The breast tumor tissues and paired normal adjacent tissues were collected from patients who underwent surgical resection at the Department of Breast and Thyroid Surgery of the Shanghai Tenth People’s Hospital (Shanghai, China) between December 2016 and February 2017. The patients were women between the ages of 32 and 71 years, with a mean age of 53 years. None of the patients had received any chemotherapy (19) or radiotherapy prior to surgery. Patients with distant metastases or a history of a previous or concomitant malignancy were excluded. The samples were immediately snap-frozen in liquid nitrogen. Tumor and normal tissues were histologically confirmed by more than one experienced pathologist according to the World Health Organization guidelines (19), using hematoxylin and eosin staining. All specimens were embedded in 10% formalin solution for 12–24 h at room temperature and cut into 5-μm thick sections. The sections were stained with hematoxylin for 3–10 min and with eosin for 60 sec at room temperature, and then observed under a light microscope at ×40 magnification. The specimen collection and use was approved by the Institutional Ethics Committees of Tongji University (Shanghai, China). All patients provided written informed consent. The data of the patients are not shown.

Human breast cancer cell lines, MDA-MB-231, HCC-1937, MCF-7, BT-549 and MDA-MB-468, and the human mammary epithelial cell line, MCF-10A, were purchased from the Chinese Academy of Sciences (Shanghai, China). The BT-549 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Enpromise, Hangzhou, China) and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). MCF-10A cells were cultured in mammary epithelial basal medium (Cambrex Corporation, East Rutherford, NJ, USA). The remaining cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 μg/ml) (both from Enpromise). All cells were incubated at 37°C in a humidified chamber containing 5% CO₂.

ASPP2 siRNA and negative control siRNA (NC siRNA) oligonucleotides were chemosynthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequence of the ASPP2 siRNA was 5′-GCCCAGUAGAAAUCCAGAATT-3′ (sense) and 5′-UUCUGGAUUUCUACUGGGCTT-3′ (antisense), while the sequence of the NC siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense).

The MDA-MB-231, MCF-7 and HCC-1937 cells (8×10⁴/well) were cultured in a 6-well plate with serum and antibiotic-free DMEM for transfection. When the confluence reached 30–50%, transfection of ASPP2 siRNA and NC siRNA was performed using the Lipofectamine^® 2000 Transfection kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer′s protocols, at working concentrations. The concentration of siRNAs used was 100 nmol/l, and the ratio of mimics to Lipofectamine 2000 was 1.25:1.00 (volume). The medium was replaced by DMEM with 10% FBS after 4–6 h of incubation. The cells were used for future analysis after 48 h of transfection.

Total cellular RNA was extracted from the transfected MDA-MB-231, MCF-7 or HCC-1937 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and stored at −80°C. For ASPP2 detection, cDNA was generated by RT using the PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan) in accordance with the manufacturer′s protocols. Conditions of the RT reaction were 37°C for 15 min, then 85°C for 5 sec. RT-qPCR was performed using SYBR-Green PCR master mix (Takara Bio, Inc.) on a 7900HT Fast RT-PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The amplification procedure was as follows: 3 min at 95°C, followed by 40 cycles at 95°C for 3 sec and 60°C for 30 sec. The relative expression was evaluated following the relative quantification 2^−ΔΔCt method (20). Each sample was tested in triplicate. The primers used in the RT-PCR were as follows: ASPP2 forward, 5′-CTGTGCAAA GAACCCGGCG-3′ and reverse, 5′-CAACTGGACGTTCAGAGCCACA-3′; and β-actin forward, 5′-CAGAGCCTCGCCTTTGCC-3′ and reverse, 5′-GTCGCCCACATAGGAATC-3′.

The transfected MDA-MB-231, MCF-7 or HCC-1937 cells were harvested and lysed in radioimmunoprecipitation assay lysis buffer (80 μl/well; Beyotime Institute of Biotechnology, Jiangsu, China) after 48–72 h of transfection. The protein concentration was quantified with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Next, equal amounts of protein (30–50 μg) were separated by 8 or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology), and then transferred to 0.45-μm nitrocellulose membranes using the cold transfer buffer (3.03 g Tris + 14.4 g glycine + 200 ml methanol + 800 ml deionized water). Subsequent to blocking at room temperature for 1 h in 5% skimmed milk diluted with phosphate-buffered saline plus Tween-20 (PBST), the membranes were hybridized overnight at 4°C with specified primary antibodies in PBST containing 5% skimmed milk. Subsequently, the membranes were washed with PBST and incubated with IRDye 680 donkey anti-mouse IgG-(H+L) (1:1,000 dilution; cat. no. 926-68072) or goat anti-rabbit IRDye 800CW secondary antibody (1:1,000 dilution; cat. no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at a room temperature. Protein bands were detected with an Odyssey Scanning system (LI-COR Biosciences).

Antibodies used were follows: Anti-ASPP2 (1:20,000 dilution; cat. no. ab181377; Abcam, Cambridge, UK), anti-β-actin (1:2,000 dilution; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-caspase-9 (1:1,000 dilution; cat. no. ab202068; Abcam), anti-caspase-3 (1:1,000 dilution; cat. no. 9662; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-poly (ADP-ribose) polymerase (PARP; 1:1,000 dilution; cat. no. ab191217; Abcam), anti-Bax (1:1,000 dilution; cat. no. 2772), anti-E-cadherin (1:750 dilution; cat. no. 3195) (both from Cell Signaling Technology, Inc.), anti-N-cadherin (1:2,000 dilution; cat. no. ab18203), anti-Snail (1:1,000 dilution; cat. no. ab82846), anti-zinc finger E-box-binding homeobox 1 (ZEB1; 1:1,000 dilution; cat. no. ab155249), anti-matrix metalloproteinase 2 (MMP2; 1:2,000 dilution; cat. no. ab37150) (all from Abcam), anti-MMP9 (1:1,000 dilution; cat. no. 54980; Arigo Biolaboratories, Hsinchu, Taiwan), anti-phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1; 1:1,000 dilution; cat. no. 13666), anti-AKT (1:1,000 dilution; cat. no. 9272), anti-phosphorylated (p-)AKT (ser-473; 1:1,000 dilution; cat. no. 4060) (all from Cell Signaling Technology, Inc.), anti-extracellular signal-regulated kinases (ERK; 1:2,000 dilution; cat. no. ab17942) and anti-p-ERK (ser-T202 and ser-T185; 1;1,000 dilution; cat. no. ab201015) (both from Abcam).

At 24 h post-transfection, the MDA-MB-231 and HCC-1937 cells were seeded in 200 μl growth medium at 5×10² cells per well in 96-well plates (BD Biosciences, Franklin Lakes, NJ, USA) and incubated overnight at 37°C in 5% CO₂. Every 24 h until 72 h, 20 μl MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution was added to each well and incubated at 37°C for 4 h. Next, 150 μl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to each well and agitated gently for 10 min to dissolve the MTT formazan crystals after removing the supernatant. Cell viability was measured by the recording absorbance at 490 nm with a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

A total of 1×10³ transfected MDA-MB-231 and HCC-1937 cells from each group were seeded in a 6-well plate in DMEM with 10% FBS. The plates were agitated to disperse the cells equally. After 7 to 10 days of culturing, or when the colonies were visible, the cell culture was terminated and the plates were washed twice with PBS. Next, the cells were fixed in 95% ethanol for 10 min, dried and stained with 0.1% crystal violet solution for 10 min at room temperature. Finally, the staining solution was washed away and the number of colonies with diameters of >1.5 mm was counted by eye. The experiments were performed in triplicate.

To assay the migratory response of breast cancer cells to ASPP2 expression, the transfected MDA-MB-231 and HCC-1937 cells were seeded into 6-well plates and cultured until the cells reached ~90% confluence. Next, a scratch was made in each well using a sterile pipette tip. Cells were washed with PBS to remove cellular debris and allowed to migrate for 48 h. The process of wound healing was observed under a light microscope and representative images were acquired at 0 and 48 h post-wounding with a digital camera system. All experiments were performed in triplicate.

The transfected MDA-MB-231 and HCC-1937 cells at a density of 5×10⁴ were suspended in serum-free DMEM (200 μl) and added into the upper chamber of the Transwell, with a Matrigel-coated (2 mg/ml) membrane containing 8-μm diameter pores, to observe invasion following transfection. Complete DMEM (500 μl) was then added to the bottom chamber of 24-well plates to serve as a chemoattractant. Subsequent to 20 h of incubation at 37°C in 5% CO₂, the non-invading cells on the upper surface were carefully removed with a cotton swab. The cells that had invaded the lower surface of the membrane were fixed with 10% formalin for 30 min prior to staining with crystal violet for 15 min at room temperature, and then counted under a light microscope at ×200 magnification. The cells were counted in five random fields on each membrane. The experiments were conducted in triplicate.

For the measurement of apoptosis, at 24 h post-transfection, the MDA-MB-231 and HCC-1937 cells (2×10⁵) were treated with 1μmol/l docetaxel for 36 h. The cells were then collected in centrifuge tubes (1,000 × g, at room temperature for 5 min), and washed in chilled PBS. Subsequently, the cells were re-suspended in 250 μl binding buffer, and Annexin V/fluorescein isothiocyanate solution and propidium iodide (PI) solution were added to the cell suspension. Following incubation for 30 min, the rate of apoptosis was detected by flow cytometry (FACSCanto™ II; BD Biosciences).

Data are presented as the mean ± standard deviation. Two-way analysis of variance or Student’s t-test was used for comparisons between groups. P<0.05 was used to indicate a statistically significant difference. GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) or the SPSS program (IBM Corp., Armonk, NY, USA) was used to perform the statistical analyses.

To analyze the mRNA levels of ASPP2 expression in breast cancer tissues compared with those in para-cancerous normal tissues, the level of ASPP2 mRNA was determined by RT-qPCR. The results showed that ASPP2 mRNA expression was suppressed in a number of the breast cancer tissues compared with that in the matched normal tissues (P<0.05; Fig. 1A). In addition, the mRNA and protein expression of ASPP2 was examined in a panel of breast cancer cell lines (BT-549, MDA-MB-231, HCC-1937, MDA-MB-468 and MCF-7) compared with the expression in the breast epithelial cell line (MCF-10A). Notably, with the exception of HCC-1937 cells, a reduction in ASPP2 mRNA expression was found in all the remaining cancer cell lines compared with that in MCF-10A, as measured by RT-qPCR (P<0.001; Fig. 1B). Meanwhile, western blot analysis showed that the majority of cancer cell lines (with the exception of HCC-1937 cells) expressed lower levels of ASPP2 protein compared with MCF-10A, which was consistent with the tendency of the RT-qPCR results (P<0.001; Fig. 1C and D). For further investigation, ASPP2 siRNA was transfected into three different cell lines (MDA-MB-231, HCC-1937 and MCF-7 cells), and the interference effect on endogenous ASPP2 expression was validated by RT-qPCR and western blotting. Following transfection with ASPP2 siRNA, the expression of ASPP2 decreased at the mRNA and protein levels in all three different breast cancer cell lines (P<0.001; Fig. 1E–G). Accordingly, siRNA transfection was considered to be effective for ASPP2 silencing in breast cancer cells.

For the determination of the impact of ASPP2 on the cell viability and cell proliferation of TNBC cells, MDA-MB-231 and HCC-1937 cells were transfected with ASPP2 siRNA and NC siRNA. Subsequently, MTT and colony formation assays were performed. As shown in Fig. 2A, as determined by MTT assay, silencing ASPP2 clearly enhanced the cell proliferation in a time-dependent manner in the MDA-MB-231 and HCC-1937 cells. Likewise, the downregulation of ASPP2 caused a significant increase in the colony formation number compared with the NC siRNA transfected cells in the two cell lines (P<0.01; Fig. 2B and C). All the results indicated that ASPP2 could promote MDA-MB-231 and HCC-1937 cellular growth.

To investigate whether ASPP2 silencing could increase cell viability by reducing cell apoptosis, docetaxel (1 μmol/l) was added to the transfected cells to induce apoptosis following 24 h of transfection. Next, 36 h later, flow cytometric analysis was performed to analyze the apoptosis in the MDA-MB-231 and HCC-1937 cells. The total apoptosis rate of the cells was reflected by the number of early and late apoptotic cells in the Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺ domains. As shown in Fig. 2D and E, in comparison with the NC groups, the siRNA transfection group significantly decreased apoptosis. Furthermore, the western blotting results showed that the levels of apoptosis-related proteins, including cleaved caspase-9, cleaved caspase-3, cleaved PARP and Bax, were significantly decreased compared with those of the NC groups (Fig. 2F and G). Caspase-9 is at the top of the caspase cascade activation response, as the most important promoter and key protease of mitochondrial apoptotic pathway, the activation of which can further activate the downstream Caspase family and then promote cell apoptosis (21). Bax can enhance the permeability of the mitochondrial membrane for entry of cytochrome c into the cytoplasm and then promote cell apoptosis (22). All the data indicated that ASPP2 silencing promoted cell proliferation and reduced cell apoptosis, perhaps through the mitochondrial death pathway.

To investigate the role of ASPP2 in cell migration and invasion, wounding-healing and Transwell invasion assays were performed. The wound-healing assay showed that ASPP2 silencing significantly promoted the migration ability of the MDA-MB-231 and HCC-1937 cells compared with the NC (both P<0.001; Fig. 3A and B). The Transwell invasion assay showed that ASPP2 silencing increased the invasion ability of the MDA-MB-231 and HCC-1937 cells (P<0.01 and P<0.001; Fig. 3C and D). These results indicated a direct association between ASPP2 and the motility of TNBC cells. On the basis of the cell functional study, research on the EMT-related proteins was performed using western blot to further investigate the effect of ASPP2 on the migration and invasion mechanism. Following depletion of ASPP2, the expression of representative epithelial marker E-cadherin significantly decreased, whereas the expression of mesenchymal marker N-cadherin and other key markers, including Snail and ZEB1, all increased. Furthermore, the levels of MMP2 and MMP9, which are involved in EMT, also increased (Fig. 3E and F). Taken together, these results suggest that ASPP2 silencing may be responsible for EMT development, and this serves a vital role in the progression of breast cancer.

Activation of the PI3K/AKT signaling pathway is regarded as a crucial emblem in breast cancer that is associated with its development, progress and metastatic spread (23). To further validate whether ASPP2 is involved in the p53-independent pathway in TNBC, the effect of ASPP2 on the PI3K/AKT pathway was investigated. The important molecular markers associated with the PI3K/AKT pathway were then detected. As shown in Fig. 4, the downregulation of ASPP2 resulted in the decreased expression of PIK3R1 (p85α) and the increased expression of p-AKT, whereas it had no influence on the expression of p-ERK.

PIK3R1 (p85α) is the regulatory subunit of PI3K and negatively regulates the PI3K pathway (24). The present results suggested that the downregulation of ASPP2 was able to activate the PI3K/AKT pathway in TNBC cells.