The human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF7, T-47D, SK-BR-3, MDA-MB-231, MDA-MB-468 and BT-549 were bought from ATCC (Rockville, MD, USA). MCF10A was cultured in MEGM with100 ng/ml cholera toxin (ATCC, USA). MCF7, SK-BR-3 and MDA-MB-468 were cultured in DMEM with 10% fetal bovine serum (FBS, Biowest). T-47D and BT-549 were cultured in RPMI 1640 with 10% FBS. MDA-MB-231 was cultured in L-15 with 10% FBS. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Quantitative real-time PCR assay (qRT-PCR)
Trizol reagent (Sangon, Shanghai, China) was used to extract total RNA from different cells following the manufacturer’s instructions. Semiquantitative reverse transcription-PCR (RT-PCR) was performed using the one-step RT-PCR kit (Takara, Japan). QRT-PCR assay was carried out in 20μL solution with 100 ng cDNA and 0.2 μM of each forward and reverse primer and 10 μL of 2 × SYBR green Mix (Takara, Japan). Samples were run using the StepOnePlus real-time PCR system (ABI). The following primers were used for the specific amplification of GAPDH, ADAM10, NOTCH1, CD44 and PRNP: GAPDH forward primer: 5’-catcaagaaggtggtgaagc-3’, and reverse primer: 5’-ggaaattgtgagggagatgc-3’; ADAM10 forward primer: 5’-GCAGACTCGTGGGAAGTTGT-3’, and reverse primer: 5’-ACAGGACACAGGAAGAACCG-3’; NOTCH1 forward primer: 5’-GGACGTCAGACTTGGCTCAG-3’, and reverse primer: 5’-ACATCTTGGGACGCATCTGG-3’; CD44 forward primer: 5’-CAGCAACCCTACTGATGATGACG-3’, and reverse primer: 5’-GCCAAGAGGGATGCCAAGATGA-3’; PRNP forward primer: 5’-AGTGGAACAAGCCGAGTAAGC-3’, and reverse primer: 5’-GTCACTGCCGAAATGTATGATG-3’.
Western blot analysis and co-immunoprecipitation
Total protein was extracted from cells using RIPA lysis buffer. Cytoplasmic and nuclear proteins were extracted from cells using NE-PER Nuclear and Cytoplasmic Extracion Reagents (Thermo, USA). For Western blots, 30μg protein extracts were electrophoresed, transferred to PVDF (Millipore, USA) membranes, blocked in 5% non-fat milk for 2h, and incubated overnight with antibodies against ADAM10 (ab1997, Abcam, USA), PrPc (P0110, Sigma, USA), CD44 (3570, Cell Signaling Technology, USA), Notch1(3608, Cell Signaling Technology, USA), Cleaved-Notch1 (4147, Cell Signaling Technology, USA), Cyclin D3 (2936, Cell Signaling Technology, USA), p21 Waf1/Cip1 (2947, Cell Signaling Technology, USA), HES1 (11988, Cell Signaling Technology, USA) and c-Myc (13987, Cell Signaling Technology, USA), GAPDH (sc-47724, Santa Cruz, USA), TBP (sc-74596, Santa Cruz, USA), respectively. And then membranes were incubated for 1h at room temperature with the appropriate HRP-conjugated secondary antibodies (Proteintech, USA). Detection of protein expression levels by enzyme-linked chemiluminescence (ECL; Pierce, Rockford, USA) was performed according to the manufacturer's protocol.
For co-immunoprecipitation, cells were collected，washed with PBS, and lysed in TNT buffer supplemented with 1 tablet/50 mL of Complete Protease Inhibitor Cocktail (Roche Molecular Biochemical). Lysates were cleared by centrifugation (10,000 × g for 15 min at 4°C) and incubated on ice for 2 h with 10μg anti-ADAM10, anti-CD44 or anti-PrPc. The antigen sample/antibody mixture was added to a 1.5mL microcentrifuge tube containing pre-washed Protein G Magnetic Beads (Pierce, Germany) and incubated at room temperature for 1 hour with mixing. The beads were retrieved by centrifugation and washed (by vortex and short spin) three times with Wash Buffer. Proteins bound to the beads were eluted by boiling in 2 × electrophoresis sample buffer. Then Western blots were performed as described above.
Inhibition of ADAM10 expression by RNA interference
2×105 cells per well in 2ml antibiotic-free normal growth medium supplemented with FBS, were seeded in 6-well plates in triplicates. After an overnight incubation, the cells were transfected with different dilutions of siRNA using transfection Reagent (sc-29528, Santa Cruz, USA) as suggested by the manufacturer’s instructions. The small interference RNA (sc-41410, Santa Cruz, USA) was used to target ADAM10 mRNA sequence, while control siRNA (sc-37007, Santa Cruz, USA) was used as negative control. After 24h, 48h or 72h, total RNA was extracted and RT-PCR was performed. Real-time PCR was carried out to detect the mRNA of ADAM10. At 48h or 72h after transfection, total protein was extracted and protein expression was determined by Western blot.
Cell migration and invasion assays
The migration and invasion assays of cells were performed as previously described , using transwell chambers with 8-μm pore size membranes (Corning Costar, USA) without or with Matrigel (BD Biosciences, San Jose, USA).
Cell proliferation assay and drug sensitivity assay
Cell proliferation was assessed using CCK8 (Dojindo, Tokyo, Japan). The cells were seeded on 96-well microplates at a density of 5×103 cells per well. At 0–4 days after transfection with ADAM10 siRNA, the cells were incubated with 10 μl of CCK8 for 3h. Then the OD of each sample was measured at a 450nm test wavelength with an ELISA multi-well spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, USA).
For drug sensitivity assay, cells transfected with ADAM10 siRNA or negative control were seeded in 96-well plates at a density of 3×104 cells per well and incubated with serially diluted paclitaxel (0, 2, 4, 6 and 8 μg/ml), or adriamycin (0, 0.2, 0.4, 0.6 and 0.8 μg/ml) for 24h followed by 2h incubation with CCK-8 solution. The OD of each well was measured at a 450 nm test wavelength. The cell survival rate was calculated based on the OD of the negative control cells. The 50% inhibitory concentration (IC50) values were determined as the drug concentration causing 50% cell growth inhibition.
Flow cytometry analysis
For cell cycle analysis, cells were harvested, washed with PBS, and fixed. Prior to the analysis, the cells were incubated with fresh propidium iodide containing RNase for 30 min at 37 °C. DNA content was determined by fluorescence-activated cytometry (FACS) analysis of the propidium iodide-stained cells using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Apoptosis analysis was carried out by dual dye staining using Annexin V and 7-AAD. Cells were harvested, washed twice with PBS, and stained with PE Annexin V apoptosis detection kit according to the manufacturer’s instructions. The stained cells were subjected to a FACSCalibur flow cytometer and the results were analyzed using the Flow Jo 7.6.1 software (Tree Star Inc., USA).
Patient characteristics and immunohistochemistry
Paraffin-embedded tissue samples from 94 primary breast cancer patients, which were diagnosed as “invasive carcinoma of no special type” at the Department of Pathology, Huashan Hospital of Fudan University between 2011 and 2013, were collected. Prior to radical mastectomy, the patients received neoadjuvant chemotherapy treatment (NACT) with cyclophosphamide, epirubicin/epidoxorubicin and taxol combination therapy at the Department of Breast Surgery in Huashan Hospital. These patients were graded into G1-G5 based on the Miller-Payne grading system  according to their response to NACT, which were evaluated by two pathologists (Tang F and Bao Y). Then the 94 cases were divided into two cohorts, including Cohort 1 (G1 and G2) with poor response to NACT and Cohort 2 (G3, G4 and G5) with good response to NACT. From each patient, a core-needle biopsy of the tumor was taken before NACT and a post-NACT radical mastectomy was excised. The specimens were paraffin-embedded for subsequent immunohistochemical staining. Slides were dehydrated in xylene and graded alcohols. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 at 95℃ for 10 min. Then slides were incubated with diluted primary antibody anti-ADAM10 (sc-48400, Santa Cruz, USA) in 1:100 dilution for 12 h followed by incubations with biotinylated secondary antibody for 1h, and diaminobenzidine (DAKO, Denmark) for 10 min. Slides were again counterstained with Mayer’s hematoxylin.
The saturation and intensity of the immunostained cells were evaluated over three visual fields, at a power of × 200 under a light microscope (Nikon, Japan). ADAM10 immunoreactivity was detected mainly in the cytoplasm and cytomembrane. According to H scoring system , the total staining of ADAM10 was based on the intensity score (0, 1, 2, 3) multiplying the percentage of positive cells, giving a possible range of 0 to 300 (%). Then ADAM10 low expression group ranged from 0 to 200 (including 200), and the high expression group ranged from 200 to 300.
Statistical analyses were performed using IBM SPSS, version 21.0. All experiments were repeated at least three times and the results are presented with mean ± standarderrors (SEM). The differences were analyzed by using the Student’s t-test. To correlate ADAM10 expression with clinicopatholgical factors, we used Chi-Square or Mann-Whitney U tests, respectively, for categorical and non-categorical variables. Correlation analysis was performed using Spearman’s rank correlation coefficients. In multivariate analyses, all the data sets were pooled and the odds ratios and P values were estimated with logistic regression model stratified by study. Overall survival was calculated using the Kaplan-Meier analysis and differences between groups were assessed using log-rank tests. Univariate and multivariate Cox regression analysis was performed to evaluate differences of clinicopatholgical factors in the risk of death. For all tests, a p< 0.05 (two-tailed) was defined as statistically significant.