Cytoplasmic-only Expression of Maspin Predicts Poor Prognosis in Patients With Oral Squamous Cell Carcinoma

Patients and Methods

Patients and tumor specimens. From January 2005 to December 2015 at Tottori University Hospital (Yonago, Japan), 127 consecutive patients underwent curative surgical resection of OSCC. A total of 47 patients were excluded for the following reasons: in nine cases, induction chemotherapy was performed before surgery and the effect was more than grade 2 according to the criteria of general rules for clinical and pathological studies on oral cancer (18); in 29 cases, radiation therapy and/or superselective intra-arterial chemotherapy were performed before surgery; and in nine cases, invasive lesion was not found in immunostained slides. Thus, 80 OSCC cases were included in the present study. The clinicopathological data of the patients were obtained from their hospital medical records. Tumor stage was determined based on the 7th edition of the American Joint Committee on Cancer staging system (19). Informed consent was obtained using the opt-out method by publishing at the Tottori University Hospital (Yonago, Japan) website, and the present study was approved by the Ethics Committee of the Faculty of Medicine, Tottori University (approval no.:18A046; May 11, 2018).

Immunohistochemistry. All specimens were fixed in 10% neutrally buffered formalin and embedded in paraffin. Sections (4 μm-thick) were deparaffinized, treated with hydrogen peroxide to inhibit endogenous peroxidase activity, and then microwaved in citrate buffer (0.01 M, pH 6.0) for 15 min. Next, we performed immunohistochemical staining using a monoclonal anti-human maspin antibody (clone EAW24, diluted 1: 150; Leica Biosystems, Newcastle upon Tyne, UK), as described previously (7).

Evaluation of immunohistochemical findings. Cells were considered positive if strong staining, defined as a staining intensity equal to that in mammary myoepithelial cells, was observed only in the cytoplasm. Maspin expression in the normal oral mucosa varies from sample to sample; therefore, myoepithelial cells of normal breast tissue were used as an internal positive control. If positive cells accounted for more than 10% of the tumor, the case was considered maspin-positive, as previously described (20). The subcellular localization of maspin was classified into four categories: cytoplasmic-only, pancellular (combined nuclear and cytoplasmic), nuclear-only, and no staining. All slides were independently evaluated by M.K. and Y.U., without knowledge of the clinicopathological data of the patients.

Cell culture. HSC2, HSC3, HSC4, Ca9-22, and SAS cell lines were purchased from the Riken Bio Resource Center Cell Bank (Riken RBC Cell Bank, Ibaraki, Japan). HSC2, HSC3, and Ca9-22 cells were maintained in Gibco Minimum Essential Media (Thermo Fisher Scientific, Waltham, MA, USA), whereas HSC4 and SAS cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific). All cell lines were maintained at 37° in a humidified incubator containing 5% CO₂. Each medium was adjusted by adding 10% fetal bovine serum (FBS; Biological Industries Ltd. Kibbutz Beit Haemek, Israel), 5% glutamine, and 1% penicillin-streptomycin.

RNA extraction and quantitative polymerase chain reaction. Total RNA from HSC2, HSC3, HSC4, Ca9-22, and SAS cells was extracted using TRIzol reagent (Thermo Fisher Scientific) and reverse-transcribed into complementary DNA (cDNA) using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Gene expression levels were measured using the TaqMan Gene Expression Assays (Thermo Fisher Scientific) with the following gene-specific primers: β-actin (ACTB) (Hs01060665_g1, Thermo Fisher Scientific) and maspin (Hs00985285_m1, Thermo Fisher Scientific).

Western blot analysis. Whole-cell lysates were prepared using RIPA lysis buffer with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Roche Applied Science). Subcellular protein fractions were obtained using a subcellular protein fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.45 μm pore size polyvinylidene difluoride membrane (Merck Millipore, Burlington, MA, USA). Blocking was performed with 5% ECL prime blocking agent (Cytiva, Tokyo, Japan) for 90 min. The primary antibodies used were mouse monoclonal anti-human maspin antibody (clone EAW24; 1:1000 dilution, Leica Biosystems), mouse monoclonal anti-β-actin (ACTB) antibody (8H10D10; 1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-HSP90 antibody (C45G5; 1:1,000 dilution, Cell Signaling Technology), and rabbit polyclonal anti-HDAC1 antibody (10E2; 1:1,000 dilution, Cell Signaling Technology). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat. No.NA934; 1:3,000 dilution, GE Healthcare, Boston, MA, USA) or HRP-conjugated anti-rabbit IgG (cat. No. NA931; 1:3,000 dilution, GE Healthcare) was used as the secondary antibody. The signals were visualized using the Immobilon Western chemiluminescent substrate (Millipore, Billerica, MA, USA) and quantified using the Image Quant LAS 4000 mini (GE Healthcare).

Plasmid and siRNA transfection. HSC3 and HSC4 cells were seeded in 6-well plates and incubated for 24 h at 60-80% confluence at transfection. Cells in each well were transfected with 10 nM siRNA targeting maspin (Silencer Select siRNA, s10468, Thermo Fisher Scientific), or control siRNA (Silencer Negative Control #1; Thermo Fisher Scientific) using 9 μl Lipofectamine RNAiMAX (cat. No. 13778030, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Cell invasion assays. Cell invasion assays were performed using the CytoSelect 24-well cell invasion assay (Cell Biolabs, Inc., San Diego, CA, USA) according to manufacturer’s instructions. HSC3 and HSC4 cells (1.8×10⁵ cells/well) were cultured in a medium containing 10% FBS for 24 h, transfected with siRNA, and further cultured for 24 h. The medium was replaced with a serum-free medium, and the cells were further cultured for 24 h. Cells transfected with siRNA were seeded in serum-free medium in the upper chamber containing polycarbonate membrane inserts (8 μm pore size) in a 24-well transwell plate. The lower chamber was filled with medium containing 10% FBS. After incubation for 48 h, the infiltrating cells at the bottom of the chamber were removed with a stripping solution. Invasive cells in the striping solution were read with a fluorescence plate reader using CyQuant GR Dye (Cell Biolabs). Three independent experiments were performed, and the mean values were statistically analyzed.

Statistical analysis. All statistical analyses were performed using the SPSS ver.25 software program (IBM SPSS, IBM, Armonk, NY, USA). The association between maspin status and clinicopathological factors were analyzed using non-parametric tests. The chi-square test or Fisher’s exact test were used when there were two categorical variables of interest, whereas the Kruskal-Wallis test was applied when there were three variables. Disease-free survival (DFS) was defined as the period from the date of initial surgery to the date of clinical or pathological cancer recurrence or last visit. DFS rates were calculated using the Kaplan-Meier method and compared using log-rank tests. Multivariate analysis of factors associated with DFS was performed using the Cox hazard test to define independent prognostic factors depending on the maspin status. Relative maspin mRNA expression was compared using one-way analysis of variance and the Tukey’s honest significant difference test. Differences in cell invasion assays were evaluated using the Student’s t-test. Differences were considered statistically significant when the p value was less than 0.05. All continuous values are presented as the mean±standard deviation.