Human breast cancer cell lines MCF-7 (ER+/PR+/HER2-), MDA-MB-231 (ER-/PR-/HER2-), MDA-MB-468 (ER-/PR-/HER2-) and BT549 (ER-/PR-/HER2-) were obtained from American Type Culture Collection (Manassas, VA, USA). MCF-7 and BT549 cells were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Chalfont, UK) and antibiotics, and incubated in a humidified atmosphere with 5% CO₂ at 37°C. MDA-MB-231 and MDA-MB-468 cells were maintained in Leibovitz's-15 (L-15) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and antibiotics and incubated in a humidified atmosphere without CO₂ at 37°C.

Expression of the CIP2A gene was examined by quantitative polymerase chain reaction (PCR) normalized to the expression of GAPDH. Total RNA was extracted from cell lines or patients' cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol (14). RT-qPCR analysis of CIP2A was performed with 2 µg of total RNA and ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan). The reverse transcription conditions were as follows: 37°C for 15 min and 98°C for 5 min, followed by storage at −20°C. For qPCR, the following primers were used: CIP2A forward, 5′-5′-TGCGGCACTTGGAGGTAATTTC-3′ and reverse, 5′-AGCTCTACAAGGCAACTCAAGC-3′; and GAPDH forward, 5′-TGTTGCCATCAATGACCCCTT-3′ and reverse 5′-CTCCACGACGTACTCAGCG-3′. RT-qPCR was performed in an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction mix contained: 10 µl SYBR Green PCR Master mix, 200 nm forward and reverse primers, 100 ng cDNA template and ddH₂O up to 20 µl volume. The PCR cycling conditions consisted of the following: 94°C for 3 min for denaturation, 94°C for 30 sec for annealing and 58°C for 40 sec for extension, for a total of 40 cycles. The threshold cycle for each sample was selected from the linear range and converted to a starting quantity by interpolation from a standard curve generated on the same plate for each set of primers. The CIP2A mRNA levels were normalized for each well to GAPDH mRNA levels using the 2^−ΔΔCq method (15). Each experiment was repeated three times.

Cell pellets were lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and 1% protease inhibitors cocktail (Merck Millipore). Protein extracts were quantified and loaded (25 µg) on 8–12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to a polyvinylidene difluoride membrane (Merck Millipore). The membrane was blocked with 5% skimmed milk at room temperature for 1 h. The membrane was incubated with primary antibodies overnight at 4°C, followed by washing with TBST. Subsequently, membranes were incubated with goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no., E030120-01 and E030110-01; EarthOx Life Sciences, Millbrae, CA, USA) at room temperature for 1.5 h. Detection was performed by using a SuperSignal^® West Pico Trial kit (Pierce Biotechnology, Inc., Rockford, IL, USA) (16). The defined sections of the film were scanned for image capture and quantification using Adobe Photoshop software (CS4; Adobe Systems, Inc., San Jose, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used were anti-CIP2A (1:1,000; cat. no. sc-80662; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phospho-Akt (1:500; cat. no. sc-7985; Santa Cruz Biotechnology, Inc.), anti-Akt (1:500; cat no. sc-8312; Santa Cruz Biotechnology, Inc.), anti-c-Myc (1:500; cat. no. sc-788; Santa Cruz Biotechnology, Inc.) anti-caspase-3 (1:1,000; cat. no. 9662; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-poly ADP ribose polymerase (PARP) (1:1,000; cat. no. 9542; Cell Signaling Technology, Inc.), anti-LC-3 (1:1,000; cat no. 2775; Cell Signaling Technology, Inc.), anti-phospho-P70S6K (1:1,000; cat. no. 2983; Cell Signaling Technology, Inc.), anti-P70S6K (1:1,000; cat. no. 2708; Cell Signaling Technology, Inc.), anti-phospho-mTOR (1:1,000; cat. no. 5536), anti-mTOR (1:1,000; cat. no. 2973; Cell Signaling Technology, Inc.) and anti-GAPDH (1:5,000; cat. no. AB10016; Sangon Biotech Co., Ltd., Shanghai, China).

Two siRNA targeting CIP2A were designed and synthesized by Shanghai GenePharma Co. (Shanghai, China), referred to as siRNA1 and siRNA2. The siRNA sequences were as follows: 5′-CUGUGGUUGUGUUUGCACUTT-3′ (CIP2A siRNA1), 5′-ACCAUUGAUAUCCUUAGAATT-3′ (CIP2A siRNA2) and 5′-UUCUCCGAACGUGUCACGUTT-3′ [negative control (NC) siRNA].

Using lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, MDA-MB-231 and MDA-MB-468 cells were transfected with 100 nM siRNA. And 48 h transfection, the cells were harvested for western blot, cell viability, soft-agar colony formation assay, invasion assay and wound healing assay, as previously described (5,17).

Cell viability was estimated by trypan blue dye exclusion, as previously described (18). Briefly, a 0.4% solution of trypan blue was prepared in phosphate-buffered saline (PBS; pH 7.2–7.3). Then, 0.1 ml trypan blue stock solution was added to 1 ml cell suspension. A hemacytometer was then loaded with the samples and they were examined immediately under a microscope at low magnification (IX70; Olympus Corporation, Tokyo, Japan). The number of blue stained cells and the number of total cells were counted. Cell viability should be ≥95% for healthy log-phase cultures. Percentage viable cells = [1.00 - (Number blue cells / number total cells)] × 100. To calculate the number of viable cells per ml of culture, the following formula was used: Number of viable cells × (10⁴ × 1.1) = cells/ml culture.

Cells were suspended in 1 ml L-15 containing 0.3% low-melting-point agarose (Amresco Inc., Farmingham, MA, USA) and 10% FBS, and plated on a bottom layer containing 0.6% agarose and 10% FBS in 6-well plate in triplicate. After 2 weeks, plates were stained with 0.2% gentian violet and the colonies were counted under light microscope (5).

An invasion assay was performed using a 24-well plate (Corning). A polyvinyl-pyrrolidone-free polycarbonate filter (8 µm pore size) (Corning, Inc., Corning, NY, USA) was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The lower chamber was filled with medium containing 20% FBS as a chemoattractant agent. The coated filter and upper chamber were laid over the lower chamber. Cell suspension (1×10⁴ cells/well) was seeded onto the upper chamber wells. After incubation for 20 h at 37°C, the filter was fixed and stained with 2% ethanol containing 0.2% crystal violet (15 min). After drying, the stained cells were enumerated under a light microscope at 10x objective. For quantification, the invaded stained cells on the other side of the membrane were extracted with 33% acetic acid. The absorbance of the eluted stain was determined at 570 nm.

Cells (4×10⁵ cells/2 ml) were seeded in a 6-well plate and incubated at 37°C until 90 to 100% confluent. After the confluent cells were scratched with a 200 µl pipet tip, followed by washing with PBS, they were then re-suspended in complete medium. After 24 h incubation, the cells were fixed and stained with 2% ethanol containing 0.2% crystal violet powder (15 min), and randomly chosen fields were photographed under a light microscope at 4x objective. The number of cells migrated into the scratched area was calculated.

All experiments were repeated at least three times and the data are presented as the mean ± standard deviation unless noted otherwise. Data were analyzed using SPSS version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA). All experiments were repeated at least 3 times and the data are presented as the mean ± standard deviation unless noted otherwise. Differences between data groups were evaluated for significance using Student's t-test of unpaired data of one way analysis of variance, followed by the Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

CIP2A expression was studied by RT-qPCR and western blot analysis in TNSC cell lines MDA-MB-231, MDA-MB-468, and BT-549 cells. The poorly invasive ER+ breast cancer line MCF-7 was used as a positive control. The results show that CIP2A is expressed in BNSC cell lines at mRNA level. Interestingly, highly invasive TNSC breast cancer cells have increased CIP2A expression compared with poorly invasive ER+ MCF-7 cells (Fig. 1A). Consistent with mRNA expression, western blot analysis of the protein level of CIP2A revealed that highly invasive TNSC cells have increased CIP2A expression compared with poorly invasive ER+ MCF-7 cells (Fig. 1B). The results show that CIP2A is overexpressed in TNSC cells, and that higher expression of CIP2A in highly invasive TNSC cells may contribute towards invasion.

To evaluate the role that CIP2A overexpression serves in TNSC cells, MDA-MB-231 and MDA-MB-468 cells were transfected with siRNA1 or siRNA2 targeting CIP2A (Fig. 2A). CIP2A silencing significantly inhibited proliferation of MDA-MB-231 and MDA-MB-468 cells (Fig. 2B; P<0.01). The proliferation rate was determined by trypan blue dye exclusion assay. Consistent with the trypan blue dye exclusion results, colony formation assay showed that CIP2A knockdown in MDA-MB-231 cells led to a significant decrease in focus numbers (Fig. 2C; P<0.01). These data demonstrate that CIP2A serves a critical role in TNSC cell proliferation.

To determine which cell death pathway was induced by CIP2A depletion, MDA-MB-231 and MDA-MB-468 cells were treated with CIP2A siRNA for 48 h and apoptosis proteins were measured using western blot analysis. As presented in Fig. 3A, the results showed that PARP cleavage, as well as cleaved-caspases-3, were detected in CIP2A depleted MDA-MB-231 and MDA-MB-468 cells. These results indicate that the apoptosis pathway is primarily activated along with caspase-dependent apoptosis in BNSC cells following CIP2A depletion. To assess whether autophagy is also involved in CIP2A siRNA-induced cell death, the expression level of LC-3 II in cells treated with CIP2A siRNA was subsequently analyzed. Interestingly, a marked increase in LC-3 II was observed following CIP2A siRNA treatment for 48 h (Fig. 4B). These findings suggest that CIP2A is associated with proliferation, apoptosis and autophagy.

Considering that highly invasive TNSC cells have higher CIP2A expression than poorly invasive MCF-7 cells, it was determined whether CIP2A depletion inhibited the invasive behavior of breast cancer cells. An invasion assay was performed in highly invasive MDA-MB-231 cells using Matrigel-coated 24-well microchemotaxis chambers. As shown in Fig. 3A, MDA-MB-231 cells were treated with CIP2A siRNA (100 nM), and cell invasion was determined after 20 h. CIP2A depletion markedly suppressed the invasion of MDA-MB-231 cells. In addition, the effect of CIP2A depletion on migration was explored. MDA-MB-231 cells were treated with CIP2A siRNA (100 nM), and cell migration was determined after 48 h. As shown in Fig. 3B, CIP2A depletion significantly decreased MDA-MB-231 cell migration. To investigate the mechanism underlying invasion and migration inhibition, the effect of CIP2A knockdown on c-Myc, Akt, mTOR and P70S6K levels was explored. The results indicated that depletion of CIP2A by siRNA resulted in inhibition of c-Myc protein expression, Akt, mTOR and P70S6K phosphorylation in MDA-MB-231 cells (Fig. 4C). These results indicated that CIP2A promotes TNSC cell invasion through the c-Myc upregulatory effect on c-Myc expression, and through the activation of the Akt/mTOR/P70S6K signaling pathway.