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EI24 regulates epithelial-to-mesenchymal transition and tumor progression by suppressing TRAF2-mediated NF-κB activity.

Choi JM, Devkota S, Sung YH, Lee HW - Oncotarget (2013)

Bottom Line: Decreased expression levels of EI24 in epithelial tumor cells induced EMT in association with increased cell motility and invasiveness and resistance to anoikis.Overexpression of EI24 resulted in the opposite cell biological characteristics and suppressed in vivometastatic behavior.EI24 attenuated NF-κB activity by binding to the Complex I component TRAF2 and inducing its lysosome-dependent degradation, leading to transcriptional alterations of EMT- and inflammation-related genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.

ABSTRACT
Tumor metastasis is a multistep process that requires the concerted activity of discrete biological functions. The epithelial-to-mesenchymal transition (EMT) is the most critical mechanism implicated in tumor progression that is controlled by the inflammatory microenvironment. Understanding how an inflammatory microenvironment is maintained and contributes to tumor progression will be crucial for the development of new effective therapies. Here, we report that etoposide induced 2.4 (EI24) has a multifaceted role against tumor progression that is regulated by both EMT and inflammation. Decreased expression levels of EI24 in epithelial tumor cells induced EMT in association with increased cell motility and invasiveness and resistance to anoikis. Overexpression of EI24 resulted in the opposite cell biological characteristics and suppressed in vivometastatic behavior. EI24 attenuated NF-κB activity by binding to the Complex I component TRAF2 and inducing its lysosome-dependent degradation, leading to transcriptional alterations of EMT- and inflammation-related genes. Analysis of clinical samples demonstrated that reduced EI24 expression and copy number was positively correlated with tumor malignancy and poor prognosis. Collectively, these findings establish EI24 as a critical suppressor of tumor progression and implicate EI24 expression level in malignant tumors as a useful therapeutic and diagnostic marker.

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Related in: MedlinePlus

Knockdown of EI24 promotes malignant characteristics that accompany EMT(A) The indicated cell lines were added to Matrigel-coated transwell inserts, and invading cells were visualized after 16 hours by crystal violet staining. Scale bar represents 200 μm. (B) Formation of invasive acini was evaluated using the 3D matrigel assay. The data are representative of at least two independent experiments. Scale bar represents 100 μm. (C) NMuMG Parental, NMu-Ctrl and NMu-shEi24 cells were maintained in suspension using ultra-low-attachment plates and cell viability was analyzed at the indicated time points. (D) and (E) F10-Ctrl or F10-EI24 cells were intravenously injected into C57BL/6NTac mice and specific metastatic ability was analyzed by counting nodules in the lungs; n ≥7; one-way analysis of variance, p <0.0001. (F) and (G) F1-Ctrl or F1-EI24 cells were intravenously injected into C57BL/6NTac mice and their specific metastatic ability was analyzed by counting nodules in the liver; n ≥3; one-way analysis of variance, p <0.01.
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Figure 3: Knockdown of EI24 promotes malignant characteristics that accompany EMT(A) The indicated cell lines were added to Matrigel-coated transwell inserts, and invading cells were visualized after 16 hours by crystal violet staining. Scale bar represents 200 μm. (B) Formation of invasive acini was evaluated using the 3D matrigel assay. The data are representative of at least two independent experiments. Scale bar represents 100 μm. (C) NMuMG Parental, NMu-Ctrl and NMu-shEi24 cells were maintained in suspension using ultra-low-attachment plates and cell viability was analyzed at the indicated time points. (D) and (E) F10-Ctrl or F10-EI24 cells were intravenously injected into C57BL/6NTac mice and specific metastatic ability was analyzed by counting nodules in the lungs; n ≥7; one-way analysis of variance, p <0.0001. (F) and (G) F1-Ctrl or F1-EI24 cells were intravenously injected into C57BL/6NTac mice and their specific metastatic ability was analyzed by counting nodules in the liver; n ≥3; one-way analysis of variance, p <0.01.

Mentions: Since EMT is a characteristic feature of malignant tumors, we investigated cellular properties that are accompanied by EMT in the context of EI24 dysregulation. Matrigel invasion assays revealed that EI24 overexpression inhibited the invasive capacity of malignant B16F10 cells (Figure 3A, top panel). In contrast, ZR-shEI24 cells showed increased invasiveness (Figure 3A, bottom panel). ZR-shEI24 cells in 3D-culture consistently displayed invasive characteristics compared with the rounded morphology of the control cells (Figure 3B). We next examined the effect of EI24 on anchorage independent survival by measuring the cell viability after inducing the anoikis. After 72 hours, the EI24 knockdown cells survived approximately 15% more than the control cells (Figure 3C). This result indicates that loss of EI24 protects cells from anoikis. Taken together, acquisition of invasive characteristics and resistance to anoikis upon reduced levels of EI24 facilitates the enhanced malignancy of tumor cells.


EI24 regulates epithelial-to-mesenchymal transition and tumor progression by suppressing TRAF2-mediated NF-κB activity.

Choi JM, Devkota S, Sung YH, Lee HW - Oncotarget (2013)

Knockdown of EI24 promotes malignant characteristics that accompany EMT(A) The indicated cell lines were added to Matrigel-coated transwell inserts, and invading cells were visualized after 16 hours by crystal violet staining. Scale bar represents 200 μm. (B) Formation of invasive acini was evaluated using the 3D matrigel assay. The data are representative of at least two independent experiments. Scale bar represents 100 μm. (C) NMuMG Parental, NMu-Ctrl and NMu-shEi24 cells were maintained in suspension using ultra-low-attachment plates and cell viability was analyzed at the indicated time points. (D) and (E) F10-Ctrl or F10-EI24 cells were intravenously injected into C57BL/6NTac mice and specific metastatic ability was analyzed by counting nodules in the lungs; n ≥7; one-way analysis of variance, p <0.0001. (F) and (G) F1-Ctrl or F1-EI24 cells were intravenously injected into C57BL/6NTac mice and their specific metastatic ability was analyzed by counting nodules in the liver; n ≥3; one-way analysis of variance, p <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926834&req=5

Figure 3: Knockdown of EI24 promotes malignant characteristics that accompany EMT(A) The indicated cell lines were added to Matrigel-coated transwell inserts, and invading cells were visualized after 16 hours by crystal violet staining. Scale bar represents 200 μm. (B) Formation of invasive acini was evaluated using the 3D matrigel assay. The data are representative of at least two independent experiments. Scale bar represents 100 μm. (C) NMuMG Parental, NMu-Ctrl and NMu-shEi24 cells were maintained in suspension using ultra-low-attachment plates and cell viability was analyzed at the indicated time points. (D) and (E) F10-Ctrl or F10-EI24 cells were intravenously injected into C57BL/6NTac mice and specific metastatic ability was analyzed by counting nodules in the lungs; n ≥7; one-way analysis of variance, p <0.0001. (F) and (G) F1-Ctrl or F1-EI24 cells were intravenously injected into C57BL/6NTac mice and their specific metastatic ability was analyzed by counting nodules in the liver; n ≥3; one-way analysis of variance, p <0.01.
Mentions: Since EMT is a characteristic feature of malignant tumors, we investigated cellular properties that are accompanied by EMT in the context of EI24 dysregulation. Matrigel invasion assays revealed that EI24 overexpression inhibited the invasive capacity of malignant B16F10 cells (Figure 3A, top panel). In contrast, ZR-shEI24 cells showed increased invasiveness (Figure 3A, bottom panel). ZR-shEI24 cells in 3D-culture consistently displayed invasive characteristics compared with the rounded morphology of the control cells (Figure 3B). We next examined the effect of EI24 on anchorage independent survival by measuring the cell viability after inducing the anoikis. After 72 hours, the EI24 knockdown cells survived approximately 15% more than the control cells (Figure 3C). This result indicates that loss of EI24 protects cells from anoikis. Taken together, acquisition of invasive characteristics and resistance to anoikis upon reduced levels of EI24 facilitates the enhanced malignancy of tumor cells.

Bottom Line: Decreased expression levels of EI24 in epithelial tumor cells induced EMT in association with increased cell motility and invasiveness and resistance to anoikis.Overexpression of EI24 resulted in the opposite cell biological characteristics and suppressed in vivometastatic behavior.EI24 attenuated NF-κB activity by binding to the Complex I component TRAF2 and inducing its lysosome-dependent degradation, leading to transcriptional alterations of EMT- and inflammation-related genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.

ABSTRACT
Tumor metastasis is a multistep process that requires the concerted activity of discrete biological functions. The epithelial-to-mesenchymal transition (EMT) is the most critical mechanism implicated in tumor progression that is controlled by the inflammatory microenvironment. Understanding how an inflammatory microenvironment is maintained and contributes to tumor progression will be crucial for the development of new effective therapies. Here, we report that etoposide induced 2.4 (EI24) has a multifaceted role against tumor progression that is regulated by both EMT and inflammation. Decreased expression levels of EI24 in epithelial tumor cells induced EMT in association with increased cell motility and invasiveness and resistance to anoikis. Overexpression of EI24 resulted in the opposite cell biological characteristics and suppressed in vivometastatic behavior. EI24 attenuated NF-κB activity by binding to the Complex I component TRAF2 and inducing its lysosome-dependent degradation, leading to transcriptional alterations of EMT- and inflammation-related genes. Analysis of clinical samples demonstrated that reduced EI24 expression and copy number was positively correlated with tumor malignancy and poor prognosis. Collectively, these findings establish EI24 as a critical suppressor of tumor progression and implicate EI24 expression level in malignant tumors as a useful therapeutic and diagnostic marker.

Show MeSH
Related in: MedlinePlus