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Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/β-catenin signaling.

He W, He S, Wang Z, Shen H, Fang W, Zhang Y, Qian W, Lin M, Yuan J, Wang J, Huang W, Wang L, Ke Z - BMC Cancer (2015)

Bottom Line: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens.Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT.Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Guangzhou, 510080, Province Guangdong, Peoples' Republic of China. heweiling@mail.sysu.edu.cn.

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/β-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors.

Methods: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/β-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry.

Results: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients.

Conclusions: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.

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Related in: MedlinePlus

AEG-1 activated β-catenin, which could reverse AEG-1-siRNA-mediated MET. (A) AEG-1 promoted β-catenin nuclear translocation. Slu-01 cells were transfected with pcDNA3.1-AEG-1. The subcellular localization of β-catenin was visualized through immunofluorescence (magnification × 400) and Western blotting. (B) Total β-catenin mRNA was detected by RT-PCR. (C) AEG-1 increased β-catenin/TCF transcriptional activity. NCI-H226 cells treated with AEG-1-siRNA, and Slu-01 cells treated with pcDNA3.1-AEG-1 were transfected with TCF-responsive promoter reporter (TOP-flash) or nonresponsive control reporter (FOP-flash); then, luciferase activity was measured as the ratio of TOP/FOP. Relative luciferase activity is presented as the mean ± SD. (error bars) from each sample after normalizing to the control. The asterisk indicates statistical significance (p < 0.01). (D) The morphology characteristics of NCI-H226 and Slu-01 cells were observed through phase-contrast microscopy (magnification × 200). (E) and (F) β-catenin overexpression reverses AEG-1-siRNA-mediated MET. An increasing amount of β-catenin was transfected in NCI-H226 (E) and Slu-01 (F) cells for 24 hours. Total cell lysates were probed with antibodies against E-cadherin, Vimentin, and β-catenin.
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Fig2: AEG-1 activated β-catenin, which could reverse AEG-1-siRNA-mediated MET. (A) AEG-1 promoted β-catenin nuclear translocation. Slu-01 cells were transfected with pcDNA3.1-AEG-1. The subcellular localization of β-catenin was visualized through immunofluorescence (magnification × 400) and Western blotting. (B) Total β-catenin mRNA was detected by RT-PCR. (C) AEG-1 increased β-catenin/TCF transcriptional activity. NCI-H226 cells treated with AEG-1-siRNA, and Slu-01 cells treated with pcDNA3.1-AEG-1 were transfected with TCF-responsive promoter reporter (TOP-flash) or nonresponsive control reporter (FOP-flash); then, luciferase activity was measured as the ratio of TOP/FOP. Relative luciferase activity is presented as the mean ± SD. (error bars) from each sample after normalizing to the control. The asterisk indicates statistical significance (p < 0.01). (D) The morphology characteristics of NCI-H226 and Slu-01 cells were observed through phase-contrast microscopy (magnification × 200). (E) and (F) β-catenin overexpression reverses AEG-1-siRNA-mediated MET. An increasing amount of β-catenin was transfected in NCI-H226 (E) and Slu-01 (F) cells for 24 hours. Total cell lysates were probed with antibodies against E-cadherin, Vimentin, and β-catenin.

Mentions: Based on the critical role of the Wnt/β-catenin pathway in metastasis, we then explored whether AEG-1 activates Wnt/β-catenin signaling and if the Wnt/β-catenin pathway mediates AEG-1-induced EMT. In the canonical Wnt/β-catenin pathway, the hallmark of Wnt signaling activation is β-catenin’s nuclear translocation, where it forms a complex with a specific T-cell factor/lymphoid enhancer factor (Tcf/Lef) [18]. After up-regulating AEG-1 expression in Slu-01 cells with pcDNA3.1-AEG-1, we observed a substantial accumulation of β-catenin in the nucleus, suggesting that AEG-1 might contribute to the activation of Wnt signaling (Figure 2A). However, the total β-catenin mRNA level did not change significantly after AEG-1 overexpression in Slu-01 cells (Figure 2B). As expected, luciferase assays also demonstrated that AEG-1 overexpression noticeably increased the transcriptional activity of β-catenin/TCF in Slu-01 cells, as determined by the β-catenin reporter system (TOP/FOP) (Figure 2C). In contrast, transfection of AEG-1 siRNA could reduce the β-catenin/TCF transcriptional activity in NCl-H226 cells (Figure 2C).Figure 2


Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/β-catenin signaling.

He W, He S, Wang Z, Shen H, Fang W, Zhang Y, Qian W, Lin M, Yuan J, Wang J, Huang W, Wang L, Ke Z - BMC Cancer (2015)

AEG-1 activated β-catenin, which could reverse AEG-1-siRNA-mediated MET. (A) AEG-1 promoted β-catenin nuclear translocation. Slu-01 cells were transfected with pcDNA3.1-AEG-1. The subcellular localization of β-catenin was visualized through immunofluorescence (magnification × 400) and Western blotting. (B) Total β-catenin mRNA was detected by RT-PCR. (C) AEG-1 increased β-catenin/TCF transcriptional activity. NCI-H226 cells treated with AEG-1-siRNA, and Slu-01 cells treated with pcDNA3.1-AEG-1 were transfected with TCF-responsive promoter reporter (TOP-flash) or nonresponsive control reporter (FOP-flash); then, luciferase activity was measured as the ratio of TOP/FOP. Relative luciferase activity is presented as the mean ± SD. (error bars) from each sample after normalizing to the control. The asterisk indicates statistical significance (p < 0.01). (D) The morphology characteristics of NCI-H226 and Slu-01 cells were observed through phase-contrast microscopy (magnification × 200). (E) and (F) β-catenin overexpression reverses AEG-1-siRNA-mediated MET. An increasing amount of β-catenin was transfected in NCI-H226 (E) and Slu-01 (F) cells for 24 hours. Total cell lysates were probed with antibodies against E-cadherin, Vimentin, and β-catenin.
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Fig2: AEG-1 activated β-catenin, which could reverse AEG-1-siRNA-mediated MET. (A) AEG-1 promoted β-catenin nuclear translocation. Slu-01 cells were transfected with pcDNA3.1-AEG-1. The subcellular localization of β-catenin was visualized through immunofluorescence (magnification × 400) and Western blotting. (B) Total β-catenin mRNA was detected by RT-PCR. (C) AEG-1 increased β-catenin/TCF transcriptional activity. NCI-H226 cells treated with AEG-1-siRNA, and Slu-01 cells treated with pcDNA3.1-AEG-1 were transfected with TCF-responsive promoter reporter (TOP-flash) or nonresponsive control reporter (FOP-flash); then, luciferase activity was measured as the ratio of TOP/FOP. Relative luciferase activity is presented as the mean ± SD. (error bars) from each sample after normalizing to the control. The asterisk indicates statistical significance (p < 0.01). (D) The morphology characteristics of NCI-H226 and Slu-01 cells were observed through phase-contrast microscopy (magnification × 200). (E) and (F) β-catenin overexpression reverses AEG-1-siRNA-mediated MET. An increasing amount of β-catenin was transfected in NCI-H226 (E) and Slu-01 (F) cells for 24 hours. Total cell lysates were probed with antibodies against E-cadherin, Vimentin, and β-catenin.
Mentions: Based on the critical role of the Wnt/β-catenin pathway in metastasis, we then explored whether AEG-1 activates Wnt/β-catenin signaling and if the Wnt/β-catenin pathway mediates AEG-1-induced EMT. In the canonical Wnt/β-catenin pathway, the hallmark of Wnt signaling activation is β-catenin’s nuclear translocation, where it forms a complex with a specific T-cell factor/lymphoid enhancer factor (Tcf/Lef) [18]. After up-regulating AEG-1 expression in Slu-01 cells with pcDNA3.1-AEG-1, we observed a substantial accumulation of β-catenin in the nucleus, suggesting that AEG-1 might contribute to the activation of Wnt signaling (Figure 2A). However, the total β-catenin mRNA level did not change significantly after AEG-1 overexpression in Slu-01 cells (Figure 2B). As expected, luciferase assays also demonstrated that AEG-1 overexpression noticeably increased the transcriptional activity of β-catenin/TCF in Slu-01 cells, as determined by the β-catenin reporter system (TOP/FOP) (Figure 2C). In contrast, transfection of AEG-1 siRNA could reduce the β-catenin/TCF transcriptional activity in NCl-H226 cells (Figure 2C).Figure 2

Bottom Line: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens.Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT.Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Guangzhou, 510080, Province Guangdong, Peoples' Republic of China. heweiling@mail.sysu.edu.cn.

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/β-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors.

Methods: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/β-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry.

Results: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/β-catenin signaling cascade, including GSK-3β and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients.

Conclusions: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.

Show MeSH
Related in: MedlinePlus