HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

The human breast cancer cell lines SK-BR-3 and MCF7 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO). Both cell lines were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO₂.

Recombinant human HRG-β1 (purity: >97%) was purchased from R&D Systems (Minneapolis, MN). It was divided into small aliquots in phosphate-buffered saline (PBS) and stored at –70°C. The PI3k inhibitor, LY294002 and phospho-Smad2 pharmacological inhibitors, PD169316 and SB203580 were purchased from Calbiochem (San Diego, CA). The inhibitors were dissolved in dimethyl sulfoxide (DMSO). An anti-ErbB3 antibody was purchased from Santa Cruz Biotechnology Inc. (CA, USA). Anti-phospho-Smad2 (Ser465/467) and anti-Smad2 antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MO). An anti-Snail antibody was obtained from Abcam Ltd. (Cambridge, UK). Anti-E-cadherin and anti-vimentin antibodies were from BD Pharmingen (San Diego, CA). An anti-fibronectin antibody was obtained from Millipore (Billerica, MA). A monoclonal anti-β-actin antibody was obtained from Sigma (St Louis, MO).

Cells were harvested and lysed with RIPA buffer (20 mM Tris–HCl pH 7.5, 2 mM EDTA, 150 mM NaCl, 1 mM sodium vanadate, 10 mM NaF, 2.5 mM sodium pyrophosphate, 1% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with a protease inhibitor (1 mM phenylmethylsulfonyl fluoride) and a protease inhibitor cocktail (Roche, Mannheim, Germany). The cell lysates was cleared by centrifugation at 14,000 rpm for 20 min at 4°C, and the supernatants were used as total cellular protein extracts. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). The protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA). The blocked membranes with 5% skim milk were incubated with the indicated primary antibodies, followed by incubation with horseradish peroxidase-labeled secondary antibodies. Antibody-bound proteins were detected using the Enhanced Chemiluminescence (ECL) reagent (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. The levels of protein expression were quantified using ImageJ software (NIH, Bethesda, MD) and then normalized by the corresponding expression level in control cells for each group.

Nuclear translocation of phospho-Smad2 and Snail was examined by immunofluorescence staining. Approximately 2 × 10⁴ cells/well were seeded onto 2-well Lab-Tek II chamber slides (NUNC, Rochester, NY). After serum starvation, the cells were incubated with HRG-β1 and specific inhibitors. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde for 10 min. Following three washes with PBS, the cells were permeabilized with 0.1% Triton X-100 for 20 min. After washing with PBS, the cells were blocked with 3% bovine serum albumin for 1 h at room temperature and then incubated with rabbit polyclonal anti-Snail (1:500) and anti-phospho-Smad2 (1:100) primary antibodies overnight at 4°C. After three washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-goat IgG secondary antibodies (Invitrogen, Grand Island, NY). The cells were then washed, mounted with mounting medium containing DAPI (VECTOR Laboratories, Burlingame, CA), and observed using an LSM700 confocal laser scanning microscope (Carl Zeiss, Thornwood, NY). The expressions of E-cadherin and vimentin were evaluated with specific antibodies as described above and incubated with a DyLight 488-conjugated anti-mouse IgG secondary antibody (VECTOR Laboratories).

For scratch wound healing assays, cells were seeded into 12-well plates and grown to confluence. After serum starvation, the confluent monolayers were scratched with a plastic tip, washed with PBS to remove the detached cells, and incubated with HRG-β1 and the indicated inhibitors for 24 h. The cell migration into the wounded area was monitored at the indicated time points using a light microscope (Olympus BX51 Tokyo, Japan). Quantification of the closure of the monolayers was determined using an NIH image analysis program and the results were presented as the relative percentages of wound closure compared with control monolayers. The assays were repeated three times independently.

For invasion assay, serum free medium (500 μl) treated with or without HRG-β1 was added to the lower chambers of a 24 transwell plate (8.0 μm pore size, Corning, NY) and untransfected or transfected with control (Ctrl), Smad2 and ErbB3 siRNA cells (2 × 10⁵ cells in 200 μl medium) were seeded in upper chamber which was coated with Matrigel (BD Biosciences). After 48 h of incubation, non-migrating cells were removed with a cotton swab and cells on the bottom surface of the membrane were stained with Diff-Quick Staining kit (Biochemical Sciences, Swedesboro, NJ). The invaded cells were photographed randomly with microscope and quantified by counting the number of cells in three independent experiments.

For transfection, the cells were grown to confluence in 6-cm plates and a Smad2 siRNA (Santa Cruz Biotechnology Inc.) and a ErbB3 siRNA at 60 pmol (Santa Cruz Biotechnology Inc.) were transfected using a siRNA transfection reagent (Santa Cruz Biotechnology Inc.) according to the manufacturer’s instructions. A nonspecific siRNA (Santa Cruz Biotechnology Inc.) was transfected as a control. After incubation for 6 h, the medium was replaced with the standard culture medium described above. After another 24 h of incubation, the transfected cells were treated with HRG-β1 and then used in subsequent evaluations.

All experiments were performed in triplicate. The data were expressed as means ± SD. Statistical analyses were performed using Student’s t-test. Values of P < 0.05 were considered to indicate statistical significance.

Cheng et al. have previously published that Snail is induced by HRG-β1 in SK-BR-3 cells [19]. As shown in Figure 1a, HRG-β1 increased the expression of Snail after 2 h and maintained its expression until 24 h in SK-BR-3 cells. We identified a few of the common acquired markers during EMT. Vimentin and fibronectin are commonly used to identify cells undergoing EMT in cancers. In SK-BR-3 cells, vimentin and fibronectin were expressed in a time-dependent manner after HRG-β1 treatment, while E-cadherin expression was decreased after 48 h of HRG-β1 treatment. We further examined the expression of E-cadherin by immunofluorescence staining, and found that E-cadherin was decreased in the HRG-β1-treated cells at 48 h compared with control cells (Figure 1b). In MCF7 cells, the expressions of Snail, vimentin, and fibronectin were increased after treatment with HRG-β1, while E-cadherin expression was suppressed at 72 h (Figure 2a). Immunofluorescence staining revealed that the expression of vimentin was increased in HRG-β1-treated cells compared with control cells (Figure 2b). These findings indicated that HRG-β1 upregulated Snail, vimentin, and fibronectin and suppressed E-cadherin in SK-BR-3 and MCF7 cells.

We examined the effects of the EGF family peptide HRG-β1 on the activation of Smad2 phosphorylation. HRG-β1 at 25 ng/ml induced the phosphorylation of Smad2 in a time-dependent manner in SK-BR-3 and MCF7 cells (Figure 3a, b). The level of phospho-Smad2 (Ser465/467) reached its maximum at 2–8 h after treatment and remained for 24 h without affecting the total Smad2 expression. Commonly, TGF-β1 induces phosphorylation of Smad2 within a few minutes of stimulation. Here, we found that HRG-β1 prolonged the phosphorylation of Smad2 compared with TGF-β1.

As shown in Figure 4, knockdown of ErbB3 expression by siRNA transfection suppressed the expressions of phospho-Smad2, Snail, and fibronectin by HRG-β1, whereas the expression of E-cadherin was increased in ErbB3 siRNA-transfected cells (ErbB3 siR) compared with control siRNA-transfected SK-BR-3 cells (Ctrl siR). On this basis, HRG-β1/ErbB3 signaling induced EMT in the SK-BR-3 and MCF7 breast cancer cell lines.

First, we identified that HRG-β1-induced Smad2 phosphorylation was inhibited by pretreatment with the PI3k inhibitor LY294002 (data not shown). It is known that HRG-β1 phosphorylates Smad2 via the PI3k/Akt signaling pathway [19]. Therefore, to investigate the possible involvement of Smad2 in HRG-β1-induced Snail gene expression, SK-BR-3 and MCF7 cells were pretreated with two known inhibitors of Smad2 phosphorylation, PD169316 and SB203580 [20, 21]. PD169316 inhibited HRG-β1-induced Smad2 phosphorylation in SK-BR-3 cells and SB203580 had a more efficient inhibitory effect in MCF7 cells (Figure 5a, c). We pretreated the cells with LY294002, PD169316, or SB203580 alone and combinations of LY294002 and PD169316 or SB203580 prior to HRG-β1 stimulation to both cell types. As shown in Figure 5b, d, the HRG-β1-induced expressions of phospho-Smad2 and Snail were inhibited by treatment with the above inhibitors, indicating that HRG-β1 induced expression of Snail through activation of Smad2 via the PI3k/Akt signaling pathway. Since these Smad2 phosphorylation inhibitors are also known to block p38 phosphorylation, the role of Smad2 was further explored by the more specific genetic approach of RNA interference (siRNA).

HRG-β1 treatment for 24 h induced nuclear colocalization of phospho-Smad2 and Snail in SK-BR-3 cells, and this translocation to the nucleus was inhibited by pretreatment with LY294002 and PD169316 before HRG-β1 stimulation (Figure 6). In MCF7 cells, HRG-β1 induced nuclear colocalization of phospho-Smad2 and Snail, and pretreatment with LY294002 and SB203580 suppressed the nuclear translocation induced by HRG-β1 (data not shown). The mean percentages of fluorescence of phospho-Smad2 and Snail are also shown in Figure 6.

As mentioned earlier, HRG-β1 increased the expressions of vimentin and fibronectin during EMT in SK-BR-3 and MCF7 cells. As shown in Figure 7a, b, the HRG-β1-induced expressions of vimentin and fibronectin were inhibited by the indicated inhibitors. Taken together, HRG-β1 induced EMT through phospho-Smad2-mediated expression of Snail via the PI3k/Akt signaling pathway in both breast cancer cell lines.

SK-BR-3 and MCF7 cells were transfected with control and Smad2 siRNAs. As shown in Figure 8a, b, the HRG-β1-increased expressions of Snail and fibronectin in control siRNA-transfected cells (Ctrl siR) compared with untreated control cells (untreated ctrl) were downregulated in Smad2 siRNA-transfected cells (Smad2 siR). Taken together, Smad2 activation plays roles in the expression of Snail and induction of EMT by HRG-β1 in SK-BR-3 and MCF7 cells.

We performed in vitro wound healing assays. Pretreatment with LY294002 and PD169316 or SB203580 inhibited the cell migration of SK-BR-3 and MCF7 cells in the presence of HRG-β1 (Figure 9a, b). In cell invasion assay, knockdown of ErbB3 and Smad2 by siRNA transfection inhibited the cell invasive ability of SK-BR-3 and MCF7 cells under HRG-β1 stimulation in matrigel-coated chamber (Figure 9c, d). Collectively, these data suggested that HRG-β1 induced cancer cell migration and invasion through induction of EMT via PI3k/Akt-phospho-Smad2-Snail signaling pathway.