Patients and methods
All breast samples were obtained from patients diagnosed and treated at the A. C. Camargo Hospital, São Paulo, Brazil. TMAs were arranged from a series of 1040 cases of primary breast carcinoma from patients diagnosed between 1980 and 1999. Duplicate 1 mm cores from tumors were used in the TMAs. The median age of the patients was 55 years (range: 26–90 years). The tumor tissues were removed at surgery. The A. C. Camargo Hospital Ethics Committee approved this research (Process number: 1648/12) and waived the need for informed consent term.
Vimentin and pan-cytokeratin were used as tissue immunoreactivity and staining controls. RECK staining was performed using a monoclonal antibody diluted 1:200 (Cell Signaling, Beverly, MA, USA). Several patient and tumor characteristic data were available, including age, menopausal status, tumor size, lymph node stage, SBR stage (Scarff-Bloom-Richardson, histological classification method) and TNM tumor stage. The expression state of several biomarkers, such as estrogen receptor (ER), progesterone (PR), Her-2 and cytokeratins (CK5, CK6, CK8, CK14 and CK18), was also evaluated by two distinct pathologists.
The ScanScope XT device was used for slide scanning. The percentage of cells positively stained for RECK, and its staining pattern in the TMA core, was determined using the Spectrum Plus computational program, by a trained person. For each subject, at least three measurements were obtained, from two distinct slides, with samples represented in duplicate. The average of these three values was considered as the RECK immunoreactivity for each subject. RECK immunoreactivity values greater than the median were considered as positive (or high expression).
Cell lines and reagents
Six breast cell lines (S1, MCF-7, T47D, T4-2, MDA-MB-231 and Hs578T) with distinct tumorigenic and aggressiveness levels were used in this study . The broad-spectrum MMP inhibitor (GM6001) was purchased from Millipore (Billerica, MA, USA).
Viral transduction and shRNA experiments
For the inhibition of RECK expression, five different specific shRNA sequences (sequentially numbered from shRECK23 through shRECK27) were tested. These vectors and the structural ones belonged to the Mission® shRNA system (Sigma-Aldrich, St. Louis, MO, USA). Viral recombinant particles were generated by co-transfection of the 293FT virus packer cell line with these recombinant lentiviral vectors. Transduced cells were selected by the addition of 2 μg/mL puromycin.
Quantitative RT-PCR studies
Total RNA was extracted from cell lines using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). Its integrity and quantity were evaluated using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, the SuperScript® III Reverse Transcriptase Kit was used (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using QuantiTect SYBR Green RT-PCR Kit (Qiagen). The primers used to amplify RECK, MMP-9 and the endogenous control (18S ribosomal RNA) were obtained from a validated primer panel available from Qiagen (QuantiTect Primer Assays). Each experiment was carried out in duplicate. The relative expression levels were calculated according to the 2-∆∆Ct method .
Total extracts were prepared from each cell culture line. Equal protein amounts from each extract were fractionated by SDS-PAGE and then electro-transferred to nitrocellulose membranes. After blockade with BSA, these membranes were incubated with RECK (Cell Signaling) and Lamin A/C (Santa Cruz, Santa Cruz, CA, USA) antibodies, diluted at 1:1000 and 1:200, respectively. Immunoreactive proteins were detected with the appropriate secondary horseradish peroxidase-conjugated antibody (GE Healthcare, Little Chalfont, UK) and visualized using the SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific). Quantitative densitometry of the electrophoretic band images was carried out using AlphaEaseFC software.
Gelatin zymography assays
Gelatin zymography of the conditioned media was used to assess the in vitro enzymatic activity levels of MMP-9 in RECK-silenced and scrambled MDA-MB-231 cells. These samples were fractionated by 10 % SDS-polyacrylamide gel electrophoresis co-polymerized with the enzyme substrate, 0.1 % denatured type I collagen (gelatin type A; Sigma, St. Louis, MO, USA). After electrophoresis, the gels were washed at room temperature with 2.5 % Triton X-100, incubated for 48 h at 37 °C in substrate buffer containing 50 mM Tris buffer (pH 8.5) and 10 mM CaCl2. Gels were stained with Coomassie Blue R-250 (Sigma) and destained with 40 % methanol (Merck) and 10 % acetic acid (Merck) in water. Gelatinolytic activity was visualized as negatively staining bands. Each independent experiment was performed in duplicate. Quantitative densitometry of the electrophoretic band images was performed using the AlphaEaseFC software.
In vitro invasion assays
To assess the role of RECK in cellular invasiveness potential, RECK-deficient or scrambled control MDA-MB-231 cells were plated on top of Matrigel-coated 8-μm-pore transwells (BD Bioscience, Franklin Lakes, NJ, USA). These cells were maintained in the presence of GM6001 or its vehicle (DMSO) in serum-free medium. The medium present in the bottom chamber was supplemented with 10 % bovine serum, used as a chemo-attractant. Cells were allowed to invade for 36 h. Cells remaining at the top chamber were removed, and those present at the bottom of the filter were stained and fixed with Coomassie Blue 0.125 % in methanol:acetic acid:H2O (45:10:45, v/v/v) for 15 min. Using the ImageJ® program, the relative cellular invasion was quantified from the images obtained (10× objective lens) under each experimental condition. Non-tumorigenic S1 mammary cells were used as a negative control. Triplicate wells were utilized per condition in each independent experiment.
Statistical analysis of data from patients was performed using R (http://www.r-project.org/) . Chi-square tests were used to assess the association between RECK immunoreactivity and different clinico-pathological parameters. P-values were corrected for multiple tests by the False Discovery Rate procedure . Kaplan-Meier (KM) plots were used for overall (OS) and disease-free (DFS) survival analysis, and the log-rank test was used to compare curves separated according to RECK expression. For KM analysis, 3 of 1040 subjects were identified as being outliers by Tukey’s criterion; thus, they were removed from the data analysis. Cox proportional hazards regression was used to estimate hazard ratios (HRs). Subjects for whom no information on the analyzed covariates was available, were removed, with 940 of the 1040 subjects remaining for the Cox regression analysis.
Data obtained using the cell lines model were analyzed using the GraphPad Prism 5.0 program. In this case, statistical significance was determined using the one way ANOVA variance analysis and the Tukey-Kramer test. The results were presented as the mean ± standard error of the mean. A p-value less than 0.05 was considered to be statistically significant.