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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 phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
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Figure 3: HRG-β1 induces phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.

Mentions: 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.


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 phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750857&req=5

Figure 3: HRG-β1 induces phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, significant difference.
Mentions: 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.

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