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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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Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β-actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, significant difference.
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Figure 8: Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β-actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, significant difference.

Mentions: SK-BR-3 and MCF7 cells were transfected with control and Smad2 siRNAs. As shown in Figure 8a, b, the HRG-β1-increased expressions of Snail and fibronectin in control siRNA-transfected cells (Ctrl siR) compared with untreated control cells (untreated ctrl) were downregulated in Smad2 siRNA-transfected cells (Smad2 siR). Taken together, Smad2 activation plays roles in the expression of Snail and induction of EMT by HRG-β1 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)

Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β-actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, significant difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β-actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, significant difference.
Mentions: SK-BR-3 and MCF7 cells were transfected with control and Smad2 siRNAs. As shown in Figure 8a, b, the HRG-β1-increased expressions of Snail and fibronectin in control siRNA-transfected cells (Ctrl siR) compared with untreated control cells (untreated ctrl) were downregulated in Smad2 siRNA-transfected cells (Smad2 siR). Taken together, Smad2 activation plays roles in the expression of Snail and induction of EMT by HRG-β1 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