fetoprotein; OS, overall survival; DFS, disease-free survival.