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

Kim J, Jeong H, Lee Y, Kim C, Kim H, Kim A - BMC Cancer (2013)

Bottom Line: 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.Knockdown of ErbB3 and Smad2 also decreased SK-BR-3 and MCF7 cell invasion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Korea University Guro Hospital, #97 Gurodong-gil, Guro-gu, Seoul 152-703, Korea.

ABSTRACT

Background: 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.

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-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.

Results: 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.

Conclusions: 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.

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HRG-β1 induces upregulation of the transcription factor Snail and EMT markers in SK-BR-3 cells. (a) The cells were incubated with 25 ng/ml of HRG-β1 for different times after serum starvation. The expressions of Snail, mesenchymal markers including vimentin and fibronectin, and epithelial marker E-cadherin were examined by western blotting. β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference. (b) Immunofluorescence staining of E-cadherin protein. Cells were treated with or without 25 ng/ml of HRG-β1 for 48 h. The green color represents staining of E-cadherin and the blue color represents nuclear DNA staining by DAPI (magnification, ×400). The data were analyzed as the percentages of the control cells (**P < 0.01).
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Figure 1: HRG-β1 induces upregulation of the transcription factor Snail and EMT markers in SK-BR-3 cells. (a) The cells were incubated with 25 ng/ml of HRG-β1 for different times after serum starvation. The expressions of Snail, mesenchymal markers including vimentin and fibronectin, and epithelial marker E-cadherin were examined by western blotting. β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference. (b) Immunofluorescence staining of E-cadherin protein. Cells were treated with or without 25 ng/ml of HRG-β1 for 48 h. The green color represents staining of E-cadherin and the blue color represents nuclear DNA staining by DAPI (magnification, ×400). The data were analyzed as the percentages of the control cells (**P < 0.01).

Mentions: 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.


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

Kim J, Jeong H, Lee Y, Kim C, Kim H, Kim A - BMC Cancer (2013)

HRG-β1 induces upregulation of the transcription factor Snail and EMT markers in SK-BR-3 cells. (a) The cells were incubated with 25 ng/ml of HRG-β1 for different times after serum starvation. The expressions of Snail, mesenchymal markers including vimentin and fibronectin, and epithelial marker E-cadherin were examined by western blotting. β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference. (b) Immunofluorescence staining of E-cadherin protein. Cells were treated with or without 25 ng/ml of HRG-β1 for 48 h. The green color represents staining of E-cadherin and the blue color represents nuclear DNA staining by DAPI (magnification, ×400). The data were analyzed as the percentages of the control cells (**P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: HRG-β1 induces upregulation of the transcription factor Snail and EMT markers in SK-BR-3 cells. (a) The cells were incubated with 25 ng/ml of HRG-β1 for different times after serum starvation. The expressions of Snail, mesenchymal markers including vimentin and fibronectin, and epithelial marker E-cadherin were examined by western blotting. β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference. (b) Immunofluorescence staining of E-cadherin protein. Cells were treated with or without 25 ng/ml of HRG-β1 for 48 h. The green color represents staining of E-cadherin and the blue color represents nuclear DNA staining by DAPI (magnification, ×400). The data were analyzed as the percentages of the control cells (**P < 0.01).
Mentions: 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.

Bottom Line: 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.Knockdown of ErbB3 and Smad2 also decreased SK-BR-3 and MCF7 cell invasion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Korea University Guro Hospital, #97 Gurodong-gil, Guro-gu, Seoul 152-703, Korea.

ABSTRACT

Background: 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.

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-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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus