Clinical Significance of Wnt-induced Secreted Protein-1 (WISP-1/CCN4) in Esophageal Squamous Cell Carcinoma

Patients and Methods

Tissue samples and cell lines. One-hundred and ninety patients with thoracic esophageal carcinoma underwent transthoracic subtotal esophagectomy at Kumamoto University Hospital, between 2005 and 2009. Any patients who had undergone previous therapy were excluded and 105 resected specimens of esophageal cancer from the patients who did not receive any radiotherapy or chemotherapy prior to surgical resection of the esophagus were included in this study. Diagnosis and routine histopathological examinations of hematoxylin and eosin-stained sections were performed at the Department of Surgical Pathology, Kumamoto University Hospital. Alcohol-fixed, paraffin-embedded samples were cut into 4 μm sections for immunohistochemical staining. All of the samples were histopathologically diagnosed as ESCC, and the following information was recorded for each patient: age, gender, clinicopathological staging (TNM system) according to the Unio Internationalis Contra Cancrum (UICC), depth of invasion, tumor size, tumor type, lymphatic invasion, venous invasion and lymph node status. The patients were routinely followed-up clinically after surgery for a median period of 36 months.

The human esophageal cancer cell lines TE1, TE4, TE6, TE8, TE9, TE10, TE11, TE14 and TE15 were obtained from Tohoku University, Sendai, Japan. These cell lines were cultured at 37°C in 5% carbon dioxide, in RPMI medium with 10% fetal bovine serum.

Immunohistochemical staining. Paraffin-embedded tissue sections were stained with rabbit polyclonal antibody to human WISP-1 (Santa Cruz, CA, USA) at 1:50 dilution. Normal rabbit immunoglobulin G was used as a negative control. Immunostaining was performed using an EnVision⁺ System kit (Dako, Copenhagen, Denmark). Sections were deparaffinized in xylene and rehydrated through alcohol. Endogenous peroxidase was quenched with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 15 min, followed by antigen retrieval with 0.01 mol/l citrate buffer (pH 6.0) for 15 min in a microwave oven. Sections were blocked with 3% bovine serum albumin (BSA) in PBS for 40 min, followed by incubation overnight at 4°C with primary antibody and EnVision⁺ horseradish peroxidase for 60 min. The slides were washed in PBS three times for 5 min after each incubation. The color reaction was performed in 3’-diaminobenzidine (DAB) for 5 min, then the slides were counterstained with hematoxylin for 30 s. The slides were dehydrated in alcohol prior to mounting. Immunoreactivity was graded as: 0, negative; 1+, weak homogenous cytoplasmic staining; 2+, strong staining in <30% of tumor cells; 3+, strong staining in >30% of tumor cells. 0 and 1+ were defined as WISP1-negative; 2+ and 3+ as WISP-1-positive. All of the slides were independently examined by two experienced investigators.

RT-PCR analysis. Total RNA extraction was performed using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using SuperScript III (Invitrogen), according to the manufacturer's instructions. The initial denaturing was performed at 94°C for 3 min, followed by 27 cycles of amplification (denaturing at 94°C for 30 s, annealing at 57°C for 30 s and extension at 72°C for 30 s) and terminal extension at 72°C for 3 min. The primers used for RT-PCR amplification were: human WISP-1; 5’-GAT GTG CGC TCA GCA GCT TG-3’ (forward) and 5’-TAT GGA TGT CCA CAT CGC ATG-3’ (reverse); human GAPDH; 5’-TGA CCA CAG TCC ATG CCA TC-3’(forward) and 5’-CCA CCC TGT TGC TGT AGC C-3’ (reverse).

Construction of the WISP-1 expression vector and transfection. The PCR amplified WISP-1 was cloned into a pcDNA3.1/V5-His TOPO vector (Invitrogen). The primer sets for full-length WISP-1 amplification were: human WISP-1; 5’-GAG GTG GTC GGA TCC TCT G -3’ (forward) and 5’-GGT AAC TAA GGC TCA TTG GTG-3’ (reverse). The ligated vector was transformed using a One Shot chemical competent cell kit (Invitrogen). Plasmid DNA was extracted using a QIA filter Plasmid Maxi Kit (QIAGEN, Tokyo, Japan), and sequenced using an ABI 310 auto-sequencer (Applied Biosystems). The expression vectors were transfected using the standard protocol with Lipofectamine 2000 reagent (Invitrogen). The transfected cells were transferred into 25 cm² flasks after 24 h and used for further analysis. The WISP-1-transfected cell line was designated TE6+WISP-1 and the LacZ (control)-transfected cell line was designated TE6+LacZ.

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Figure 1.

Representative immunohistochemical staining of WISP-1 positive ESCC tissue (×400, ×50).

Western-blot analysis. Proteins were extracted using RIPA buffer (0.5% sodium deoxycholate, 1M Tris, 5M NaCl, 1% NP-40, 0.1% sodium dodecyl sulphate) containing protease inhibitor and phosphatase inhibitor. Each lane of an 11% SDS-PAGE gel was loaded with 20 μg of protein. The proteins were separated by electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in PBS/Tween 20 (0.1%) at room temperature for 1 h and then incubated with anti-rabbit human WISP-1 (SC-25441; Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was rinsed twice for 10 min with PBS/Tween 20 and then incubated with anti rabbit IgG (Santa Cruz) for 1 h at room temperature. The membrane was rinsed twice for 10 min with PBS/Tween 20 and for 10 min with PBS, incubated with ECL-Plus (GE healthcare) and then exposed to X-ray film and developed.

Growth assay. TE6+LacZ and TE6+WISP-1 cells (2×10³) were plated in a 96-well plate and cultured for 72 h. Cell numbers were measured using a Cell Counting Kit-8 assay kit (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer's protocol.

Invasion assay. A Matrigel Invasion Chamber (Becton Dickinson Labware, Bedford, MA, USA) was used to examine the invasion rate. The TE6+LacZ and TE6+WISP-1 cell suspensions were diluted to 1×10⁵ cells/well in a 24-well Matrigel Invasion Chamber using chemoattractant (growth medium supplemented with 10% fetal bovine serum) in the well. The invasive cells were counted 22 h after seeding the cells.

Statistical analysis. The data were analyzed using the Mann–Whitney U-test or Chi-square test. Overall survival (OS) was calculated from the date of esophagectomy to the date of death. Univariate survival analyses were performed using the Kaplan–Meier method and log-rank tests. The Cox proportional hazards regression model was used for the multivariate analysis. The level of significance was defined as being a p-value of 0.05 or less. All the statistical analyses were performed using the STATVIEW5.0 software package (SAS Institute Inc., Cary, NC, USA).