Subcellular Localization of Maspin Correlates with Histone Deacetylase 1 Expression in Human Breast Cancer

Patients and Methods

Patients and tumors specimens. From January 2008 to December 2011 at Tottori University Hospital, Japan, 227 consecutive female patients underwent a surgical resection of breast cancer diagnosed as invasive carcinoma of no special type according to the World Health Organization classification of breast cancer (13). Sixty-three cases were excluded due to a history of neoadjuvant treatment (38 cases), microinvasive carcinoma (9 cases), distant metastasis (5 cases), unavailable tumor specimen (5 cases), bilateral breast cancer (4 cases) and short follow-up period (2 cases). Thus, 164 patients were included in this analysis. The patients' clinicopathological data were obtained from their hospital medical records. The histopathological parameters including lymph node metastasis, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) statuses were retrieved from the patients' pathology reports. Histological grades were determined according to the criteria of Elston and Ellis (14), and the grading was independently evaluated by M.W. and Y.U. Tumor stage was determined based on the seventh tumor–node–metastasis (TNM) classification of breast cancer (15). The study was approved by the ethics committee of the Faculty of Medicine, Tottori University (approval number: 1608A083; 15 August 2016).

Immunohistochemistry. All specimens were fixed in 10% neutrally buffered formalin and embedded in paraffin. The sections (4 μm-thick) were deparaffinized and endogenous peroxidase activity was blocked. The specimens were then pretreated in citrate buffer (0.01 M, pH 6.0) in a microwave oven (IMB-T17-5/6; Irisohyama, Sendai, Japan) for 15 min at 700W for maspin, or a water bath at 95°C for 10 min for HDAC1. After the pretreated specimens had cooled to room temperature, non-specific binding was blocked by blocking buffer (Block Ace; Megmilk Snow Brand, Sapporo, Japan). Next, the sections were incubated at 4°C overnight with a monoclonal antibody to maspin (clone EAW24, diluted 1:200; Leica Biosystems, Newcastle, UK) or at room temperature for 30 min with polyclonal antibody to HDAC1 (clone ab19845, diluted 1:3600; Abcam, Cambridge, MA, USA). The sections for maspin were incubated with biotinylated anti-mouse IgG antibody (BA-2000, diluted 1:150; Vector Laboratories, Burlingame, CA) for 20 min, followed by streptavidin biotinylated horseradish peroxidase (HRP) conjugate (diluted 1:150; Invitrogen, Camarillo, CA, USA) for 20 min. The sections for HDAC1 were incubated with the EnVision+ System HRP (Dako, Glostrup, Denmark). Finally, all of the sections were incubated with diaminobenzidine (DAB) solution (liquid DAB+ substrate, imidazole-HCI buffer, pH 7.5, containing hydrogen peroxide and an antimicrobial agent; Dako) for 4 min, and counterstained with hematoxylin.

Evaluation of immunohistochemical findings. The cells were considered positive for maspin expression when strong cytoplasmic-only staining was identified. Strong staining was defined as the staining intensity equal to that of myoepithelial cells in normal breast tissue, and it served as an internal positive control if included. Tumors with >10% positive cells were considered maspin-positive as described previously (8). The subcellular localization of maspin was classified into four categories; cytoplasmic-only, pancellular (combined nuclear and cytoplasmic), nuclear-only, and no staining. The evaluation of HDAC1 expression status was performed according to the methods described by Seo et al. (16). Briefly, the intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the proportion of positive cells (0, negative; 1, <10%; 2, 10-33%; 3, 33-66%; 4, ≥66%) scores were calculated through multiplication and cases were divided into two groups according to their scores as follows: low expression (scores, 0-6) and high expression (scores, 8-12). All slides were evaluated by M.W. and Y.U. independently who were blinded to the patients' clinicopathological data.

Cell culture. The human mammary epithelial cell line MCF10A and the human breast cancer cell line MDA-MB-231 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF10A cells were maintained in mammary epithelial cell growth medium (MEGM Bullet Kit; Lonza, Walkersville, MD, USA) supplemented with 100 ng/mL cholera toxin at 37°C with 5% CO2. MDA-MB-231 cells were maintained in Leibovitz's L-15 Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries Ltd. Kibbutz Beit Haemek, Israel) and L-glutamine at 37°C with 0% CO₂. The human breast cancer cell line MCF7 was purchased from the Riken BioResource Center Cell Bank (Riken BRC Cell Bank, Ibaraki, Japan) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS, glucose, and L-glutamine at 37°C with 5% CO₂.

Immunofluorescence analysis. MCF10A (4.0×10⁴ cells/cm²), MCF7 (2.0×10⁴ cells/cm²), and MDA-MB-231 (4.0×10⁴ cells/cm²) cells were separately seeded in 8-well chamber slides (Nalgen Nunc International, Rochester, NY, USA) and incubated for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with ice-cold 100% methanol at −20°C for 10 min. These samples were blocked in 10% goat serum/ phosphate-buffered saline (PBS) at room temperature for 60 min and subsequently incubated with the anti-Maspin primary antibody (G167-70, diluted 1:10; BD Pharmingen, Franklin Lakes, NJ, USA) at 4°C overnight, followed by incubation with Alexa Fluor conjugated anti-mouse secondary antibody (Molecular Probes, Leiden, Netherlands). Nuclear counterstaining was performed using DAPI (Molecular Probes, Leiden, Netherlands). Immunofluorescence images were detected using an FV1000 confocal microscope (Olympus, Tokyo, Japan).

RNA extraction and quantitative polymerase chain reaction. Total RNA from MCF10A, MCF7, and MDA-MB-231 cells was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and reverse-transcribed into complementary DNA (cDNA) using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's protocol. We measured the gene expression levels by performing TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) with the following gene-specific primers: GAPDH, Human GAPDH Endogenous Control (Hs99999905_m1, Thermo Fisher Scientific, Waltham, MA, USA) and HDAC1, TaqMan Gene Expression Assays (Hs00606262_g1, Thermo Fisher Scientific, Waltham, MA, USA).

Statistical analysis. All statistical analyses were performed using the SPSS version 23 software (IBM SPSS Statistics; IBM Corporation, Armonk, NY, USA). The associations between maspin expression status and clinicopathological factors, and the correlation between the subcellular localization of maspin and HDAC1 expression status were evaluated by the Chi-square test. We compared the relative HDAC1 mRNA expression by using a one-way analysis of variance (ANOVA) and the Tukey honest significant difference (HSD) test. For the survival analyses, cancer relapse (local recurrence or distant recurrence) was used to calculate disease-free survival (DFS). DFS was defined as the period from the date of primary surgery to the date of clinical or pathological cancer relapse. In the calculation of DFS, patients were censored at the time of their last cancer-free follow-up or at the time of death due to a reason unrelated to cancer. Survival curves were plotted by the Kaplan-Meier method and differences in DFS were tested using the log-rank test. All tests were two-sided, and differences were considered significant at p-values<0.05.

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Figure 1.

Immunohistchemical staining patterns of maspin in invasive carcinoma of the breast. A: Cytoplasmic-only expression of maspin. B: Pancellular (combined nuclear and cytoplasmic) expression of maspin. Immunohistchemical staining of HDAC1 in invasive carcinoma of the breast. C: High expression of HDAC1 (intensity score: 3, proportion score: 4). D: Low expression of HDAC1 (intensity score: 2, proportion score: 2).