Sample collection
We obtained 13 BC cell lines (BT-20, BT-474, BT-549, HCC1419, HCC1954, Hs578T, MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-468, SK-BR-3, and ZR-75-1) and two non-cancerous breast epithelial cell lines (MCF-10A and MCF-12A). BT-549, HCC1419, HCC1954, and Hs578T cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). BT-474, MCF7, and MCF-12A were kindly provided by Prof. David Sidransky from Johns Hopkins University (Baltimore, MD, USA). Other cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were stored at –80°C using a cell preservation solution (Cell Banker; Mitsubishi Chemical Medicine Corporation, Tokyo, Japan) and cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and incubated in an atmosphere of 5% carbon dioxide at 37°C [12].
We also collected primary BC and non-cancerous specimens from 167 patients histologically diagnosed with BC after undergoing surgery at Nagoya University Hospital from March 2002 to May 2007. Surveillance data for more than five years after surgery for all 167 patients were available. All specimens were immediately resected to a diameter of approximately 1.5 mm and stored at –80°C. Non-cancerous specimens were resected ≥3 cm from the edge of the tumor [13]. All specimens were histologically diagnosed as BC and classified using the Union for International Cancer Control (UICC) staging system (8th edition). Postoperative adjuvant therapy was determined on the basis of the patient’s condition, pathological features, cancer subtype, and physicians’ discretion [13].
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
PFKP mRNA expression levels were evaluated by qRT-PCR. RNA was extracted from cell line (8.0 × 106 cells per cell line), as well as from BC and non-cancerous specimens from 167 patients. cDNA was synthesized as described previously [12]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were quantified to normalize expression levels. The primers specific for each gene were as follows: PFKP: forward 5'-GGGCCAAGGTGTACTTCATC-3' and reverse 5'-TGGAGACACTCTCCCAGTCG-3', which generated a 90-bp product. GAPDH: forward 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse 5'-GAAGATGGTGATGGGATTTC-3', which generated a 226-bp product [13]. qRT-PCR was performed using an ABI StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, USA) as described previously [12]. The mRNA expression level of PFKP was obtained dividing each sample’s value by the corresponding GAPDH value [12].
PCR array analysis
To determine the correlation between the expression levels of PFKP and 84 cancer-related genes in BC cell lines, PCR array analysis was conducted using RT2 Profiler PCR Array Human Oncogenes & Tumor Suppressor Genes (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
PFKP knockdown using PFKP-specific small interfering RNAs (siRNAs)
For PFKP knockdown, MCF7, SK-BR-3, and MDA-MB-231 cell lines were transfected with three kinds of siRNAs specific for PFKP, named “siPFKP”. Their sequences were siPFKP-1: 5'-UAUUAAUGUCAAUAAUACGUG-3'; siPFKP-2: 5'-GGAGCAAUUGAUACCCAAATT-3'; and siPFKP-3: 5'-GGAUCACUGCAAAACUCAATT-3' (Hokkaido System Science, Sapporo, Japan). AccuTargetTM Negative Control siRNA Fluorescein-labeled (Cosmo Bio Co. Ltd., Tokyo, Japan) served as control nontargeting siRNA, named “siControl”. BC cells were seeded in antibiotic-free RPMI-1640 supplemented with 10% FBS; 24 h after seeding, cells were transfected the corresponding siRNAs in the presence LipoTrust EX Oligo (Hokkaido System Science). After transfection, cells were cultured in antibiotic-free RPMI-1640 with 10% FBS for 72 h. Knockdown efficiency was determined using qRT-PCR.
Simple Western analyses
Simple Western analyses was performed using the WES machine (ProteinSimple, San Jose, CA, USA) according to the manufacturer’s recommendations. Cells were incubated in RIPA lysis buffer, and the lysates were stored at –30°C. Protein concentration was measured using a BCA protein assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples, biotin ladder, primary antibody, secondary antibody, blocking reagent, chemiluminescent substrate, and wash buffer were prepared and dispensed into the assay plate. Then, the assay plate was loaded into the instrument, and the protein was separated into individual capillaries. Protein separation and detection was performed automatically on individual capillaries. Anti-PFKP antibody (1:50 dilution) (Cell Signaling Technology, Beverly, MA, USA) and anti-beta actin antibody (1:50 dilution) (Abcam, Cambridge, UK) were used as primary antibodies. Streptavidin HRP and anti-mouse or anti-rabbit secondary antibodies (ProteinSimple) were selected according to the corresponding primary antibody [14,15].
Proliferation assay
Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). MCF7 (3.0 × 103 cells per well), SK-BR-3 (3.0 × 103 cells per well), and MDA-MB-231 (3.0 × 103 cells per well) cells, which transfected with siPFKP or siControl, were seeded into 96-well plates with RPMI-1640 containing 10% FBS and 1% antibiotic. Each sample was applied to six wells, and the optical density of each well was measured 2 h after adding 10 μL of CCK-8 solution up to 5 days after seeding [13].
Invasiveness assay
Invasiveness in Matrigel was determined using BioCoat Matrigel Invasion Chambers (Corning Inc., Corning, NY, USA) according to the manufacturer's protocol. After transfection, MCF7 (2.5 × 104 cells per well), SK-BR-3 (2.5 × 104 cells per well), and MDA-MB-231 (2.5 × 104 cells per well) cells were suspended in serum-free RPMI-1640 and seeded in the upper chambers. After 72 h of incubation, cells on the membrane surfaces were fixed, stained, and counted in ten randomly selected microscope fields [13].
Migration assay
Migration of MCF7, SK-BR-3, and MDA-MB-231 cells was determined using a wound-healing assay. After transfection, MCF7 (4.9 × 104 cells per well), SK-BR-3 (4.9 × 104 cells per well), and MDA-MB-231 (4.9 × 104 cells per well) cells were seeded into each well of a 35-mm dish with culture insert (Ibidi, Martinsried, Germany) using RPMI-1640 containing 10% FBS and 1% antibiotic. After 24 h, the insert was removed, and wound widths were measured 20 times per well at 100-μm intervals [13].
Kaplan–Meier survival analysis using Kaplan–Meier Plotter
We used the website of the Kaplan–Meier Plotter (http://kmplot.com/analysis/index.php?p=background) to analyze relapse-free survival (RFS) and OS for patients with BC with respect to expression of PFPK by classifying its expression levels into the upper quartile and others [16].
Statistical analysis
Numeric variables between two groups were compared using the Mann–Whitney test. Spearman’s rank correlation test was performed to evaluate the correlation between PFKP and cancer-related gene expression levels in the PCR array analysis. We analyzed the association between PFKP mRNA expression and clinicopathological factors using the χ2 test. Disease-free survival (DFS) and OS were calculated using the Kaplan–Meier method, and survival curves were compared using the log-rank test. All statistical analyses were performed using JMP 15 software (SAS Institute Inc., Cary, NC, USA), and statistical significance was defined as P < 0.05.