Reduced expression of P120 catenin in cholangiocarcinoma correlated with tumor clinicopathologic parameters

Abstract

AIM: To investigate the relationship between the expression of P120 and the clinicopathologic parameters in intrahepatic cholangiocarcinoma (ICC).

METHODS: An immunohistochemical study of E-cadherin and P120 catenin was performed on 42 specimens of ICC with a Dako Envision kit.

RESULTS: The expression of E-cadherin and P120 was reduced in 27 cases (64.3%) and 31 cases (73.8%), respectively. Both E-cadherin and P120 expressions were significantly correlated with the tumor histological grade (χ² = 9.333, P = 009 and χ² = 11.71, P = 0.003), TNM stage (χ²= 8.627, P = 0.035 and χ² = 13.123, P = 0.004), intrahepatic metastasis (χ²= 7.292, P = 0.007 and χ² = 4.657, P = 0.041, respectively) and patients’ survival (χ²= 6.351, P = 0.002 and χ² = 4.023, P = 0.000, respectively). In addition, the expression of P120 was in concordance with that of E-cadherin (χ² = 13.797, P = 0.000), indicating that the expression of P120 may be dependent on that of E-cadherin. Finally, only P120 expression was found to be an independent prognostic factor in Cox regression model (r = 0.088, P = 0.049).

CONCLUSION: Down-regulated expression of E-cadherin and P120 occurs frequently in ICC and contributes to the progression and development of tumor. Both of them may be valuable biologic markers for predicting tumor invasion, metastasis and patients’ survival, but only P120 is an independent prognostic factor for ICC.

Key Words: P120, Intrahepatic cholangiocarcinoma, Clinicopathologic feature, Invasion and metastasis, Survival

INTRODUCTION

P120-catenin is a member of the Armadillo (ARM)/β-catenin gene family and is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness[12]. Cadherin, one of the transmembrane cell-cell adhesion receptors involved in development, and morphogenesis of ICC[3], is necessary and sufficient for P120 targeting cell-cell junctions.

A main function of P120 is to stabilize cadherins at the cell membrane by regulating cadherin turnover and degradation. In this way, P120 level acts as a set point mechanism underlying cell-cell adhesive interactions. P120 may function as a “cap” to bind to the cadherin cytoplasmic tail and prevent cadherin interactions with endocytic membrane trafficking machinery. Alternatively, P120 may stabilize cell junctions or regulate membrane trafficking machinery through interactions with small GTPases, such as Rho A, Rac and Cdc42. Through these mechanisms, P120 exerts its influence over a wide range of biological processes that are dependent upon tight regulation of cell surface cadherin levels[4].

Intrahepatic cholangiocarcinoma (ICC) is the second most common tumor of primary liver cancers in adults worldwide, accounting for about 15% of liver cancers, and its incidence has increased in recent years[5]. Despite improved diagnostic and operative techniques, the prognosis of ICC remains poor. In addition, molecular events involving the development of ICC are not well understood. Some studies examined the expression of E-cadherin/catenin complex in ICC, but the conclusion is still controversial. Moreover, to our knowledge, no study has demonstrated the expression characteristics of P120 and the relationship between the expression of P120, and the clinicopathologic parameters in ICC. Therefore, in the present study, we used immunohistochemical staining for the E-cadherin/P120 complex in primary ICC to correlate its expression with its clinicopathologic features.

MATERIALS AND METHODS

Selection of patients and definition of clinicopathologic parameters

In this study, we selected 42 specimens of intrahepatic cholangiocarcinoma collected and diagnosed at the Eastern Hepatobilliary Surgery Hospital, the Second Military Medical University from October 1997 to March 2004. The patients were consisted of 32 men and 10 women. Their age ranged from 27 to 73 years, with an average age of 51 years. Cancer tissue and non-tumorous liver tissue were obtained from each patient for pathological examination. The detailed pathologic data were obtained from the Department of Pathology of Eastern Hepatobilliary Surgery Hospital. Background liver showed cirrhosis in 19 (45.2%) patients, and chronic hepatitis in 15 (35.7%) patients.

Clinicopathologic parameters included histological grade, pTNM stage, tumor size, capsular and vascular invasion, satellite nodules, intrahepatic metastasis, lymph node status and patients’ survival. Histological grade of ICC was sub-classified into well, moderately and poorly differentiated ICC based on the criteria for a liver cancer study in Japan[6]. Tumor staging was performed according to the pTNM staging system of the International Union against Cancer UICC[7].

Immunohistochemistry

Formalin-fixed and paraffin-embedded tissues were cut to 5-&mgr;m thick sections. Immunohistochemical staining for E-cadherin and P120 was performed with a Dako EnVisionTM kit (Dakocytomation Company, Denmark). The sections were dewaxed, incubated with methanol containing 30% H₂O₂ for 20 min to block endogenous peroxidase activity, immersed in 0.01 mol/L citrate buffer(pH 6.0), heated at 100°C in a microwave oven for 20 min, washed three times with distilled water and blocked with 1% BSA for 30 min. The sections were then incubated overnight at 4°C with rabbit polyclonal IgG of E-cadherin (H-297, SC-7870, Santa Clauze Corporation, USA ) and rabbit polyclonal IgG of P120 (H-90, SC-13957, Santa Clauze Corporation, USA) at a 1:200 dilution. A subsequent reaction was carried out using second antibodies (Dakocytomation Company, Denmark) at 37°C for 30 min. Then, the sections were washed three times with phosphate-buffered saline (PBS) and subsequently the color was displayed with DAB (Dakocytomation Company, Denmark) for about 5 min. Nuclei were lightly counterstained with hematoxylin. No staining was obtained when immune serum or PBS was used instead of primary antibodies, thus confirming the specificity of each primary antibody.

Evaluation of immunostaining

A scoring system was used to semiquantitatively evaluate the immunoexpression of E-cadherin and P120 in ICC as described previously[8]. The expression of E-cadherin and P120 in nontumorous tissue was used as an internal control. Briefly, immune activities of E-cadherin and P120 were assessed by the extent (broadness) and intensity (color strength). Depending on the percentage of positive cells, the extent was scored as follows: 0 = no positive cells or less than 5%, +1 = 5%-25% positive cells, +2 = 26%-50% positive cells, +3 = 51%-75% positive cells, and +4 = 76%-100% positive cells. The intensity was also scored as follows: 0 = no immunoreaction, +1 = mild immunoreaction, +2 = moderate immunoreaction, +3 =marked immunoreaction. E-cadherin or P120 expression was defined as positive when the composite score was 6 or 7, and as “absent or loss” when the total score was 0.

Statistical analysis

Results from immunohistochemistry were analyzed by χ² or Fisher’s exact test. P < 0.05 was considered statistically significant. Survival analysis was performed using the log-rank test (P < 0.05). Survival curves were plotted according to the method of Kaplant and Meier. The prognosis value of E-cadherin and P120 for ICC was evaluated with univariate (log-rank test) and multivariate analysis (Cox regression model). SPSS 10.1 software package for Windows (SPSS, Inc., Chicago, IL) was used.

RESULTS

Observation under microscope

In nontumorous liver tissue, both E-cadherin and P120 were expressed strongly on cell membranes, but the staining intensity was gradually decreased. In addition, these molecules were normally expressed on cell membranes of bile ducts, proliferating ductules and intra-hepatic vessels. No expression was found in other types of cells in the liver.

In ICC, the expression of E-cadherin and P120 catenin was reduced in 27 (64.3%) and 31 cases (73.8%), being absent in 8 and 10 cases, respectively. In addition, P120 was expressed in 17 cases (40.5%) (Figure 1).

Figure 1 Immunoreactivity of E-cadherin and P120 in intrahepatic cholangiocarcinomas. “Preserved type”(+) (A, D), “reduced type”(-) (B, E), and “complete absent” (C, F) of E-cadherin and P120 induced type”(-) and staining, respectively (× 200).

Relationship between expression of E-cadherin/P120 and histological features of ICC

As shown in Table 1, the membranous expression of E-cadherin and P120 was significantly correlated with the tumor grade (P = 0.009 and P = 0.003, respectively). The expression of E-cadherin and P120 tended to be reduced in poorly-differentiated tumors compared with well- and moderately-differentiated tumors. In addition, the expression of E-cadherin and P120 was inversely associated with the pTNM stage of tumors (P = 0.035 and P = 0.004, respectively).

Table 1 Relationship between expressions of E-cadherin/P120 catenin and histological features of ICC n (%).

	E-cadherin				P120 catenin
	n	+	-	P value	+	-	P value
Differentiation grade
Well	3	3 (100)	0 (0)	0.009	3 (100)	0 (0)	0.003
Mediate	14	7 (50.0)	7 (50.0)		5 (35.7)	9 (64.3)
Poor	25	5 (20.0)	20 (80.0)		3 (12.0)	22 (88.0)
pTNM
I	2	2 (100)	0 (0)	0.035	2 (100)	0 (0)	0.004
II	9	5 (55.6)	4 (44.4)		5 (55.6)	4 (44.4)
III	25	8 (32.0)	17 (68.0)		4 (16.0)	21 (84.0)
IV	6	0 (0)	6 (100)		0 (0)	6 (100)

Relationship between expression of E-cadherin and P120 and clinical parameters of ICCs

As shown in Table 2, the expression of E-cadherin or P120 was significantly associated with intra-hepatic matestasis of ICC (P = 0.007 and P = 0.041, respectively). No statistically significant difference was observed between the expression level of E-cadherin or P120 and tumor size, capsular and vascular invasion, lymph node permission and satellite nodules.

Table 2 Relationship between expressions of E-cadherin/P120 catenin and clinical parameters of ICC n (%).

	E-cadherin				P120 catenin
	n	+	-	P value	+	-	P value
Size
< 5 cm	17	7 (41.2)	10 (58.8)	0.826	6 (35.3)	11 (64.7)	0.584
5-10 cm	16	5 (31.3)	11 (68.7)		3 (18.8)	12 (81.2)
> 10 cm	9	3 (33.3)	6 (66.7)		2 (22.2)	7 (77.8)
Capsular invasion
+	6	4 (66.7)	2 (33.3)	0.164	3 (50)	3 (50)	0.391
-	36	11 (30.6)	25 (69.4)		8 (22.2)	28 (77.8)
Satellite nodules
+	11	4 (36.4)	7 (65.6)	1	3 (27.3)	8 (72.7)	0.314
-	31	11 (35.5)	20 (64.5)		8 (25.8)	23 (74.2)
Vascular invasion
+	13	2 (15.4)	11 (84.6)	0.089	1 (7.7)	12 (92.3)	0.127
-	29	13 (44.8)	16 (55.2)		10 (34.5)	19 (65.5)
L.N.P
+	7	1 (14.3)	6 (85.7)	0.39	0 (0)	7 (100)	0.161
-	35	14 (40)	21 (60)		11 (31.4)	24 (68.6)
I.M.
+	10	0 (0)	10 (100)	0.007	0 (0)	10 (100)	0.041
-	32	15 (46.9)	17 (53.1)		11 (34.4)	21 (65.6)

Relationship between expressions of E-cadherin and P120 in ICC

As shown in Table 3, positive and negative expression of E-cadherin and P120 was found in 9 and 25 cases, respectively. However, negative expression of P120 was observed in 7 cases. There was a significant concordance between the expressions of E-cadherin and P120 (P = 0.000).

Table 3 Relationship between expression of E-cadherin and P120 catenin in ICC.

	P120
E-cadherin	+	-	P value
+	9	6	0.000
-	2	25

Relationship between expression of E-cadherin/P120 and survival of ICC patients

The patients were followed up for 4-67 months. The overall survival rate of patients according to the expression of E-cadherin and P120 in tumor is shown in Figure 2. Analysis of the survival of all patients showed that abnormal expression of E-cadherin and P120 was significantly correlated with the poor survival of patients (P = 0.024 and P = 0.004, respectively). However, when the expression of E-cadherin or P120 and the clinicopathological parameters were analyzed by the Cox regression model, abnormal expression of P120 was found to be an independent prognostic factor for ICC patients (P = 0.049) (Table 4).

Figure 2 Kaplan-Meier survival curves. A: Expression of P120 induced type”(-) and B: Expression of E-cadherin.

Table 4 Cox multivariate analysis for survival of 37 patients.

	Sig	RR	95% CI
			Lower	Upper
E-cadherin expression	0.724	1.525	0.147	15.827
P120 expression	0.049	0.088	0.008	0.991
Differentiation	0.194	0.407	0.105	1.583
pTNM stage	0.073	2.898	0.904	9.288
Tumor size	0.037	0.387	0.159	0.944
Capsular invasion	0.052	17.046	0.981	6.166
Satellite nodules	0.709	1.597	0.137	18.578
Vascular invasion	0.948	0.961	0.284	3.247
Limph node invasion	0.087	4.72	0.8	27.855
Intrahepatic metastasis	0.786	1.352	0.154	11.899

Sig: Significance; RR: Relative risk; CI: Confidence interval.

DISCUSSION

Usually, ICC is an adenocarcinoma and may arise from the large intra-hepatic bile ducts near the hepatic hilus or from the bile ducts at the border of hepatic parenchyma. It was reported that altered expression of E-cadherin/catenins complex in ICC occurs frequently and is significantly correlated with tumor histological features and/or vascular invasion and metastasis[9–14].

It was recently reported that P120 plays a role in the occurrence of various cancers, and that P120 may behave either as a tumor suppressor or as a metastasis promoter, depending on the loss of E-cadherin and P120. If E-cadherin is lost first, P120 may directly and actively promote metastasis. If P120 is lost first, E-cadherin levels would fall significantly, which is likely to be parallel to the reduced levels of α-and β-catenins[15]. P120 down-regulation results in a striking dose-dependant loss of endogenous cadherins, indicating that P120 is essential for cadherin stability. Moreover, P120 down-regulation occurs frequently in almost all carcinomas[16]. P120 loss is often associated with the stage and poor prognosis of tumors, suggesting that its loss may be associated with biological aggressiveness and progression of tumors. Nevertheless, to our knowledge, no report is available on the expression of P120 in human intrahepatic chola-giocarcinoma.

The present study showed that reduced or absent expression of E-cadherin and P120 was associated with the histological grade of tumors, which is consistent with reported data[17–21]. In well-differentiated tumors, there were obvious and strong staining along the cell-cell boundaries, whereas in poorly- differentiated tumors, the immunohistostaining was focally and heterogeneously distributed, with patchy or spotty features along the cell-cell boundaries, indicating that the staining of E-cadheri and P120 is related with the differentiation of ICC, namely both E-cadherin and P120 may be regarded as differentiation markers of tumor. In addition, the staining intensity of the E-cadheri and P120 complex was gradually decreased, suggesting that P120 may play a critical role in ICC progression.

Microscopy revealed that E-cadherin was located on the membrane either in non-tumor tissues or in tumor cells, whereas P120 was expressed on the membrane or in cytoplasm of tumor cells. However, it was reported that P120 is also in nuclei[22], suggesting that P120 plays an important role in cell signal transduction. P120 has an intrinsic nucleocytoplasmic shuttling activity that is modulated, in part, by extrinsic factors such as cadherin binding and interactions with the microtubule network[22]. Julia and his colleagues reported[23] that P120 displays up-regulation and nuclear expression in pancreatic cancer. No expression of P120 in nuclei of cancer cells, however, was observed in our study, suggesting that it is necessary to further investigate the mechanism underlying P120 expression in nuclei of cancer cells.

In this study, we observed the relationship between reduced expression of E-cadherin and P120 and several clinicopathologic parameters of ICC. The expression of P120 and E-cadherin was significantly associated with tumor pTNM stage and intrahepatic metastasis (IM), but not with tumor stage and size, capsular and vascular invasion, and lymph node invasion. Osada and his colleagues[24] revealed that E-cadherin is involved in intra-hepatic metastasis of hepatocellular carcinoma. Asayama et al[13] detected the expression of E-cadherin in hepatocellular carcinoma and cholangiocarcinoma, and found that reduced expression of E-cadherin is significantly correlated with the grade and IM of ICC. Therefore, E-cadherin and P120 may be important mediators in tumor progression, and can be considered as invasion and metastasis markers of ICC.

Several studies on other cancers have evaluated the relationship between the expression of E-cadherin/P120 and the survival of patients, but the results remain debatable[25–29]. In the present study, reduced expression of both E-cadherin and P120 was significantly related with the survival of patients. However, when the expression of E-cadherin/P120 and the clinicopathological parameters of ICC were analyzed by the Cox regression model, only the abnormal expression of P120 was found to be an independent prognostic factor for ICC, suggesting that P120 can be considered a valuable biological marker for predicting the prognosis of ICC patients.

In summary, abnormal expression of E-cadherin and P120 catenin occurs frequently in intrahepatic cholan-giocarcinoma. Reduced expression of P120 catenin and E-cadherin is correlated with tumor differentiation, pTNM stage, intrahepatic metastasis and survival of patients. Both P120 catenin and E-cadherin may play an important role in the development and progression of human intrahepatic cholangiocarcinoma.

COMMENTS

Background

P120-catenin is a member of the E-cadherin/catenin complex family and may be associated with biological aggressiveness and progression of tumors. However, no report is available on the expression of P120 catenin in human intra-hepatic cholangiocarcinoma.

Research frontiers

P120 down-regulation occurs frequently in almost all carcinomas. P120 loss is often associated with the stage and poor prognosis of tumors.

Innovations and breakthroughs

Our results suggest that down-regulated expression of E-cadherin and P120 catenin occurred frequently in intrahepatic cholangiocarcinoma (ICC) and contributed to the progression and development of tumors. Both E-cadherin and P120 catenin may be valuable biologic markers for predicting tumor invasion, metastasis and survival of patients, but only P120 catenin is an independent prognostic factor for ICC.

Applications

Because down-regulated expression of P120 contributes to the progression and development of ICC, P120 can be used as a valuable biologic marker for predicting the invasion and metastasis of ICC, and the survival of patients.

Peer review

This is an interesting report on E-cadherin and P120 catenin in human intra-hepatic cholangiocarcinoma. The study was performed on 42 specimens of ICC with a Dako Envision kit, indicating that. Both E-cadherin and P120 catenin may be valuable biological markers for predicting tumor invasion, metastasis and survival of patients. However, its clinical application should be further studied.