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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 promoted tumor metastasis in vivo. (A) Representative BLI images of mice bearing Slu-01/AEG-1-expressing tumors with metastatic lesions. Mice (n = 15) were imaged six weeks later to determine local tumor growth and metastasis. (B) Number of metastatic nodules or distant metastasis in individual dead mouse bearing con or Slu-01/AEG-1-expressing tumors. (C) AEG-1 overexpression in Slu-01 cells promoted EMT in athymic nude mice in vivo. H&E staining showed primary tumors without detectable metastasis in control mice and the lymph node metastases in mice bearing Slu-01/AEG-1-expressing tumors two weeks after injection (magnification, ×200). IHC showed that up-regulation of AEG-1 resulted in an increased in the expression of Vimentin and weak E-cadherin staining (magnification × 200).
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Fig5: AEG-1 promoted tumor metastasis in vivo. (A) Representative BLI images of mice bearing Slu-01/AEG-1-expressing tumors with metastatic lesions. Mice (n = 15) were imaged six weeks later to determine local tumor growth and metastasis. (B) Number of metastatic nodules or distant metastasis in individual dead mouse bearing con or Slu-01/AEG-1-expressing tumors. (C) AEG-1 overexpression in Slu-01 cells promoted EMT in athymic nude mice in vivo. H&E staining showed primary tumors without detectable metastasis in control mice and the lymph node metastases in mice bearing Slu-01/AEG-1-expressing tumors two weeks after injection (magnification, ×200). IHC showed that up-regulation of AEG-1 resulted in an increased in the expression of Vimentin and weak E-cadherin staining (magnification × 200).

Mentions: Because Slu-01 cells are of low metastatic potential and decreased AEG-1 expression, and show EMT inhibition status (Figure 1), we then observed the prometastatic trait of AEG-1 up-regulation in Slu-01 cells versus its corresponding vector control cells using an orthotopic mouse model. Stable luciferase activity ensured that every group had an equal level of AEG-1 expression before the injection of Slu-01/AEG-1 cells. Bioluminescent imaging (BLI) was utilized to monitor tumor growth and the onset of metastases dynamically. Strikingly, mice injected with Slu-01/AEG-1 cells displayed multiple distant metastatic lesions at various sites, whereas less metastasis lesions were found in mice injected with control Slu-01 cells (Figure 5A). Our data also showed that Slu-01/AEG-1 xenotransplants approximately generated a 4-fold increase in the number of distant metastases than that of vector control cells (Figure 5B), which was verified by H&E staining (Figure 5C). To further validate the fact that AEG-1 enhanced metastasis in vivo by regulating EMT status, immunohistochemistry(IHC) was applied to detect the expression characteristics of EMT-related molecular markers. Immunohistochemistry (IHC) revealed that the majority of tumor cells in Slu-01/AEG-1 xenotransplants strongly expressed Vimentin, but exhibited weak staining of E-cadherin (Figure 5C).Figure 5


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 promoted tumor metastasis in vivo. (A) Representative BLI images of mice bearing Slu-01/AEG-1-expressing tumors with metastatic lesions. Mice (n = 15) were imaged six weeks later to determine local tumor growth and metastasis. (B) Number of metastatic nodules or distant metastasis in individual dead mouse bearing con or Slu-01/AEG-1-expressing tumors. (C) AEG-1 overexpression in Slu-01 cells promoted EMT in athymic nude mice in vivo. H&E staining showed primary tumors without detectable metastasis in control mice and the lymph node metastases in mice bearing Slu-01/AEG-1-expressing tumors two weeks after injection (magnification, ×200). IHC showed that up-regulation of AEG-1 resulted in an increased in the expression of Vimentin and weak E-cadherin staining (magnification × 200).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: AEG-1 promoted tumor metastasis in vivo. (A) Representative BLI images of mice bearing Slu-01/AEG-1-expressing tumors with metastatic lesions. Mice (n = 15) were imaged six weeks later to determine local tumor growth and metastasis. (B) Number of metastatic nodules or distant metastasis in individual dead mouse bearing con or Slu-01/AEG-1-expressing tumors. (C) AEG-1 overexpression in Slu-01 cells promoted EMT in athymic nude mice in vivo. H&E staining showed primary tumors without detectable metastasis in control mice and the lymph node metastases in mice bearing Slu-01/AEG-1-expressing tumors two weeks after injection (magnification, ×200). IHC showed that up-regulation of AEG-1 resulted in an increased in the expression of Vimentin and weak E-cadherin staining (magnification × 200).
Mentions: Because Slu-01 cells are of low metastatic potential and decreased AEG-1 expression, and show EMT inhibition status (Figure 1), we then observed the prometastatic trait of AEG-1 up-regulation in Slu-01 cells versus its corresponding vector control cells using an orthotopic mouse model. Stable luciferase activity ensured that every group had an equal level of AEG-1 expression before the injection of Slu-01/AEG-1 cells. Bioluminescent imaging (BLI) was utilized to monitor tumor growth and the onset of metastases dynamically. Strikingly, mice injected with Slu-01/AEG-1 cells displayed multiple distant metastatic lesions at various sites, whereas less metastasis lesions were found in mice injected with control Slu-01 cells (Figure 5A). Our data also showed that Slu-01/AEG-1 xenotransplants approximately generated a 4-fold increase in the number of distant metastases than that of vector control cells (Figure 5B), which was verified by H&E staining (Figure 5C). To further validate the fact that AEG-1 enhanced metastasis in vivo by regulating EMT status, immunohistochemistry(IHC) was applied to detect the expression characteristics of EMT-related molecular markers. Immunohistochemistry (IHC) revealed that the majority of tumor cells in Slu-01/AEG-1 xenotransplants strongly expressed Vimentin, but exhibited weak staining of E-cadherin (Figure 5C).Figure 5

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