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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 expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
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Figure 5: HRG-β1 induces expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.

Mentions: 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-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 expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
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Figure 5: HRG-β1 induces expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
Mentions: 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).

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