Reduced Ncadherin expression is associated ... - Wiley Online Library

doi:10.1111/j.1440-1746.2011.06847.x

H E PAT O L O G Y

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Reduced N-cadherin expression is associated with metastatic potential and poor surgical outcomes of hepatocellular carcinoma Da-qian Zhan,*1 Shuang Wei,*1 Cong Liu,† Bin-yong Liang,* Gui-bao Ji,* Xiao-ping Chen* Min Xiong‡ and Zhi-yong Huang* *Department of Surgery, †Institute of Pathology, and ‡Department of Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Key words expression, hepatocellular carcinoma, knockdown, metastasis, N-cadherin. Accepted for publication 26 June 2011. Correspondence Dr Zhi-yong Huang, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, no. 1095, Jie Fang Da Dao, Wuhan 430030, China. Email: [email protected] 1

Equally contributing to this work.

Abstract Background and Aim: N-cadherin (N-cad), one of the classic cadherins, has been reported to be involved in tumor metastasis in some types of tumors. This study aims to investigate the expression status of N-cad in hepatocellular carcinoma (HCC) and the correlation between N-cad expression and metastatic potential, as well as the surgical outcomes of HCC. Methods: N-cad expression in HCC and adjacent liver tissues, as well as normal liver tissues, was studied by immunohistochemistry and Western blot, and the relationship between N-cad expression and the clinicopathological features of HCC was evaluated. By using RNA interference technique, the correlation of N-cad expression and metastatic potential was investigated by downregulating N-cad expression in HCCLM3 cells, and the effects of N-cad downregulation on cell aggregation, migration, and invasion were then analyzed. Furthermore, the correlation between N-cad expression and the surgical outcomes of a cohort of HCC patients was analyzed. Results: In liver tissues, N-cad was strongly expressed on cell–cell boundaries, whereas various reduced-expression patterns were observed in tumors. Of 64 HCC, 34 (53%) tumors showed reduced N-cad expression, compared with their adjacent liver tissues. The decreased expression of N-cad was significantly correlated with poorer tumor differentiation (P = 0.001) and vascular invasion (P = 0.003). N-cad knockdown in HCCLM3 cells resulted in decreased cell aggregation and increased cell migration and invasion. The decreased expression of N-cad in HCC was significantly associated with shorter postoperative disease-free survival (P = 0.039). Conclusions: N-cad expression is decreased in HCC, and the downregulation of N-cad is associated with the metastatic potential of HCC and poorer surgical prognosis.

Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies, and represents the third most common cause of cancer-related death worldwide.1 Both hepatic resection and liver transplantation are potentially curative modalities; however, most HCC patients lose the chance of hepatic resection or liver transplantation because of vascular invasion or metastasis. For patients who have undergone curative hepatectomy, long-term survival remains unsatisfactory, due to postoperative recurrence or metastasis.2 Metastasis is believed to be a complex, multistep process that involves the detachment of cancer cells from the primary tumor mass, and the development of new foci in a remote organ.3,4 Cadherins belong to a superfamily of calcium-dependent cell–cell adhesion molecules that have been implicated in this process.5 E-cadherin (E-cad), a member of the classic cadherin family, has

been identified as a tumor suppressor.6 The decreased expression of E-cad has been frequently found to be associated with dedifferentiation and invasion in a variety of human malignancies, including HCC.7,8 Our previous and other studies have indicated that T-cadherin, an atypical cadherin, also plays an important role in HCC invasion and metastasis.9–11 N-cadherin (N-cad), another classic cadherin, is usually restricted to neural tissues and some non-neural tissues.12 Recent studies have suggested that N-cad is re-expressed or upregulated in a number of human epithelial tumors, such as colon, bladder, and prostate cancers.13–15 The aberrant N-cad expression correlates with a morphological change towards a more fibroblastic phenotype, with cells becoming more motile, invasive, and metastatic.16–18 However, in some types of invasive tumors, N-cad was found to be downregulated, and was a tumor suppressor.19,20 In HCC, few studies on N-cad expression have indicated conflicting

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results. Based on an immunohistochemistry (IHC) study, Cho reported that discontinuous N-cad staining was significantly more frequent in HCC tissue than in the surrounding tissue, and discontinuous N-cad staining was correlated with an advanced disease stage and predicted a high risk of recurrence after resection of HCC.21 On the contrary, another IHC-based study indicated that N-cad was overexpressed in HCC tissues compared to corresponding adjacent liver tissues, and the N-cad overexpression was closely related to postoperative recurrences of HCC.22 These conflicting results might result from methodological limitations of IHC and the lack of further experimental evidences in the studies. In this study, to elucidate the expression status of N-cad in HCC and its correlation with metastatic potential and surgical outcomes of HCC, N-cad expression in HCC tissues and adjacent liver tissues, as well as in normal liver tissues, was studied using IHC and Western blot analysis. The effects of N-cad knockdown on cell aggregation, motility, and invasion were investigated in HCCLM3 cells in vitro. The surgical outcomes of a cohort of HCC patients were analyzed.

provided by Dr Ding-zhan Xiao (Guangdong Provincial People’s Hospital Medical Centre, Guangzhou, China). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. pSUPER.retro.puro vectors containing short hairpin RNA (shRNA) for N-cad and enhanced green fluorescent protein (eGFP) were kind gifts from Professor Keith R. Johnson (University of Nebraska Medical Center, Omaha, NB, USA). N-cad knockdown HCCLM3 cells was generated as previously described.24 Briefly, Phoenix 293 cells were transfected with pSUPER.retro.puro containing shRNA for N-cad using Lipofectamine (Gibco-BRL Life Sciences, Rockville, MD, USA) to produce retroviral particles. HCCLM3 cells were then infected with the freshly prepared viral supernatant supplemented with 4 mg /mL polybrene and selected with puromycin. pSUPER.retro.puro containing shRNA for eGFP was used as a control vector. The knockdown of N-cad expression in HCCLM3 cells was confirmed by Western blotting.

IHC

Methods Patients and tissue specimens The HCC tissues and their adjacent liver tissues were obtained from 64 patients with HCC who underwent curative resection (R0 resection) at the Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology (Wuhan, China) between October 2003 and June 2006. The diagnosis of HCC was confirmed by histology. The patients’ ages ranged from 15 to 71 years, with an average of 46 years; there were 58 male and six females. Forty-three patients were found to have liver cirrhosis. Tumor differentiation was determined according to the World Health Organization International Histological Classification. Well differentiation was found in 12 cases, moderate differentiation in 19 cases, and poor differentiation in 33 cases. Of these patients, 12 patients were found to have portal vein tumor thrombus (PVTT), which extended to the second or first portal vein branch, but not to the main portal or opposite-side portal branch by preoperative imaging studies and postoperative pathological examination. Follow-up data after liver resection were available for 54 patients. The median follow up was 52 months. During the follow up, the serum a-fetoprotein level, abdominal ultrasound, chest radiographic examination, computed tomography, and/or magnetic resonance imaging examination were employed to monitor tumor recurrence and metastases. By the end of follow up, recurrence and/or metastasis occurred in 30 patients (56%). For the patients with recurrence and/or metastasis, radiofrequency, microwave, ethanol injection, or/and transcatheter arterial chemoembolization were followed. In addition, normal liver samples from hepatic hemangioma patients were obtained. This study was approved by the medical ethics committee of Tongji Hospital. Informed consent was obtained from each patient.

Cell culture, transfection, and infection The HCC cell line, HCCLM3,23 was purchased from the Liver Cancer Institute, Fudan University (Shanghai, China). The phoenix 293 packaging cell line (Orbigen, San Diego, CA, USA) was kindly 174

An IHC analysis of N-cad expression was performed on serial sections of each sample of tumor and adjacent liver tissues, as well as normal liver tissues. After deparaffinization and rehydration, endogenous peroxidase activity was blocked by 3% H2O2 in methanol solution. The sections were then processed to unmask antigens by microwave oven heating in citrate buffer for 15 min (pH 6.0). Afterwards, the sections were incubated overnight at 4°C with an N-cad monoclonal antibody (1:200; Zymed Laboratories; San Francisco, CA, USA). After several rinses with phosphatebuffered saline (PBS), the sections were incubated with goat antimouse Envision (Dako, Glostrup, Hovedstaden, Denmark) for 30 min at room temperature. All antibody-stained sections were developed with 3,3′-diaminobenzidine-tetrachloride and counterstained with hematoxylin. The primary antibody was omitted to create a negative control. Cells were defined as positive if cells showed strong, uniform staining at the cell–cell boundary or in the cytoplasm. The N-cad expression status of the tumors was semiquantitatively evaluated according to the proportion of N-cadpositive cells in a coded manner by two pathologists who had no prior knowledge of the clinical features of the patients. When more than 90%, between 10% and 90%, and less than 10% of the cancer cells were N-cad positive, the tumors were evaluated as uniformly N-cad positive (+), heterogeneous (+/-), and uniformly N-cad negative (-), respectively. The method was similar to that described by Shimazui.25 In this study, the N-cad (+) tumors were classified as a type of preserved N-cad expression (Pr type), while the others (+/- and – tumors) were collectively classified as one type of reduced N-cad expression (Rd type).

Western blotting The cells and tissue samples were solubilized in lyses buffer (50 mM Tris-HCl, pH 8.0, 1% NP40, 0.1% sodium dodecylsulfate [SDS], 0.5% sodium deoxycholate, 0.02% sodium azide, 150 mM NaCl, and a protease inhibitor cocktail). Protein concentrations of the samples were measured using the bicinchoninic acid protein assay kit according to the manufacturer’s protocol (Pierce, Rockford, IL, USA). For immunoblotting, 50 mg protein was separated


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on 10% SDS-polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Non-specific binding to the membrane was blocked with 5% non-fat milk in Tris-buffered saline (TBS) buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membranes were then probed with specific primary antibodies against N-cad (1:1000; Zymed Laboratories, USA) and b-actin (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), followed by appropriate secondary horseradish peroxidase-conjugated antibodies (1:5000, Pierce, USA). The protein bands were visualized using an enhanced chemiluminescence detection system (Pierce, USA).

Na2HPO4, 12H2O, and 0.1% glucose) to remove the Ca2+, and resuspended at 100 000 cells per 0.5 mL HCMF. In quadruplicate, 0.5 mL of the cell suspension was seeded in the wells of a 24-well plate that was precoated with 1% bovine serum albumin. Ca2+ was added to 1 mM of the final concentration for each well, while the plates were kept on ice. The cells were allowed to aggregate for 1 h at 37°C with constant shaking at 80 rpm. Afterwards, cell aggregates were fixed in 4% paraformaldehyde. The number of aggregates and single cells were counted with a hemacytometer, and the aggregation index (Nt/N0) was calculated (Nt = total particle number, including aggregates and single cells; N0 = total cell number in cell suspension). The assay was repeated three times, and the means and standard deviations (SD) were determined.

Cell aggregation assay Cell aggregation assays were performed as described previously.26 Briefly, the cells were washed with PBS and detached with 0.05% trypsin containing 0.5 mM Ca2+. Ca2+ was used to protect cadherins from trypsin digestion. The cells were washed with HCMF (10 mM HEPES [pH 7.4], 0.137 M NaCl, 5.4 mM KCl, 0.34 mM

In vitro invasion and motility assays For motility assays, 5 ¥ 104 cells were plated into the top chamber of a Transwell membrane with the non-coated membrane (24-well insert, 8 mm pore size; Corning Costar, Lowell, MA, USA). For the invasion assays, 1 ¥ 105 cells were plated into the top chamber

a

d

b

e

c

f

Figure 1 Immunohistochemical analysis of N-cadherin (N-cad) expression in normal liver, hepatocellular carcinoma (HCC), and adjacent liver tissues. Strong and homogeneous membranous staining for N-cad was showed in normal liver (a), adjacent liver tissues (b), and cirrhotic liver tissues (c). Uniformly-positive (d), uniformly-negative (e), and heterogeneous (f) staining for N-cad in HCC were shown. Arrow, N-cad-positive cell; arrowhead, N-cad-negative cell. Magnification: ¥200.


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Figure 2 Western blot analyses for N-cadherin (N-cad) expression. Lanes NL1 and NL2 show N-cad expression in normal liver tissues. N-cad expression of the tumors, identified as N-cad (+), N-cad (+/-), and N-cad (-), are shown in cases 1, 2, and 7, cases 3 and 5, and cases 4 and 6, respectively. N, adjacent liver tissues; T, tumor tissues. b-actin was used as an internal control.

of a Transwell membrane, which was coated with Matrigel. For the motility and invasion assays, 600 mL DMEM containing 10% serum was used as a chemoattractant in the lower chamber. The cells were then incubated at 37°C for 24 h in 5% CO2, and those that did not migrate through the pores in the membrane were removed by scraping the membrane with a cotton swab. The cells that transversed this membrane were fixed in methanol and stained with crystal violet. Cells in four random fields of view at ¥100 were counted under a light microscope and expressed as the average number of cells/field of view. Three independent experiments were completed in each case. The data were represented as the average of the three independent experiments, with the SD of the average indicated.

Statistical analysis Relationships between N-cad expression and clinicopathological factors were analyzed with a c2-test or Fisher’s exact test. Diseasefree survival rates and overall survival rates were calculated using the Kaplan–Meier method and were compared between the groups with the log–rank test. The data presented as the mean ⫾ SD were analyzed with the t-test or anova. Significance was defined as P < 0.05.

Results N-cad expression was reduced in HCC tissues In normal liver tissues, strong and homogeneous membranous staining was observed in hepatocytes, particularly in areas of cell– cell contact (Fig. 1a). In adjacent liver tissues, N-cad staining was also observed at the cell–cell borders of hepatocytes (Fig. 1b), including cirrhotic liver tissues (Fig. 1c). In HCC tissues, however, three staining patterns for N-cad expression could be distinguished: uniformly-positive, uniformly-negative, and heterogeneous staining (Fig. 1d–f). Among the 64 HCC, 30 tumors (47%) were identified as uniformly-positive staining (Pr type), and 34 tumors (53%) were identified as uniformly-negative staining or heterogeneous staining (Rd type). Western blotting confirmed the expression patterns of N-cad in the HCC and adjacent liver tissues that had been determined by IHC (Fig. 2).

Reduced N-cad expression is associated with poor differentiation and vascular invasion of HCC The relationships between the N-cad expression and various clinicopathological features are shown in Table 1. In 176

Table 1 Correlation between N-cadherin expression and various clinicopathological features in 64 hepatocellular carcinoma patients Total

Pr type

Rd type

Parameters

n

+

+/-

-

Subtotal

Total Age (year) < 50 ⱖ 50 Sex Male Female HbsAg (+) Liver cirrhosis Tumor size (cm)†