Expression Analysis of Aquaporin-1 ( Aqp-1 ) in Human Biliary Tract Carcinoma

Background: Aquaporins (AQPs) are important in controlling bile water secretion. AQP is related to the invasion and metastasis of cancer. However, the relationship of biliary tract cancer is not clear. The role of AQP-1 in cancer cell is also unknown. Methhods: We analyzed AQP-1 expression using tissue microarray (TMA) in 99 samples immunohistochemically (50 gallbladder carcinoma, 39 bile duct carcinoma and 10 Papilla Vater carcinoma patients who underwent surgery at our department from 1997 to 2011). Gene expressions were evaluated by the combination of the immunohistological intensity and distribution. The expression level is compared to the clinico-pathological data of the patients. Results: In the TMA, depth of tumor invasion and histological type are associated with AQP-1 expression. The group of patients with high AQP-1 expression is associated with higher rates of disease specific survival (log-rank p = 0.013). Cox’s proportional hazard model reveals that AQP-1 expression is an independent prognostic factor (RR, 0.324; p = 0.001) in multivariate analysis. There is a correlation between AQP-1 expression and tumor invasion. Conclusions: These observations of this study suggest that AQP-1 expression may be favorable biomarkers associated with prognosis and tumor invasion in biliary tract carcinoma.


Introduction
Biliary tract carcinoma (BTC) is composed of mutated epithelial cells that originate in the bile ducts, which drain bile from the liver into the small intestine.BTC includes carcinomas of the bile duct, gallbladder, and pa-pilla of Vater.Most patients with BTC present at an advanced disease stage; thus, prognosis remains poor despite the recent development of new diagnostic modalities [1] [2].Currently, there is no consensus on a universal measure of BTC malignancy; therefore, it is necessary to identify prognostic factors indicating the biological properties of this disease.
Aquaporins (AQPs) are integral membrane proteins that facilitate the movement of water and play important roles in the control of bile formation.However, the exact role of AQPs in human biliary tract carcinogenesis has not been defined [3]- [5].AQP-1 and AQP-4 have also been implicated in the absorption of water by the intrahepatic bile duct [6] [7].Epithelial cells of the human and mouse gallbladder express AQP-1 and AQP-8 [8] [9], with AQP-1 localized on both the apical and basolateral plasma membranes of epithelial cells lining the neck of the organ [8].In addition, AQPs are reportedly distributed within cells lining the mammalian biliary tract [8] [9].

Methods
We analyzed AQP-1 expression in BTC using tissue microarray (TMA) and identified correlations among the clinicopathological parameters, and patient survival.In this study, we assessed paraffin-embedded tissues of 99 BTC samples (50, 39, and 10 from gall bladders, bile ducts, and papilla of Vater, respectively) collected from patients who had undergone surgery from 1997 to 2011 at Toyama University Hospital (Toyama, Japan).This study was approved by the Ethics Committee, University of Toyama.All samples were histologically diagnosed at the Department of Pathology.The final stage of BTC was pathologically confirmed, according to the TNM classification system of malignant tumors by the Union for International Cancer Control (seventh edition).CEA, CA19-9, tissue type one by each one cases, was not able to confirm the inspection data.Expression profiles of AQP-1 were analyzed using the TMA, as described previously [10] [11].The protein expression profiles were evaluated by combining immunohistological intensity and distribution.Selected micrographs from the TMAs immunostained with polyclonal antibodies against AQP-1 are shown in Figure 1.The following primary antibodies were used: rabbit polyclonal anti-AQP-1 (H-55: dilution, 1:100; Santa Cruz Biotech, Santa Cruz, CA, USA).Goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin-G was used as a secondary antibody, according to the manufacturer's instructions.The secondary antibodies were visualized using En Vision™ + Dual Link, Single Reagent (K4061; Dako, Tokyo, Japan), according to the manufacturer's instructions.The staining intensity of carcinoma cells was scored on a 4-point scale: 0 = no staining of carcinoma cells, 1 = weak staining, 2 = moderate staining, and 3 = marked staining, as compared to the staining of control tissues.The staining distribution within the tumor cells was graded on a 3-point scale: 0: n ≤ 10%; 1: 10% ≤ n < 50%; and 2: n ≥ 50%.AQP-1 expression in the carcinoma tissue was defined as positive when the sum total of the staining intensity and distribution was graded at a score of ≥ 3 (Figure 1).Using the Ki67 (MIB-1)-labeling index, the malignancy grade of BTC was rated on a 2-point scale (≤10 and >10%).
Statistical analysis was performed using the chi-square test and t-tests.Prognostic factors were examined by both univariate and multivariate analyses.Survival curves were estimated using the Kaplan-Meier method and differences between survival curves were analyzed using the log-rank test.Multivariate analyses were performed using the Cox proportional hazards model to assess the risk of cancer death.A p value of <0.05 was considered statistically significant.All statistical analyses were performed using JMP software for Windows (SAS Institute Inc., Cary, NC, USA).

Results
The clinicopathological backgrounds and univariate analysis of factors related to survival in patients with BTC are shown in Table 1.Depth of tumor invasion, lymph node metastasis, distant metastasis, histological type, CEA and CA19-9 were associated with prognosis.AQP-1 expression was also associated with disease-specificsurvival (p = 0.001).
Correlations between patient characteristics and expression patterns of AQP-1 in BTC are shown in Table 2. TMA analysis of the 99 tissue samples showed that AQP-1 expression was significantly associated with depth of tumor invasion and histological type (p = 0.021 and 0.005).Kaplan-Meier analysis revealed that the group of patients with high AQP-1 expression were associated with higher rates of disease specific survival (log-rank test,  p = 0.013) (Figure 2).Cox's proportional hazard model revealed that AQP-1 expression was an independent prognostic factor (RR, 0.324; p = 0.001) in multivariate analysis (Table 3).Multivariate analysis showed that Lymph node metastasis, Distant metastasis and CEA (p = 0.035, <0.001 and 0.027) were also associated with survival (Table 3).
Recently, various studies have focused on the association of AQPs with carcinoma and reported that several types of cancer express AQP-1, which may be involved in carcinogenesis and tumor progression [14]- [19].These observations suggested a potential role of AQP-1 in BTC.There was no difference in AQP-1 expression between carcinomas of the gallbladder, bile duct carcinoma, and papilla of Vater.AQP-1 is expressed in the biliary epithelium and its expression decreases with the degree of invasion of the carcinoma.This conclusion is also evident in the literature [20].AQPs are distributed in the pancreas and biliary tract and are essential for secretion and reabsorption of water in the bile and pancreatic juice.AQP-1 is strongly expressed in the intercalated ducts in humans and maintains cellular integrity to protect tissues against cancer cell invasion [6] [13].
Overexpression of AQP-1 was associated with increased proliferation and migration in colorectal and nonsmall cell lung carcinomas.The results of this study suggested that AQP-1 plays a different role in BTC, such as facilitation of bile transport and reabsorption.The roles of the biliary epithelium in BTC differ from those in cancers of the colon and lung [17]- [19].Therefore, AQP-1 expression in BTC may be an indicator of tumor cell invasion and proliferation associated with carcinoma progression.
Epithelium-mesenchyme transition (EMT) is a specialized mechanism in which the character of the tissue is undifferentiated.Previous studies have suggested that EMT through AQPs greatly contributes to regulation of malignancy [21].Involvement in the invasion and metastasis of a hypercoagulable state of adenocarcinoma through loss of cell-cell adhesion is mediated by the loss of AQP-1 function.The activation of molecules related to epithelial stromal migration and cell proliferation has been suggested.Bordering on this stage, there exists the possibility for acceleration of metastatic potential.Controlling AQP-1 may contribute to prognosis extension in BTC.

Conclusion
These observations of this study suggest that AQP-1 expression may be favorable biomarkers associated with

Figure 2 .
Figure 2. Survival rates of BTC patients with the AQP-1 expression.

Table 1 .
Univariate analysis of factors related to survival in patients with biliary tract carcinoma.

Table 3 .
Relationship between patient characteristics and prognosis in biliary tract carcinoma.(Multivariate analysis).