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

Background: Heregulin (HRG; also known as neuregulin) is a ligand for ErbB3. One of its isotypes, HRG-beta 1, 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-beta signaling. Therefore, we investigated whether the HRG-beta 1 and ErbB3 activate Smad2 signaling during process of EMT in breast cancer cells. Methods: 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-beta 1. 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-beta 1-induced EMT. Results: HRG-beta 1 induced EMT through activation of Smad2. The expression of E-cadherin was decreased after HRG-beta 1 treatment, while the expressions of Snail, vimentin, and fibronectin were increased. The HRG-beta 1-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-beta 1 was inhibited. Knockdown of Smad2 by siRNA transfection suppressed the expressions of Snail and fibronectin in response to HRG-beta 1 stimulation and knockdown of ErbB3 suppressed the expressions of phospho-Smad2, Snail, and fibronectin induced by HRG-beta 1, 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. Conclusions: Our data suggest that HRG-beta 1 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.