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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

Lung cancer cell lines showed different AEG-1 expression characteristics and AEG-1 promoted the EMT process. (A) AEG-1 protein expression levels in lung cancer cell lines. NCI-H226 cells expressed a high level of AEG-1; NCl-H460, L-78, A549 and Slu-1 cells expressed a low level of AEG-1. (B) The expression spectrum of mesenchymal and epithelial markers in AEG-1-knockdown cells and pcDNA3.1-AEG-1-transfected cells was analyzed by using the Western blotting method. GAPDH served as a control. (C) Immunofluorescence staining of AEG-1, E-cadherin and Vimentin in NCI-H226 and Slu-01 cell lines (magnification × 100). (D) Knockdown of AEG-1 reversed EMT in vitro. Morphology of NCl-H226 cells transfected with AEG-1 siRNA was observed through phase-contrast microscopy (magnification × 100). Up-regulation of AEG-1 initiated EMT in vitro. Slu-01 cells were transfected with pcDNA3.1-AEG-1 and the morphology was observed through phase-contrast microscopy (magnification × 100). (E) The effect of AEG-1 expression changes on invasion ability. NCI-H226 cells were transfected with AEG-1 siRNA, and Slu-01 cells were transfected with pcDNA3.1-AEG-1. The data represent the mean ± SD of three independent experiments (asterisk; p < 0.01).
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Fig1: Lung cancer cell lines showed different AEG-1 expression characteristics and AEG-1 promoted the EMT process. (A) AEG-1 protein expression levels in lung cancer cell lines. NCI-H226 cells expressed a high level of AEG-1; NCl-H460, L-78, A549 and Slu-1 cells expressed a low level of AEG-1. (B) The expression spectrum of mesenchymal and epithelial markers in AEG-1-knockdown cells and pcDNA3.1-AEG-1-transfected cells was analyzed by using the Western blotting method. GAPDH served as a control. (C) Immunofluorescence staining of AEG-1, E-cadherin and Vimentin in NCI-H226 and Slu-01 cell lines (magnification × 100). (D) Knockdown of AEG-1 reversed EMT in vitro. Morphology of NCl-H226 cells transfected with AEG-1 siRNA was observed through phase-contrast microscopy (magnification × 100). Up-regulation of AEG-1 initiated EMT in vitro. Slu-01 cells were transfected with pcDNA3.1-AEG-1 and the morphology was observed through phase-contrast microscopy (magnification × 100). (E) The effect of AEG-1 expression changes on invasion ability. NCI-H226 cells were transfected with AEG-1 siRNA, and Slu-01 cells were transfected with pcDNA3.1-AEG-1. The data represent the mean ± SD of three independent experiments (asterisk; p < 0.01).

Mentions: To investigate the role of AEG-1 expression in lung cancer, we comparatively analyzed AEG-1 protein profiles in lung cancer cell lines with different metastatic ability. As shown in Figure 1A, Western blot analysis revealed that AEG-1 protein was highly expressed in NCI-H226 cells (from lung squamous cell carcinoma with high metastatic ability), whereas Slu-01 cells (from lung adenocarcinoma with low metastatic ability) had undetectable AEG-1 protein expression. In cell lines (with middle metastatic ability) such as NCI-H460, L-78 and A549, the expression levels of AEG-1 protein were significantly lower than that of NCI-H226 cells, but higher than that of Slu-01 cells. We also showed that NCI-H226 cells expressed high levels of Twist1, Vimentin and E-cadherin, but low level of E-cadherin, while Slu-01 cells displayed the opposite expression pattern (Figure 1B and C). These results indicated that AEG-1 might be associated with the metastasis process of lung cancer.Figure 1


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)

Lung cancer cell lines showed different AEG-1 expression characteristics and AEG-1 promoted the EMT process. (A) AEG-1 protein expression levels in lung cancer cell lines. NCI-H226 cells expressed a high level of AEG-1; NCl-H460, L-78, A549 and Slu-1 cells expressed a low level of AEG-1. (B) The expression spectrum of mesenchymal and epithelial markers in AEG-1-knockdown cells and pcDNA3.1-AEG-1-transfected cells was analyzed by using the Western blotting method. GAPDH served as a control. (C) Immunofluorescence staining of AEG-1, E-cadherin and Vimentin in NCI-H226 and Slu-01 cell lines (magnification × 100). (D) Knockdown of AEG-1 reversed EMT in vitro. Morphology of NCl-H226 cells transfected with AEG-1 siRNA was observed through phase-contrast microscopy (magnification × 100). Up-regulation of AEG-1 initiated EMT in vitro. Slu-01 cells were transfected with pcDNA3.1-AEG-1 and the morphology was observed through phase-contrast microscopy (magnification × 100). (E) The effect of AEG-1 expression changes on invasion ability. NCI-H226 cells were transfected with AEG-1 siRNA, and Slu-01 cells were transfected with pcDNA3.1-AEG-1. The data represent the mean ± SD of three independent experiments (asterisk; p < 0.01).
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Fig1: Lung cancer cell lines showed different AEG-1 expression characteristics and AEG-1 promoted the EMT process. (A) AEG-1 protein expression levels in lung cancer cell lines. NCI-H226 cells expressed a high level of AEG-1; NCl-H460, L-78, A549 and Slu-1 cells expressed a low level of AEG-1. (B) The expression spectrum of mesenchymal and epithelial markers in AEG-1-knockdown cells and pcDNA3.1-AEG-1-transfected cells was analyzed by using the Western blotting method. GAPDH served as a control. (C) Immunofluorescence staining of AEG-1, E-cadherin and Vimentin in NCI-H226 and Slu-01 cell lines (magnification × 100). (D) Knockdown of AEG-1 reversed EMT in vitro. Morphology of NCl-H226 cells transfected with AEG-1 siRNA was observed through phase-contrast microscopy (magnification × 100). Up-regulation of AEG-1 initiated EMT in vitro. Slu-01 cells were transfected with pcDNA3.1-AEG-1 and the morphology was observed through phase-contrast microscopy (magnification × 100). (E) The effect of AEG-1 expression changes on invasion ability. NCI-H226 cells were transfected with AEG-1 siRNA, and Slu-01 cells were transfected with pcDNA3.1-AEG-1. The data represent the mean ± SD of three independent experiments (asterisk; p < 0.01).
Mentions: To investigate the role of AEG-1 expression in lung cancer, we comparatively analyzed AEG-1 protein profiles in lung cancer cell lines with different metastatic ability. As shown in Figure 1A, Western blot analysis revealed that AEG-1 protein was highly expressed in NCI-H226 cells (from lung squamous cell carcinoma with high metastatic ability), whereas Slu-01 cells (from lung adenocarcinoma with low metastatic ability) had undetectable AEG-1 protein expression. In cell lines (with middle metastatic ability) such as NCI-H460, L-78 and A549, the expression levels of AEG-1 protein were significantly lower than that of NCI-H226 cells, but higher than that of Slu-01 cells. We also showed that NCI-H226 cells expressed high levels of Twist1, Vimentin and E-cadherin, but low level of E-cadherin, while Slu-01 cells displayed the opposite expression pattern (Figure 1B and C). These results indicated that AEG-1 might be associated with the metastasis process of lung cancer.Figure 1

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