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HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells
- Jinkyoung Kim†1,
- Hoiseon Jeong†2,
- Youngseok Lee†1,
- Chungyeul Kim†1,
- Hankyeom Kim†1 and
- Aeree Kim1Email author
© Kim et al.; licensee BioMed Central Ltd. 2013
Received: 2 April 2013
Accepted: 8 August 2013
Published: 12 August 2013
Heregulin (HRG; also known as neuregulin) is a ligand for ErbB3. One of its isotypes, HRG-β1, binds to ErbB3 and forms heterodimers with other ErbB family members, thereby enhancing the proliferation and tumorigenesis of breast cancer cells. HRG stimulation may contribute to the progression of epithelial–mesenchymal transition (EMT) and tumor metastasis in breast cancer. Majority of studies regarding EMT has been concentrated on TGF-β signaling. Therefore, we investigated whether the HRG-β1 and ErbB3 activate Smad2 signaling during process of EMT in breast cancer cells.
The SK-BR-3 and MCF7 breast cancer cell lines were used. The expressions of phospho-Smad2 and EMT markers were observed by western blotting and immunofluorescence assays after treatment with HRG-β1. The cell motility and invasiveness were determined by wound healing and matrigel invasion assays. Smad2 and ErbB3 small interfering RNA (siRNA) transfections were performed to assess the involvement of ErbB3 and Smad2 in HRG-β1-induced EMT.
HRG-β1 induced EMT through activation of Smad2. The expression of E-cadherin was decreased after HRG-β1 treatment, while the expressions of Snail, vimentin, and fibronectin were increased. The HRG-β1-induced expressions of Snail, vimentin, and fibronectin, and nuclear colocalization of phospho-Smad2 and Snail were inhibited by pretreatment with a PI3k inhibitor, LY294002, or two phospho-Smad2 inhibitors, PD169316 or SB203580 and cancer cell migration by HRG-β1 was inhibited. Knockdown of Smad2 by siRNA transfection suppressed the expressions of Snail and fibronectin in response to HRG-β1 stimulation and knockdown of ErbB3 suppressed the expressions of phospho-Smad2, Snail, and fibronectin induced by HRG-β1, whereas E-cadherin was increased compared with control siRNA-transfected cells. Knockdown of ErbB3 and Smad2 also decreased SK-BR-3 and MCF7 cell invasion.
Our data suggest that HRG-β1 and ErbB3 induce EMT, cancer cell migration and invasion through the PI3k/Akt-phospho-Smad2-Snail signaling pathway in SK-BR-3 and MCF7 breast cancer cells.
Epithelial–mesenchymal transition (EMT) is a highly conserved and fundamental process that governs morphogenesis in multicellular organisms. EMT is involved in both embryonic development and progression of carcinoma toward dedifferentiated and more malignant states . It is defined by loss of the epithelial phenotype and acquisition of mesenchymal characteristics, such as migratory capacity, loss of polarity, and cell-to-cell contacts . EMT can contribute to tumor invasion, metastasis, and resistance to specific chemotherapy or hormone therapy.
EMT can be triggered by different signaling molecules, such as epidermal growth factor (EGF), fibroblast growth factor, hepatocyte growth factor, transforming growth factor (TGF)-β, bone morphogenetic proteins, WNTs, and Notch . Among them, TGF-β is a major inducer of EMT [4, 5]. Binding of TGF-β to its receptor leads to activation of the transcription factors Smad2/3, which form complexes with Smad4 and then translocate into the nucleus, where they control the transcription of target genes  in collaboration with specific transcription factors and cofactors such as Snail, Slug, and Zeb1/2 [7, 8]. In particular, the role of the Snail family of zinc finger proteins in EMT and cancer has been highlighted in several publications [9, 10].
Heregulin (HRG; also known as neuregulin) is a member of the EGF-like growth and differentiation factors, and binds with high affinity to the receptors ErbB3 and ErbB4 . ErbB3, a member of the human epidermal growth factor receptor (EGFR) family of transmembrane receptors, undergoes heterodimerization with other ErbB family members and leads to cell differentiation, migration, proliferation, and survival . Although four genes have been identified (HRG1–4), most research interests have focused on the HRG1 gene .
HRG-1 has been implicated in normal heart and nervous system development  as well as in the pathophysiological processes of psychiatric diseases, cardiac diseases, and various types of cancer [15, 16]. HRG-1 is expressed in 30% of human breast cancer patients  and is correlated with poor histological grades . Cheng et al.  demonstrated that HRG-β1 induced EMT through upregulation of Snail via the PI3k/Akt pathway in the SK-BR-3 cell line. However, the mechanism of HRG-β1 and ErbB3 for the regulation of EMT in breast cancer cells has not been documented in detail. In this study, we investigated whether HRG-β1/ErbB3 induces the process of EMT with involvement of Smad2 activation in the ErbB2-overexpressing SK-BR-3 cell line and luminal A breast cancer cell line MCF7.
Cell lines and culture
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.
Reagents and antibodies
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).
Wound healing assay
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.
Matrigel invasion assay
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.
Small interfering RNA (siRNA) transfection
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.
HRG-β1 induces Snail expression and EMT in SK-BR-3 and MCF7 cells
HRG-β1 induces activation of Smad2 in SK-BR-3 and MCF7 cells
Knockdown of ErbB3 expression suppresses HRG-β1-induced EMT in SK-BR-3 cells
HRG-β1 induces expression of Snail through activation of Smad2 via the PI3k/Akt signaling pathway
HRG-β1 induces nuclear colocalization of phospho-Smad2 and Snail
HRG-β1 induces EMT through phospho-Smad2-mediated Snail via the PI3k/Akt signaling pathway
Knockdown of Smad2 expression suppresses HRG-β1-induced expressions of Snail and fibronectin
HRG-β1 and ErbB3 induces cancer cell migration and invasion through Smad2 activation
Breast cancer is the most common malignancy among women worldwide. Understanding the mechanisms of cancer invasion and metastasis is a very important issue in cancer research. The majority of studies regarding EMT have focused on TGF-β signaling in various kinds of disease settings [5, 6, 8, 22]. Thus far, the basal-like type and triple-negative type of breast carcinomas are characterized to show mesenchymal and stem-cell features and are known to be correlated with resistance to therapy [23, 24].
It has been suggested that not only TGF-β but also various kind of signaling molecules, such as growth factors, cytokines, integrins, and Wnts, are inducers of EMT . HRG is a ligand for ErbB3 and ErbB4 and has also been reported to promote the invasive behavior of breast cancer cells in vitro . HRG-induced ErbB2/ErbB3 heterodimers are considered to induce strong downstream signaling and to activate various biological responses, such as cellular proliferation, maturation, survival, apoptosis, and angiogenesis [27–31]. Cheng et al.  demonstrated that HRG-β1 induced EMT through Snail upregulation via the PI3k/Akt pathway in the ErbB2-overexpressing SK-BR-3 cell line. Various kinds of cancer cells, such as breast cancer cells, glial cells, neural tissues, and hepatocytes, are known to secrete HRG . Although the tumor cells can be stimulated by HRG in autocrine or paracrine manners, small numbers of circulating tumor cells can be activated by nearby HRG-secreting organs, such as the liver and central nervous system, where cancer cells move to and settle down. Blockade of HRG expression inhibits tumorigenesis and metastasis of breast cancer cells .
In this study, we have obtained evidence that HRG plays an important role in breast cancer.
It is a novel observation that the induction of EMT by HRG-β1 via upregulation of Snail involved the Smad2 signaling pathway, which is one of TGF-β signaling molecules. We found that phospho-Smad2 inhibitors (PD169316 or SB203580) and Smad2 siRNA transfection inhibited Snail expression and EMT, which were induced by HRG-β1. Furthermore, we identified that HRG-β1 induced cancer cell migration and invasion through Smad2 activation by wound healing assays and matrigel invasion assays. Overall, HRG-β1 induced EMT through Snail expression by activation of Smad2 not only in the SK-BR-3 cell line, but also in the MCF7 cell line, which expresses ErbB2 at basal levels. This dynamic and reversible emergence of the mesenchymal phenotype can be triggered by a variety of tumor microenvironments in the non-basal-like phenotypes of breast cancer cell lines.
Activation of RTK signaling caused by HRG-associated heterodimerization of ErbB3 and ErbB2 may be a critical step in tumor progression. We identified that the ErbB2 interaction with ErbB3 is required for the HRG-β1-induced EMT process. Specific siRNA transfection is a useful tool for evaluating the biologic effects of a target gene. In the presence of HRG-β1, knockdown of ErbB3 resulted in suppression of phospho-Smad2, Snail, and fibronectin expressions, whereas the expression of E-cadherin was increased in SK-BR-3 cells. Taken together, ErbB3 contributed to the HRG-β1-induced EMT process and cell migration through phospho-Smad2-mediated expression of Snail via the PI3k/Akt signaling pathway in SK-BR-3 and MCF7 breast cancer cells.
These findings are important for defining the tumorigenic roles of ErbB receptors and HRG as well as Smad2 activation in breast cancers, because HRG-β1 can overcome the inhibitory effects of anti-EGFR therapies on cell growth and activate invasion in tamoxifen-resistant cells through promotion of ErbB3/ErbB2 heterodimerization and activation of the PI3k/Akt signaling pathway .
In conclusion, we have demonstrated a downstream signal transduction pathway of HRG-β1-induced EMT that occurred in the SK-BR-3 and MCF7 breast cancer cell lines. Therefore, we suggest that blockade of the EMT mechanisms by HRG, including ErbB3 and not only Snail but also Smad2, might be a useful therapeutic target in breast cancer.
This study was supported by the National Research Foundation of Korea from the Ministry of Education, Science and Technology, Republic of Korea (Grant No. 20110026186).
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