MCF10A, MDA-MB-231, MDA-MB-435, MCF-7 and T47D cell lines were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and MDA-MB-435 cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA) while MCF-7 and T47D cell lines were supplemented with Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) with 10% FBS. MCF10A cells were maintained in DMEM/F12 medium (HyClone) supplemented with 5% horse serum (Gibco), 10 mg/ml insulin, 0.5 mg/ml hydrocortisone (both from Sigma-Aldrich, St. Louis, MO, USA), 20 ng/ml EGF (R&D Systems, Minneapolis, MN, USA), and 100 ng/ml cholera toxin (Sigma-Aldrich). All of the cell lines were supplemented with 1% penicillin/streptomycin (Gibco), in a 5% CO₂ and humidified atmosphere at 37°C.

The anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT, anti-FAK, anti-phospho-FAK and anti-Snail antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-E-cadherin, anti-cytokeratin 19, anti-N-cadherin, anti-Slug, anti-FYN, anti-vimentin and anti-nestin antibodies were purchased from Abcam (Cambridge, MA, USA). The siRNAs targeting FYN or the control were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FGF2 was purchased from R&D Systems. PP1, BGJ398, AZD8931, LY294002 and PD98059 were acquired from Selleck Chemicals (Houston, TX, USA).

Cells were seeded into 6-well plates. The cells were then transfected with the siRNA or plasmid using FuGENE HD (Roche, Mannheim, Germany) according to the manufacturer's instructions.

The total RNA was extracted with RNA Plus (Takara Bio, Inc., Otsu, Shiga, Japan) according to the manufacturer's recommendations. Then, the cDNA was synthesized from RNA by PrimerScript RT Reagent kit (Takara) according to the manufacturer's protocol. RT-qPCR was carried out using a SYBR-Green PCR mix (Takara) on a Bio-Rad CFX96 Real-Time PCR system. Quantification of the target gene expression was calculated by normalizing the averaged Ct value of the target gene to the averaged Ct value of the housekeeping gene β-actin (ΔCt), and determined as 2^−ΔCt.

Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail. Protein lysates were resolved by 10% SDS/PAGE and the separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Non-specific binding was blocked by 5% skimmed milk in Tris-buffered saline and Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with the primary antibody at an appropriate dilution overnight at 4°C and then incubated with a suitable HRP-conjugated anti-rabbit or anti-mouse secondary antibody at an appropriate dilution at room temperature for 1 h. The blots were visualized with enhanced chemiluminescence (ECL) reagent (Millipore).

The MTT assay was used to evaluate the ability of cell proliferation. In brief, 5x10³ cells were seeded into 96-well plates/well. After incubation for the indicated time, the cells were incubated with 10 µl MTT (0.5 mg/ml; Sigma-Aldrich) at 37°C for 4 h. The medium was then removed, and precipitated formazan was dissolved in 150 µl dimethyl sulfoxide (DMSO). The absorbance at 570 nm was detected using a microplate autoreader.

The Transwell assay (BD Biosciences, San Diego, CA, USA) was used to evaluate the ability of cell migration and invasion. Briefly, relevant cells were seeded into the top chambers with or without Matrigel (BD). Medium without serum was added to the top chambers, while complete medium was added to the bottom wells. The cells were fixed with 4% neutral formalin and stained with crystal violet after incubation. The number of migrated cells was counted under a microscope in five predetermined fields.

Cells were seeded onto glass coverslips in 12-well plates. After adherence, the cells were washed three times with phosphate-buffered saline (PBS), then fixed with 4% neutral formalin and permeabilized with 0.1% Triton X-100. Non-specific binding was blocked by 3% BSA in PBS for 1 h at room temperature and then the membranes were incubated with primary antibody at an appropriate dilution overnight at 4°C. After being washed with PBS, the membranes were incubated with a suitable FITC-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) at an appropriate dilution at room temperature for 1 h, then stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Jiangsu, China) and glass coverslips were observed with a fluorescence microscope.

Cells were transiently transfected with luciferase reporter constructs, mixtures of expression plasmids encoding FOXO1, along with the pRL-TK vector (Promega, Madison, WI, USA). Luciferase activity was measured after 24 h by the Dual Luciferase Reporter Assay (Promega).

ChIP assay was performed using kits from Active Motif (Carlsbad, CA, USA). Cells were fixed in formaldehyde. Cells were lysed and nuclei were pelleted by centrifugation. Nuclei were resuspended and sheared using a sonicator (Misonix, Inc., Farmingdale, NY, USA). Sheared chromatin was immunoprecipitated with the anti-FOXO1 or control IgG antibody. The cross-links were reversed overnight at 65°C and deproteinated with 20 g/ml proteinase K. The DNA samples were semi-quantified by PCR using the following primer sets containing the FOXO1 binding region on the FYN promoter: forward, 5′-CCTTGTTTACTTC-3′ and reverse, 5′-TGTTGTTTACNTT-3′.

Data are presented as the mean ± SEM, and were analyzed by one-way analysis of variance (ANOVA). All experiments were carried out at least three times independently to confirm the conclusions. Statistical significance was set at P<0.05.

Previous studies indicate that FYN is overexpressed in prostate cancer (22), and induces proliferation and migration in the HEK 293T cell line (26). To investigate the role of FYN in breast cancer, we determined the expression of FYN in breast cancer cell lines and the normal breast epithelial MCF10A cells by western blotting (Fig. 1A). The results showed that FYN expression was higher in the MDA-MB-231 and MDA-MB-435 cell lines and in contrast, it was lower in the MCF-7 an MCF10A cell lines.

To further investigate the role of FYN in breast cancer development and progression, we overexpressed FYN by transfecting the FYN plasmid into the MCF10A cell line. We found that FYN expression was significantly higher in the FYN-transfected MCF10A cells by western blotting (Fig. 1B). We next examined the migration and invasion abilities of the the FYN-overexpressed and the control MCF10A cells. MTT analysis showed that MCF10A-FYN cells grew much faster than the control cells (Fig. 1C). We also observed that the number of migrated cells was much higher in the FYN-transfected MCF10A cells than that in the control cells (Fig. 1D).

Conversely, to determine the effect of decreased FYN expression on breast cancer progression, we silenced FYN with siRNAs targeting FYN or inhibited FYN with FYN inhibitor PP1 in the MBA-MB-231 cell line. The FYN expression was significantly decreased in the FYN siRNA-transfected MDA-MB-231 cells when compared to the level in the control cells (Fig. 1E). The MTT assay showed that depletion of FYN inhibited cell proliferation in the MDA-MB-231 cells (Fig. 1F). The Transwell assay showed that depletion of FYN inhibited breast cancer cell migration and invasion in the MDA-MB-231 cells (Fig. 1G), suggesting that FYN promotes breast cancer cell proliferation, migration and invasion.

As previously described, FYN promotes breast cancer cell migration and invasion. We therefore speculated that FYN may be involved in the process of EMT consequently promoting breast cancer metastasis. To test this contention, we next investigated the role of FYN in breast cancer cell EMT. EMT results in loss of epithelial markers and concomitant acquisition of mesenchymal markers. Our results showed that the mRNA expression of E-cadherin was decreased, whereas the expression of N-cadherin and vimentin was significantly increased in the FYN-transfected MCF10A cells by RT-qPCR (Fig. 2A). In addition, the expression of EMT-related transcription factors Slug and Snail was significantly elevated in the FYN-overexpressed MCF10A cells when compared with that of the control cells (Fig. 2A). Furthermore, overexpression of FYN in the MCF10A cells led to decreased expression of epithelial markers E-cadherin and cytokeratin 19 and increased the expression of mesenchymal markers N-cadherin and vimentin by western blotting (Fig. 2B).

In contrast, the expression of mesenchymal markers vimentin and N-cadherin was downregulated and the expression of epithelial marker E-cadherin was upregulated in the FYN-depleted MDA-MB-231 cells by RT-qPCR (Fig. 2C) and western blotting (Fig. 2D). In addition, the expression of EMT-related transcription factors Slug and Snail was significantly reduced in the FYN-depleted MDA-MB-231 cells compared with that of the control cells by RT-qPCR (Fig. 2C) and western blotting (Fig. 2D). Immunofluorescence staining also revealed that the E-cadherin expression was decreased in the FYN-overexpressing MCF10A cells and increased in the FYN-depleted MDA-MB-231 cells, whereas the vimentin expression was increased in the FYN-overexpressing MCF10A cells and decreased in the FYN-depleted MDA-MB-231 cells (Fig. 2E). Moreover, EGF-mediated remodeling of the cytoskeleton from cortical actin to stress fibers was noted, as determined by phalloidin staining (Fig. 2E).

Previous studies have indicated that FYN is a downstream target of the TGFTβ/Smad pathway and can be upregulated by TGFβ-1 (27). In addition, the TGFβ/Smad pathway, the receptor tyrosine kinase pathway can also promote cell proliferation, migration and induce EMT. Thus, EGF and FGF2, and their inhibitors AZD8931 and BGJ398 were used to determine whether FYN is a downstream target of the receptor tyrosine kinase pathway. After treatment with EGF or FGF2 for 2 days, the expression of FYN was increased in the MCF10A cell line, while the FYN expression was decreased after treatment with their inhibitors (AZD8931 and BGJ398) (Fig. 3A). Furthermore, FYN expression was decreased in the MDA-MB-231 cells after treatment with AZD8931 or BJ398 (Fig. 3B). Moreover, the FYN expression was negatively related to the E-cadherin expression in the MCF10A cells (Fig. 3A), whereas it was positively related to N-cadherin expression in the MDA-MB-231 cells (Fig. 3B). To further investigate the role of FYN in FGF2 and EGF-induced EMT, we depleted the FYN expression by FYN siRNA and inhibitor PP1 after EGF or FGF2 treatment. The results showed that FYN depletion reduced the EGF and FGF2 induced-EMT (Fig. 3C). The PI3K/AKT and ERK/MAPK pathways are involved in FGF2-induced EMT (28). To further investigate the role of FYN in the PI3K/AKT and MAPK/ERK pathways, we examined the FYN expression after treatment with PI3K inhibitor LY294002 or MEK 1/2 inhibitor PD98059. The results showed that FYN expression was decreased after treatment with LY294002 or PD98059 (Fig. 3D).

Transcription factor FOXO1 can suppress tumor progression, while PI3K/AKT or ERK/MAPK pathway activation can suppress FOXO1 expression (29). We designed a FYN promoter containing the FOXO1 binding site (Fig. 4A) for luciferase assay to explore the relationship between FYN and FOXO1. ChIP and luciferase assay confirmed that the FOXO1 protein can combine with FYN DNA fragments (Fig. 4B) and inhibit FYN transcription (Fig. 4C). Furthermore, FOXO1 transfection decreased the expression of FYN at both the mRNA (Fig. 4D) and protein levels (Fig. 4E). Thus, we can conclude that FGF2 and EGF can decrease FOXO1 expression through the PI3K/AKT and ERK/MAPK pathways, which suppress the transcriptional inhibition of FYN and induce EMT (Fig. 4F).