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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/Arf6 reduces Wnt5a expression and promotes cell EMT through P-ERK(A–F) Cells were incubated for 2 h in the absence or presence of 10 μmol/L U0126 prior to EGF treatment (20 ng/mL for 48 h), the extracts of (A) cytoplasm and (B) nucleus were subjected to immunoblotting analysis to detect P-ERK. GAPDH or Histone 3 was as control for cytoplasm or nucleus part. (C) Total mRNA or (D) protein extracts for Wnt5a were analyzed by qPCR and immunoblotting. GAPDH was used as control. **P < 0.01 in the cultures with EGF or U0126 relative to the cultures without EGF. ##P < 0.01 in the cultures with EGF plus U0126 relative to the cultures with EGF alone. (E) The cell images were captured by phase-contrast microscopy. Scale bar, 100 μm. (F) The extracts of whole cell protein were subjected to immunoblotting analysis to detect E-cadherin. (G) SGC-7901 cells transfected with either an empty vector or an Arf6-T27N expression vector were stimulated with 20 ng/mL EGF for 48 h and ERK activity was examined.
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Figure 5: EGF/Arf6 reduces Wnt5a expression and promotes cell EMT through P-ERK(A–F) Cells were incubated for 2 h in the absence or presence of 10 μmol/L U0126 prior to EGF treatment (20 ng/mL for 48 h), the extracts of (A) cytoplasm and (B) nucleus were subjected to immunoblotting analysis to detect P-ERK. GAPDH or Histone 3 was as control for cytoplasm or nucleus part. (C) Total mRNA or (D) protein extracts for Wnt5a were analyzed by qPCR and immunoblotting. GAPDH was used as control. **P < 0.01 in the cultures with EGF or U0126 relative to the cultures without EGF. ##P < 0.01 in the cultures with EGF plus U0126 relative to the cultures with EGF alone. (E) The cell images were captured by phase-contrast microscopy. Scale bar, 100 μm. (F) The extracts of whole cell protein were subjected to immunoblotting analysis to detect E-cadherin. (G) SGC-7901 cells transfected with either an empty vector or an Arf6-T27N expression vector were stimulated with 20 ng/mL EGF for 48 h and ERK activity was examined.

Mentions: A recent study has reported that ERK acts as a transcriptional repressor for several genes [25]. We hypothesized that P-ERK might also be involved in the down-regulation of Wnt5a in gastric cancer cells in response to EGF. Therefore, we first determined whether EGF could activate ERK phosphorylation in SGC-7901 cells. Immunoblotting assay showed visible cytoplasmic phosphorylation of ERK, which was further increased by EGF stimulation (Figure 4A). Importantly, we were able to detect phosphorylated ERK in the nucleus, which reached peak levels at 24–48 h after EGF treatment as evidenced by immunoblotting (Figure 4B) and immunofluorescence staining assays (Figure 4C). Pre-treatment with MEK kinase inhibitor U0126 inhibited the EGF-induced phosphorylation of ERK both in cytoplasm and nucleus (Figure 4C & Figure 5A–5B).


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/Arf6 reduces Wnt5a expression and promotes cell EMT through P-ERK(A–F) Cells were incubated for 2 h in the absence or presence of 10 μmol/L U0126 prior to EGF treatment (20 ng/mL for 48 h), the extracts of (A) cytoplasm and (B) nucleus were subjected to immunoblotting analysis to detect P-ERK. GAPDH or Histone 3 was as control for cytoplasm or nucleus part. (C) Total mRNA or (D) protein extracts for Wnt5a were analyzed by qPCR and immunoblotting. GAPDH was used as control. **P < 0.01 in the cultures with EGF or U0126 relative to the cultures without EGF. ##P < 0.01 in the cultures with EGF plus U0126 relative to the cultures with EGF alone. (E) The cell images were captured by phase-contrast microscopy. Scale bar, 100 μm. (F) The extracts of whole cell protein were subjected to immunoblotting analysis to detect E-cadherin. (G) SGC-7901 cells transfected with either an empty vector or an Arf6-T27N expression vector were stimulated with 20 ng/mL EGF for 48 h and ERK activity was examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: EGF/Arf6 reduces Wnt5a expression and promotes cell EMT through P-ERK(A–F) Cells were incubated for 2 h in the absence or presence of 10 μmol/L U0126 prior to EGF treatment (20 ng/mL for 48 h), the extracts of (A) cytoplasm and (B) nucleus were subjected to immunoblotting analysis to detect P-ERK. GAPDH or Histone 3 was as control for cytoplasm or nucleus part. (C) Total mRNA or (D) protein extracts for Wnt5a were analyzed by qPCR and immunoblotting. GAPDH was used as control. **P < 0.01 in the cultures with EGF or U0126 relative to the cultures without EGF. ##P < 0.01 in the cultures with EGF plus U0126 relative to the cultures with EGF alone. (E) The cell images were captured by phase-contrast microscopy. Scale bar, 100 μm. (F) The extracts of whole cell protein were subjected to immunoblotting analysis to detect E-cadherin. (G) SGC-7901 cells transfected with either an empty vector or an Arf6-T27N expression vector were stimulated with 20 ng/mL EGF for 48 h and ERK activity was examined.
Mentions: A recent study has reported that ERK acts as a transcriptional repressor for several genes [25]. We hypothesized that P-ERK might also be involved in the down-regulation of Wnt5a in gastric cancer cells in response to EGF. Therefore, we first determined whether EGF could activate ERK phosphorylation in SGC-7901 cells. Immunoblotting assay showed visible cytoplasmic phosphorylation of ERK, which was further increased by EGF stimulation (Figure 4A). Importantly, we were able to detect phosphorylated ERK in the nucleus, which reached peak levels at 24–48 h after EGF treatment as evidenced by immunoblotting (Figure 4B) and immunofluorescence staining assays (Figure 4C). Pre-treatment with MEK kinase inhibitor U0126 inhibited the EGF-induced phosphorylation of ERK both in cytoplasm and nucleus (Figure 4C & Figure 5A–5B).

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