Calycosin inhibits breast cancer cell migration and invasion by suppressing EMT via BATF/TGF-β1

Calycosin inhibits in vitro proliferation, migration and invasiveness of breast cancer cells

CCK-8 assay showed that calycosin inhibited proliferation of T47D and MCF-7 breast cancer cells in a dose- and time-dependent manner (Figure 1A). Previous studies have shown that higher expression of CD147, MMP-2, and MMP-9 is associated with breast cancer progression [19, 20]. RT-qPCR and western blot results showed that calycosin treatment downregulated the mRNA and protein levels of CD147, MMP-2, and MMP-9 in T47D and MCF-7 breast cancer cells (Figure 1B, 1C). Wound healing and Transwell invasion assay results showed that calycosin treatment decreased migration and invasion rates of breast cancer cells, respectively, in a dose-dependent manner (Figure 1D, 1E). These results showed that calycosin inhibited in vitro breast cancer cell progression in a dose-dependent manner.

Calycosin inhibits breast cancer cell progression by suppressing BATF expression

RT-qPCR and western blot analyses showed that calycosin treatment downregulated BATF mRNA and protein levels in T47D and MCF-7 breast cancer cells (Figure 2A, 2B). Therefore, we analyzed the effects of BATF overexpression or knockdown on in vitro breast cancer cell migration and invasiveness. BATF mRNA and protein levels were significantly upregulated in BATF-overexpressing T47D and MCF-7 breast cancer cells and downregulated in si-BATF-transfected T47D and MCF-7 breast cancer cells (Figure 2C, 2D). The levels of CD147, MMP-2 and MMP-9 transcripts and proteins were significantly increased in BATF-overexpressing breast cancer cells and significantly reduced in BATF-silenced breast cancer cells (Figure 2E, 2F). Wound healing and Transwell invasion assays showed that breast cancer cell migration and invasiveness was significantly increased by BATF overexpression and significantly reduced by BATF silencing (Figure 2G, 2H). These results demonstrated that BATF overexpression induced breast cancer cell progression.

Calycosin counteracts pro-metastatic effects of BATF in breast cancer cells

We then analyzed if calycosin suppressed invasion and migration of BATF-overexpressing breast cancer cells. Calycosin treatment decreased the expression of CD147, MMP-2, and MMP-9 transcripts and proteins in BATF-overexpressing breast cancer cells (Figure 3A, 3B). Furthermore, calycosin treatment significantly reduced migration and invasiveness of BATF-overexpressing breast cancer cells (Figure 3C, 3D). These results showed that calycosin inhibited pro-metastatic effects of BATF in breast cancer cells.

Calycosin downregulates expression of TGFβ1 by suppressing BATF

ChIP-seq online database [21] analysis potential BATF binding sites in TGFβ1 (Figure 4A). We also identified the binding motif of BATF using the JASPAR database (Figure 4B). Calycosin treatment significantly downregulated the levels of BATF and TGFβ1 transcripts and proteins in breast cancer cells (Figure 4C, 4D). Furthermore, TGFβ1 mRNA and protein levels were significantly increased by BATF overexpression and significantly reduced by BATF knockdown in breast cancer cells (Figure 4C, 4D). Next, we identified 3 potential BATF-binding sequences in the TGFβ1 promoter sequence (P1: -544~-44, P2: -44~456, P3: 456~956) using the JASPAR database (Figure 4E). We cloned P1, P2, or P3 sequences of the TGFβ1 gene promoter into luciferase reporter vectors and transfected the recombinant vectors into breast cancer cells. Dual luciferase reporter assay results showed that BATF significantly increased luciferase activity in cells transfected with luciferase reporter vector containing P1, but luciferase activity was not observed in cells transfected with luciferase reporter vectors containing P2 or P3 (Figure 4E). This suggested potential BATF-binding sites in the P1 sequence of the TGFβ1 promoter. We then constructed recombinant luciferase reporter vectors containing either wild-type or mutated P1 sequence of TGFβ1 (P1WT or P1M1). Dual luciferase reporter assay results showed that luciferase activity was significantly higher in cells transfected with luciferase reporter vector containing wild type P1 sequence, but luciferase activity was not observed in cells transfected with the luciferase reporter vector containing mutated P1 sequence (P1MUT) (Figure 4F, 4G). These results confirmed that BATF binds to the P1 sequence of the TGFβ1 promoter. We also performed ChIP-PCR to confirm direct binding between BATF and TGFβ1. ChIP-PCR results showed that TGFβ1 levels were significantly reduced in the BATF-knockdown T47D cells and significantly increased in the BATF-overexpressing T47D cells (Figure 4H). These results confirmed that calycosin downregulated the expression of TGFβ1 by suppressing BATF.

Calycosin suppresses breast cancer cell progression by inhibiting EMT

TGFβ1 is a crucial regulator of epithelial-mesenchymal transition (EMT), a key process in tumor metastasis [22]. Hence, we investigated the effects of calycosin on EMT in breast cancer cells. Calycosin significantly increased the expression of E-cadherin (epithelial cell biomarker) and decreased the expression levels N-cadherin and Vimentin (mesenchymal cell biomarkers) as well as CD147, MMP-2, and MMP-9 (pro-metastatic proteins) in the breast cancer cells (Figure 5A). Conversely, TGFβ1 treatment decreased E-cadherin levels and increased N-cadherin, Vimentin, CD147, MMP2 and MMP9 expression levels in the breast cancer cells (Figure 5B). TGFβ1 treatment also significantly increased the migration and invasiveness of breast cancer cells, but calycosin reduced the migration and invasiveness of TGFβ1-treated breast cancer cells (Figure 5C, 5D). These results showed that calycosin suppressed migration and invasiveness of breast cancer cells by inhibiting EMT via BATF/ TGFβ1.

Calycosin inhibits breast cancer cell progression by suppressing BATF/ TGFβ1 axis

The above findings confirmed that BATF/TGFβ1 axis positively regulated the expression of pro-metastatic proteins, CD147, MMP-2, and MMP-9, in breast cancer cells. Therefore, we further investigated the relationship between BATF and TGFβ1. We observed that overexpression of BATF upregulated TGFβ1 mRNA and protein levels in breast cancer cells, whereas, TGFβ1 knockdown decreased BATF mRNA and protein levels in BATF-overexpressing breast cancer cells (Figure 6A, 6B). Furthermore, calycosin treatment significantly reduced the levels of CD147, MMP-2, and MMP-9 in TGFβ1-overexpressing breast cancer cells (Figure 6A, 6B). Transwell invasion assay results showed that TGFβ1 knockdown significantly reduced the invasiveness of BATF-overexpressing breast cancer cells (Figure 6C). However, overexpression of TGFβ1 significantly increased the invasiveness of calycosin-treated breast cancer cells (Figure 6C). Wound healing assay results showed that TGFβ1 knockdown significantly reduced the migration of BATF-overexpressing breast cancer cells compared to the corresponding controls (Figure 6D). However, overexpression of TGFβ1 significantly increased the migration of calycosin-treated breast cancer cells (Figure 6D). In addition, to further determine the effect of Calycosin inhibits breast cancer cells growth in vivo, nude mice subcutaneous tumor mode was constructed (Figure 7A). The results revealed that Calycosin in T47D cells markedly decreased the growth rate of subcutaneous xenograft tumors (Figure 7B–7D). In summary, our data demonstrated that calycosin inhibited in vitro migration, invasiveness and growth of breast cancer cells by suppressing BATF/ TGFβ1.

Cell culture

The human breast cancer cell lines, T47D and MCF-7, were purchased from Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China), and cultured in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% FBS (Gibco, CA, USA) and 1% Penicillin-Streptomycin solution (Gibco, CA, USA) in a humidified chamber maintained at 37° C and 5% CO₂. The cells were treated with different concentrations of calycosin and TGFβ1 (Sigma Aldrich, St. Louis, MO, USA).

SiRNA-mediated knockdown of BATFs and TGFβ1

We purchased the following siRNAs (GenePharma, Shanghai, China) that specifically target BATF-1, BATF-2, and TGFβ1:

si-BATF-1 sense: 5’-GCAGAAGAGUAUUAAGAAAGA-3’,

si-BATF-1 antisense: 5’-UUUCUUAAUACUCUUCUGCAU-3’;

si-BATF-2 sense: 5’-GGCCCAAUGCAGAAGAGUAUU-3’,

si-BATF-2 antisense: 5’-UACUCUUCUGCAUUGGGCCUG-3’;

si-TGFβ1 sense: 5’-GCAUAUAUAUGUUCUUCAACA-3’,

si-TGFβ1 antisense: 5’-UUGAAGAACAUAUAUAUGCUG-3’.

We used scrambled siRNAs as negative controls. We used the siRNAs with highest silencing effect for further studies.

CCK-8 cell proliferation assay

We seeded breast cancer cells (3000 cells per well) in 96-well plates at 37° C and 5% CO₂ for 0, 24, 48, and 72 h. Then, the cells were incubated with 10% CCK-8 (Dojindo; Kumamoto, Japan) in normal culture medium for 4 h. Subsequently, we measured optical density at 450 nm in a plate reader according to manufacturer’s instructions.

Real-time quantitative PCR (RT-qPCR)

We extracted total RNA from T47D and MCF-7 breast cancer cells using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). We assessed quantity and quality of RNA samples using A260/A280 ratio in a microplate reader. Then, we performed cDNA synthesis using 100 ng of total RNA per sample followed by qPCR using specific primers as listed in Table 1 and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of specific mRNAs were calculated using 2^−∆∆Ct method.

Western blotting

The cells were lysed using RIPA buffer (08714-04, Nacalai Tesque Inc., Kyoto, Japan) and the protein concentration of samples was quantified using BCA assay. Equal amounts of proteins (120 μg) were separated by SDS-PAGE and the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 30 mins, and then incubated overnight at 4° C with primary antibodies against BATF (ab236876, dilution 1:1000; Abcam, Cambridge, MA, USA), TGFβ1 (ab92486, 1:1000; Abcam, Cambridge, MA, USA), CD147 (ab188190, dilution 1:5000; Abcam, Cambridge, MA, USA), MMP-2 (ab110258, dilution 1:1000; Abcam), MMP-9 (ab97779, dilution 1:1000; Abcam, Cambridge, MA, USA), and GAPDH (5174S, dilution 1:2000; Cell Signaling Technology, Inc. Danvers, CO, USA). Then, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (ab6789 or ab6721, dilution 1:500, Abcam) for 30 min at room temperature. The blots were developed and visualized using ECL Plus Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK).

Dual luciferase reporter assay

The TGFβ1 mRNA sequence containing BATF binding site was amplified using PCR Amplification kit (TaKaRa Biotechnology, Dalian, China). We generated three recombinant luciferase reporter vectors with P1, P2, or P3 promoter sequences of TGFβ1 with potential BATF-binding domains. We also generated a recombinant luciferase reporter vector containing mutated P1 promoter sequence of TGFβ1 gene (P1 MUT). We measured relative luciferase activity using the Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA) according to manufacturer's protocol.

Wound healing assay

The breast cancer cells were grown to 70%-80% confluence. Then, the monolayer was scratched with a pipette tip and further cultured for 24 h. The width of the scratch was measured at 0 and 24 h to determine the migration rates of different experimental groups of breast cancer cells.

Transwell invasion assay

We seeded T47D or MCF-7 cells (1 × 10⁴ cells/well) in 150 μL serum-free DMEM medium into the upper chamber of Transwell chambers coated with matrigel (BD Biosciences, USA). We then added 700 μL of DMEM medium with 10% FBS into the lower chamber. The transwell plates were incubated at 37° C for 24 h. The cells invading the matrigel were fixed with cold methanol, stained with crystal violet, and counted under a light microscope to determine the number of invading cells in each experimental group.

ChIP-PCR

Chromatin immunoprecipitation experiments were performed using ChIP-IT Express kit (Active Motif) according to manufacturer’s instructions. Briefly, breast cancer cells were incubated in fixing solution containing 1% formaldehyde and protease inhibitor cocktail (PIC1; 1 μg/mL leupeptin, 1.4 μg/mL pepstatin, 0.2 mg/mL PMSF (Sigma), 1 mM EGTA, and 1 mM EDTA) for 10 min with gentle rotation. The cross-linking reaction was quenched by adding Glycine Stop-Fix solution. Then, the cells were pelleted, resuspended in ice-cold lysis buffer, and dounce-homogenized on ice initially with ~10 to 15 strokes followed by 30 strokes to extract nuclei. The nuclear pellets were obtained by centrifugation and resuspended in 6 mL homogenization buffer (10mM HEPES, pH 7.6, 25 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 M sucrose, 10% glycerol, 0.15 mM spermine, and protease inhibitor cocktail, PIC1). The buffer containing nuclei was layered on top of a 3 mL layer of homogenization buffer, and centrifuged at 5000×g for 1 h in a Beckman SW41 rotor. The nuclear pellets were then re-suspended in 0.3 mL nuclear lysis buffer (50 mM Tris pH 7.6, 10 mM EDTA and 1% SDS), and diluted with 0.6 mL immunoprecipitation (IP) dilution buffer (0.01% SDS, 1.1% Triton ×100, 167 mM NaCl, 16.7 mM Tris pH 7.6, 1.2 mM EDTA). Then, 0.3 mL of the nuclear lysate was sonicated for 25-30 cycles in 30 seconds at 4° C using BioRuptor twin sonicator (Diagenode). Sonicated chromatin was further diluted to 1 mL using IP dilution buffer. TGFβ1 was conjugated overnight at 4° C to Dynabeads Protein G (Life Technologies) using PBS containing 0.5% BSA and gentle rotation. Then, the sonicated DNA (0.33 mL made up to 1 mL in IP dilution buffer or 10% of input DNA) was incubated overnight with TGFβ1 beads at 4° C with gentle rotation. The samples were then washed twice with wash buffer I (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton ×100, 2 mM EDTA), thrice with alternate wash buffer III (10 mM Tris, pH 7.4, 250 mM LiCl, 1% NP-40 alternate, 0.7% deoxycholate, 1 mM EDTA), twice with 0.2% Triton ×100 TE buffer, and twice with TE buffer containing 50 mM NaCl. The DNA was recovered from the beads by incubating samples at 65° C overnight in 0.3 M NaCl followed by Proteinase K digestion. The DNA was purified using QIAquick PCR purification kit (Qiagen) and subjected to real-time qPCR.

Functional assay in vivo

Subcutaneous xenograft tumor model was adopted to assess the effects of Calycosin on tumorigenicity in nude mice. The procedures of animal experiments were done according to the Institutional Animal Care and Use Committee guidelines in Southern Medical University. 2×10⁶ T47D Cells were injected subcutaneously into the right flank for 4 weeks of male nude mice (Guangdong Animal Center, China). 7 weeks later, the tumors were isolated and weighted. The size of tumor was calculated as: V (mm³) = L×W²×0.5 (W: tumor short axis, L: tumor long axis). All subsequent procedures were performed using sterile technique.

Statistical analysis

All experiments were repeated at least three times. Statistical analysis was performed using the IBM SPSS 21.0 statistical software. The data were represented as means ± standard deviation. The differences between groups were analyzed using analysis of variance (ANOVA) or Student's t-test. P<0.05 was considered statistically significant.