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Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/╬▓-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/╬▓-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/╬▓-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/╬▓-catenin signaling cascade, including GSK-3╬▓ and CKI╬┤. 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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AEG-1 was associated with CKI╬┤and modulated the GSK-3╬▓/╬▓-catenin signaling pathway. (A) Slu-01 cells stably overexpressing AEG-1 were established. GSK-3╬▓ was immunoprecipitated from cell lysates, and its expression was confirmed by immunoblotting with the indicated antibodies. (B) CKI╬┤ was critical for AEG-1-mediated regulation of GSK-3╬▓/╬▓-catenin signaling and EMT. AEG-1 complexes were associated with both GSK-3╬▓ and CKI╬┤. (C) CKI╬┤played a role in AEG-mediated regulation of EMT and Ser-9 phosphorylation of GSK-3╬▓. Slu-01 cells transfected with pcDNA3.1-AEG-1 were co-transfected with CKI╬┤-specific siRNA. Cell lysates were then subjected to Western blotting.
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Fig3: AEG-1 was associated with CKI╬┤and modulated the GSK-3╬▓/╬▓-catenin signaling pathway. (A) Slu-01 cells stably overexpressing AEG-1 were established. GSK-3╬▓ was immunoprecipitated from cell lysates, and its expression was confirmed by immunoblotting with the indicated antibodies. (B) CKI╬┤ was critical for AEG-1-mediated regulation of GSK-3╬▓/╬▓-catenin signaling and EMT. AEG-1 complexes were associated with both GSK-3╬▓ and CKI╬┤. (C) CKI╬┤played a role in AEG-mediated regulation of EMT and Ser-9 phosphorylation of GSK-3╬▓. Slu-01 cells transfected with pcDNA3.1-AEG-1 were co-transfected with CKI╬┤-specific siRNA. Cell lysates were then subjected to Western blotting.

Mentions: We then investigated the molecular mechanism by which AEG-1 activates Wnt/β-catenin signaling. In the absence of Wnt signaling, cytoplasmic β-catenin undergoes sequential phosphorylation, first at Ser45(β-cat45) by casein kinase I (CKI) and then at Ser33,37/Thr41 by glycogen synthase kinase (GSK)-3β, leading to targeted ubiquitination through E3 ubiquitin ligase. In Slu-01 cells transfected with pcDNA3.1-AEG-1, immunoprecipitation experiments and Western blot analysis revealed that AEG-1 appeared to directly associate with GSK-3β and promote its phosphorylation at Ser9 (Figure 3A). In addition, co-immunoprecipitation results showed that AEG-1 could form a complex with both GSK-3β and CKIδ (Figure 3B). Moreover, after Slu-01/AEG-1 cells were treated with CKIδ-siRNA, CKIδ-siRNA treatment abolished AEG-1-mediated phosphorylation of GSK-3β at Ser9 and EMT (Figure 3C).Figure 3


Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/╬▓-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 was associated with CKI╬┤and modulated the GSK-3╬▓/╬▓-catenin signaling pathway. (A) Slu-01 cells stably overexpressing AEG-1 were established. GSK-3╬▓ was immunoprecipitated from cell lysates, and its expression was confirmed by immunoblotting with the indicated antibodies. (B) CKI╬┤ was critical for AEG-1-mediated regulation of GSK-3╬▓/╬▓-catenin signaling and EMT. AEG-1 complexes were associated with both GSK-3╬▓ and CKI╬┤. (C) CKI╬┤played a role in AEG-mediated regulation of EMT and Ser-9 phosphorylation of GSK-3╬▓. Slu-01 cells transfected with pcDNA3.1-AEG-1 were co-transfected with CKI╬┤-specific siRNA. Cell lysates were then subjected to Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358870&req=5

Fig3: AEG-1 was associated with CKI╬┤and modulated the GSK-3╬▓/╬▓-catenin signaling pathway. (A) Slu-01 cells stably overexpressing AEG-1 were established. GSK-3╬▓ was immunoprecipitated from cell lysates, and its expression was confirmed by immunoblotting with the indicated antibodies. (B) CKI╬┤ was critical for AEG-1-mediated regulation of GSK-3╬▓/╬▓-catenin signaling and EMT. AEG-1 complexes were associated with both GSK-3╬▓ and CKI╬┤. (C) CKI╬┤played a role in AEG-mediated regulation of EMT and Ser-9 phosphorylation of GSK-3╬▓. Slu-01 cells transfected with pcDNA3.1-AEG-1 were co-transfected with CKI╬┤-specific siRNA. Cell lysates were then subjected to Western blotting.
Mentions: We then investigated the molecular mechanism by which AEG-1 activates Wnt/β-catenin signaling. In the absence of Wnt signaling, cytoplasmic β-catenin undergoes sequential phosphorylation, first at Ser45(β-cat45) by casein kinase I (CKI) and then at Ser33,37/Thr41 by glycogen synthase kinase (GSK)-3β, leading to targeted ubiquitination through E3 ubiquitin ligase. In Slu-01 cells transfected with pcDNA3.1-AEG-1, immunoprecipitation experiments and Western blot analysis revealed that AEG-1 appeared to directly associate with GSK-3β and promote its phosphorylation at Ser9 (Figure 3A). In addition, co-immunoprecipitation results showed that AEG-1 could form a complex with both GSK-3β and CKIδ (Figure 3B). Moreover, after Slu-01/AEG-1 cells were treated with CKIδ-siRNA, CKIδ-siRNA treatment abolished AEG-1-mediated phosphorylation of GSK-3β at Ser9 and EMT (Figure 3C).Figure 3

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/╬▓-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/╬▓-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/╬▓-catenin signaling cascade, including GSK-3╬▓ and CKI╬┤. 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