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EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells.

Zhang Y, Du J, Zheng J, Liu J, Xu R, Shen T, Zhu Y, Chang J, Wang H, Zhang Z, Meng F, Wang Y, Chen Y, Xu Y, Gu L - Oncotarget (2015)

Bottom Line: To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression.On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells.In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Nanjing Medical University, Nanjing, Jiangsu 210029, China.

ABSTRACT
Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

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EGF induces EMT in gastric cancer SGC-7901 cells(A) SGC-7901 cells were incubated in the absence or presence of EGF (20 ng/mL), cell images were captured by phase-contrast microscopy for indicated times. Scale bar, 100 μm. (B–D) The extracts of SGC-7901 cells incubated with EGF (20 ng/mL) for 48 h, (B) representative microscopy images of SGC-7901 cells stained immunofluorescence for E-cadherin, N-cadherin and Vimentin, scale bar, 100 μm, and (C) the total cellular proteins were extracted and analyzed for expressions of E-cadherin, N-cadherin and Vimentin by immunoblotting assays. *P < 0.05, **P < 0.01 in the cultures with EGF relative to the cultures without EGF. (D) The SGC-7901 cells were scraped by a pipette tip and incubated with or without EGF for additional 48 h, a representative of wound healing assay was presented, and the quantification of cell migration rate was performed. **P < 0.01 in the cultures with EGF relative to the cultures without EGF.
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Figure 1: EGF induces EMT in gastric cancer SGC-7901 cells(A) SGC-7901 cells were incubated in the absence or presence of EGF (20 ng/mL), cell images were captured by phase-contrast microscopy for indicated times. Scale bar, 100 μm. (B–D) The extracts of SGC-7901 cells incubated with EGF (20 ng/mL) for 48 h, (B) representative microscopy images of SGC-7901 cells stained immunofluorescence for E-cadherin, N-cadherin and Vimentin, scale bar, 100 μm, and (C) the total cellular proteins were extracted and analyzed for expressions of E-cadherin, N-cadherin and Vimentin by immunoblotting assays. *P < 0.05, **P < 0.01 in the cultures with EGF relative to the cultures without EGF. (D) The SGC-7901 cells were scraped by a pipette tip and incubated with or without EGF for additional 48 h, a representative of wound healing assay was presented, and the quantification of cell migration rate was performed. **P < 0.01 in the cultures with EGF relative to the cultures without EGF.

Mentions: To assess the effect of EGF on EMT of gastric cancer cells, SGC-7901 cells were treated with EGF (20 ng/mL) and harvested at indicated time points and the cellular morphologic changes were observed by phase-contrast microscopy. We found that EGF time-dependently induced mesenchymal-like morphologies in SGC-7901 cells (Figure 1A), and led to significant induction of mesenchymal markers Vimentin and N-cadherin. Meanwhile, expression of E-cadherin, an epithelial marker, was decreased after EGF treatment, as shown by immunostaining (Figure 1B) and Western blotting analyses (Figure 1C & Figure S1A–S1B). Functionally, cell motility was increased in response to EGF (Figure 1D). In addition, Our MTT assays also showed that treatment with 20 ng/mL EGF for up to 72 h did not noticeably increase the proliferation of SGC-7901 cells (data not shown). Together, these data suggest that EGF (20 ng/mL) could induce the SGC-7901 cells to undergo EMT-like phenotypic changes. Accordingly, EGF (20 ng/mL) was used for the remainder of the experiments hereafter to identify the mechanism that accounts for the changes in the EMT of SGC-7901 cells.


EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells.

Zhang Y, Du J, Zheng J, Liu J, Xu R, Shen T, Zhu Y, Chang J, Wang H, Zhang Z, Meng F, Wang Y, Chen Y, Xu Y, Gu L - Oncotarget (2015)

EGF induces EMT in gastric cancer SGC-7901 cells(A) SGC-7901 cells were incubated in the absence or presence of EGF (20 ng/mL), cell images were captured by phase-contrast microscopy for indicated times. Scale bar, 100 μm. (B–D) The extracts of SGC-7901 cells incubated with EGF (20 ng/mL) for 48 h, (B) representative microscopy images of SGC-7901 cells stained immunofluorescence for E-cadherin, N-cadherin and Vimentin, scale bar, 100 μm, and (C) the total cellular proteins were extracted and analyzed for expressions of E-cadherin, N-cadherin and Vimentin by immunoblotting assays. *P < 0.05, **P < 0.01 in the cultures with EGF relative to the cultures without EGF. (D) The SGC-7901 cells were scraped by a pipette tip and incubated with or without EGF for additional 48 h, a representative of wound healing assay was presented, and the quantification of cell migration rate was performed. **P < 0.01 in the cultures with EGF relative to the cultures without EGF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466682&req=5

Figure 1: EGF induces EMT in gastric cancer SGC-7901 cells(A) SGC-7901 cells were incubated in the absence or presence of EGF (20 ng/mL), cell images were captured by phase-contrast microscopy for indicated times. Scale bar, 100 μm. (B–D) The extracts of SGC-7901 cells incubated with EGF (20 ng/mL) for 48 h, (B) representative microscopy images of SGC-7901 cells stained immunofluorescence for E-cadherin, N-cadherin and Vimentin, scale bar, 100 μm, and (C) the total cellular proteins were extracted and analyzed for expressions of E-cadherin, N-cadherin and Vimentin by immunoblotting assays. *P < 0.05, **P < 0.01 in the cultures with EGF relative to the cultures without EGF. (D) The SGC-7901 cells were scraped by a pipette tip and incubated with or without EGF for additional 48 h, a representative of wound healing assay was presented, and the quantification of cell migration rate was performed. **P < 0.01 in the cultures with EGF relative to the cultures without EGF.
Mentions: To assess the effect of EGF on EMT of gastric cancer cells, SGC-7901 cells were treated with EGF (20 ng/mL) and harvested at indicated time points and the cellular morphologic changes were observed by phase-contrast microscopy. We found that EGF time-dependently induced mesenchymal-like morphologies in SGC-7901 cells (Figure 1A), and led to significant induction of mesenchymal markers Vimentin and N-cadherin. Meanwhile, expression of E-cadherin, an epithelial marker, was decreased after EGF treatment, as shown by immunostaining (Figure 1B) and Western blotting analyses (Figure 1C & Figure S1A–S1B). Functionally, cell motility was increased in response to EGF (Figure 1D). In addition, Our MTT assays also showed that treatment with 20 ng/mL EGF for up to 72 h did not noticeably increase the proliferation of SGC-7901 cells (data not shown). Together, these data suggest that EGF (20 ng/mL) could induce the SGC-7901 cells to undergo EMT-like phenotypic changes. Accordingly, EGF (20 ng/mL) was used for the remainder of the experiments hereafter to identify the mechanism that accounts for the changes in the EMT of SGC-7901 cells.

Bottom Line: To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression.On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells.In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center, Nanjing Medical University, Nanjing, Jiangsu 210029, China.

ABSTRACT
Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

Show MeSH
Related in: MedlinePlus