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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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Wnt5a expression is relative with the high degree differentiation of malignant gastric cancer and has a negative correlation with P-ERK(A) The IRS was calculated as intensity of the staining reaction multiplied by the percentage of positive cells. Based on the IRS values, Wnt5a and ERK were scored as weak, medium and strong in the following parts. (B) High and low degree differentiated malignant gastric cancer tissue sections were stained against Wnt5a. Brown, Wnt5a; Blue, haematoxylin. (C) IRS scores of Wnt5a according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (D) High and low degree differentiated malignant gastric cancer tissue sections were stained against P-ERK. Brown, P-ERK; Blue, haematoxylin. (E) IRS scores of P-ERK according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (F) Scatterplot of correlated protein levels between Wnt5a and P-ERK in high and low degree differentiated malignant gastric cancer tissue (n = 50). Scale bar, 100 μm.
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Figure 8: Wnt5a expression is relative with the high degree differentiation of malignant gastric cancer and has a negative correlation with P-ERK(A) The IRS was calculated as intensity of the staining reaction multiplied by the percentage of positive cells. Based on the IRS values, Wnt5a and ERK were scored as weak, medium and strong in the following parts. (B) High and low degree differentiated malignant gastric cancer tissue sections were stained against Wnt5a. Brown, Wnt5a; Blue, haematoxylin. (C) IRS scores of Wnt5a according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (D) High and low degree differentiated malignant gastric cancer tissue sections were stained against P-ERK. Brown, P-ERK; Blue, haematoxylin. (E) IRS scores of P-ERK according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (F) Scatterplot of correlated protein levels between Wnt5a and P-ERK in high and low degree differentiated malignant gastric cancer tissue (n = 50). Scale bar, 100 μm.

Mentions: To investigate whether our experimental findings could be relevant to the pathogenesis and progression of gastric cancer in humans, we examined Wnt5a and P-ERK expression patterns in gastric cancers with varied degrees of differentiation. The immunoreactive score (IRS) was calculated as the intensity of the staining reaction multiplied by the percentage of positive cells [26, 27] (Figure 8A). Representative results of Wnt5a and P-ERK immunostaining of gastric cancer are shown in Figure 8B & 8D. Based on the analysis of a limited set of 50 gastric cancer cases, we found that the Wnt5a expression level was markedly reduced in poorly differentiated tumor tissues when compared with the well-differentiated tumor tissues, while P-ERK levels were in a reversed pattern (Figure 8C & 8E). In addition, increased nuclear p-ERK staining were observed in poorly differentiated tumor tissues (Figure S5A & S5B). Furthermore, immunostaining of Wnt5a and P-ERK in these 50 primary gastric tumors revealed a negative correlation in expression (r = −0.288, P < 0.05) (Figure 8F). Finally, increased nuclear p-ERK staining was also inversely correlated with Wnt5a expression (Figure S5A–S5C), suggesting a direct role for ERK in mediating Wnt5a transcriptional repression. Overall, our clinical data support our in vitro data that Wnt5a may function to oppose gastric cancer progression and ERK may play a role as a Wnt5a repressor (Figure 9).


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)

Wnt5a expression is relative with the high degree differentiation of malignant gastric cancer and has a negative correlation with P-ERK(A) The IRS was calculated as intensity of the staining reaction multiplied by the percentage of positive cells. Based on the IRS values, Wnt5a and ERK were scored as weak, medium and strong in the following parts. (B) High and low degree differentiated malignant gastric cancer tissue sections were stained against Wnt5a. Brown, Wnt5a; Blue, haematoxylin. (C) IRS scores of Wnt5a according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (D) High and low degree differentiated malignant gastric cancer tissue sections were stained against P-ERK. Brown, P-ERK; Blue, haematoxylin. (E) IRS scores of P-ERK according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (F) Scatterplot of correlated protein levels between Wnt5a and P-ERK in high and low degree differentiated malignant gastric cancer tissue (n = 50). Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Wnt5a expression is relative with the high degree differentiation of malignant gastric cancer and has a negative correlation with P-ERK(A) The IRS was calculated as intensity of the staining reaction multiplied by the percentage of positive cells. Based on the IRS values, Wnt5a and ERK were scored as weak, medium and strong in the following parts. (B) High and low degree differentiated malignant gastric cancer tissue sections were stained against Wnt5a. Brown, Wnt5a; Blue, haematoxylin. (C) IRS scores of Wnt5a according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (D) High and low degree differentiated malignant gastric cancer tissue sections were stained against P-ERK. Brown, P-ERK; Blue, haematoxylin. (E) IRS scores of P-ERK according to tumor histological grade. P values and tissue samples are showed above the scatter diagram. (F) Scatterplot of correlated protein levels between Wnt5a and P-ERK in high and low degree differentiated malignant gastric cancer tissue (n = 50). Scale bar, 100 μm.
Mentions: To investigate whether our experimental findings could be relevant to the pathogenesis and progression of gastric cancer in humans, we examined Wnt5a and P-ERK expression patterns in gastric cancers with varied degrees of differentiation. The immunoreactive score (IRS) was calculated as the intensity of the staining reaction multiplied by the percentage of positive cells [26, 27] (Figure 8A). Representative results of Wnt5a and P-ERK immunostaining of gastric cancer are shown in Figure 8B & 8D. Based on the analysis of a limited set of 50 gastric cancer cases, we found that the Wnt5a expression level was markedly reduced in poorly differentiated tumor tissues when compared with the well-differentiated tumor tissues, while P-ERK levels were in a reversed pattern (Figure 8C & 8E). In addition, increased nuclear p-ERK staining were observed in poorly differentiated tumor tissues (Figure S5A & S5B). Furthermore, immunostaining of Wnt5a and P-ERK in these 50 primary gastric tumors revealed a negative correlation in expression (r = −0.288, P < 0.05) (Figure 8F). Finally, increased nuclear p-ERK staining was also inversely correlated with Wnt5a expression (Figure S5A–S5C), suggesting a direct role for ERK in mediating Wnt5a transcriptional repression. Overall, our clinical data support our in vitro data that Wnt5a may function to oppose gastric cancer progression and ERK may play a role as a Wnt5a repressor (Figure 9).

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