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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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Knockdown of EI24 induces EMT(A) Phase contrast microscopy of ZR-75-1 cells stably expressing control or EI24-specific shRNA. Scale bar represents 100 μm. (B) Immunoblot analysis of changes in the expression of epithelial and mesenchymal markers in control and ZR-shEI24 cells. (C) Confocal microscopic analysis of E-cadherin and Vimentin expression in ZR-Ctrl and ZR-shEI24 cells. Scale bar represents 50 μm. (D) and (E) Real-time quantitative PCR (qPCR) analysis of transcriptional regulation of the expression of epithelial and mesenchymal markers (D) and EMT-related transcription factors (E) in control and ZR-shEI24 cells. Data are means of measurements from two independent experiments performed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.05, **p <0.01, ***p <0.0001.
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Figure 2: Knockdown of EI24 induces EMT(A) Phase contrast microscopy of ZR-75-1 cells stably expressing control or EI24-specific shRNA. Scale bar represents 100 μm. (B) Immunoblot analysis of changes in the expression of epithelial and mesenchymal markers in control and ZR-shEI24 cells. (C) Confocal microscopic analysis of E-cadherin and Vimentin expression in ZR-Ctrl and ZR-shEI24 cells. Scale bar represents 50 μm. (D) and (E) Real-time quantitative PCR (qPCR) analysis of transcriptional regulation of the expression of epithelial and mesenchymal markers (D) and EMT-related transcription factors (E) in control and ZR-shEI24 cells. Data are means of measurements from two independent experiments performed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.05, **p <0.01, ***p <0.0001.

Mentions: Because the increased cell motility, cytoskeleton rearrangements, and decreased cell-cell adhesion induced by reduced levels of EI24 are reminiscent of EMT, we examined whether EI24 ablation affected the epithelial characteristics of cancer cells. Gene set enrichment analysis (GSEA) showed a strong correlation between EI24 knockdown in ZR-75-1 cells and gene signatures that are invoked during the EMT process [15-17] (Supplemental Figure 2A-C). Additionally, gene sets characteristic of phenotypic changes and molecular signaling alterations that are prerequisites for EMT were enriched in ZR-shEI24 cells (Supplemental Table 1, 2). In this context, we investigated whether reduced expression of EI24 induces EMT. Consistent with the molecular transition, EI24 knockdown induced a morphological change of ZR-75-1 cells to the fibroblast-like scattered morphology of mesenchymal cells (Figure 2A). A significant reduction in the expression of epithelial markers such as E-cadherin, β-catenin, and γ-catenin and emergence of the mesenchymal marker vimentin further supported the induction of EMT by EI24 knockdown (Figure 2B). Moreover, expression of the epithelial marker E-Cadherin in the cell-to-cell contacts was significantly decreased, coincident with increased expression of the mesenchymal marker vimentin (Figure 2C). We consistently found that the level of CDH1 mRNA was significantly lowered whereas the mRNA levels of VIM and FN1 were considerably increased in EI24 knockdown cells (Figure 2D). Notably, mRNA levels of EMT-related transcription factors including ZEB1, SIP1, and SLUG were also significantly increased in ZR-shEI24 cells (Figure 2E). Collectively, these data indicate that EI24 plays an important role in maintaining epithelial characteristics; thus, reduced expression of EI24 promotes the initiation of EMT.


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 induces EMT(A) Phase contrast microscopy of ZR-75-1 cells stably expressing control or EI24-specific shRNA. Scale bar represents 100 μm. (B) Immunoblot analysis of changes in the expression of epithelial and mesenchymal markers in control and ZR-shEI24 cells. (C) Confocal microscopic analysis of E-cadherin and Vimentin expression in ZR-Ctrl and ZR-shEI24 cells. Scale bar represents 50 μm. (D) and (E) Real-time quantitative PCR (qPCR) analysis of transcriptional regulation of the expression of epithelial and mesenchymal markers (D) and EMT-related transcription factors (E) in control and ZR-shEI24 cells. Data are means of measurements from two independent experiments performed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.05, **p <0.01, ***p <0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Knockdown of EI24 induces EMT(A) Phase contrast microscopy of ZR-75-1 cells stably expressing control or EI24-specific shRNA. Scale bar represents 100 μm. (B) Immunoblot analysis of changes in the expression of epithelial and mesenchymal markers in control and ZR-shEI24 cells. (C) Confocal microscopic analysis of E-cadherin and Vimentin expression in ZR-Ctrl and ZR-shEI24 cells. Scale bar represents 50 μm. (D) and (E) Real-time quantitative PCR (qPCR) analysis of transcriptional regulation of the expression of epithelial and mesenchymal markers (D) and EMT-related transcription factors (E) in control and ZR-shEI24 cells. Data are means of measurements from two independent experiments performed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.05, **p <0.01, ***p <0.0001.
Mentions: Because the increased cell motility, cytoskeleton rearrangements, and decreased cell-cell adhesion induced by reduced levels of EI24 are reminiscent of EMT, we examined whether EI24 ablation affected the epithelial characteristics of cancer cells. Gene set enrichment analysis (GSEA) showed a strong correlation between EI24 knockdown in ZR-75-1 cells and gene signatures that are invoked during the EMT process [15-17] (Supplemental Figure 2A-C). Additionally, gene sets characteristic of phenotypic changes and molecular signaling alterations that are prerequisites for EMT were enriched in ZR-shEI24 cells (Supplemental Table 1, 2). In this context, we investigated whether reduced expression of EI24 induces EMT. Consistent with the molecular transition, EI24 knockdown induced a morphological change of ZR-75-1 cells to the fibroblast-like scattered morphology of mesenchymal cells (Figure 2A). A significant reduction in the expression of epithelial markers such as E-cadherin, β-catenin, and γ-catenin and emergence of the mesenchymal marker vimentin further supported the induction of EMT by EI24 knockdown (Figure 2B). Moreover, expression of the epithelial marker E-Cadherin in the cell-to-cell contacts was significantly decreased, coincident with increased expression of the mesenchymal marker vimentin (Figure 2C). We consistently found that the level of CDH1 mRNA was significantly lowered whereas the mRNA levels of VIM and FN1 were considerably increased in EI24 knockdown cells (Figure 2D). Notably, mRNA levels of EMT-related transcription factors including ZEB1, SIP1, and SLUG were also significantly increased in ZR-shEI24 cells (Figure 2E). Collectively, these data indicate that EI24 plays an important role in maintaining epithelial characteristics; thus, reduced expression of EI24 promotes the initiation of EMT.

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