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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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NF-κB activation and upregulation of genes involved in inflammation-induced tumor progression by EI24 knockdown(A) The transcriptional activity of NF-κB in control and EI24-overexpressing MDA-MB-231 cells was evaluated using a NF-κB luciferase reporter assay. Data shown are mean ± S.D. n = 6, **p <0.0001. (B) Immunoblot analysis of p65 expression levels in nuclear extracts from control and EI24-overexpressing MDA-MB-231 cells. (C) and (D) GSEA for gene signatures in EI24 knockdown cells indicating genes that contain p65-binding sites in their promoter regions (p <0.001; C) and genes that are targets of NF-κB and are associated with tumor invasiveness (p <0.001; D). NES, normalized enrichment score. (E) Relative mRNA expression levels of the indicated genes in control and ZR-shEI24 cells analyzed by real-time qPCR. Data shown are the mean ± S.D. of two independent experiments with each sample assayed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.005, **p <0.0001, ***p <0.0005. (F) Cell migratory potential was measured after treatment of ZR-shEI24 cells with an NF-κB inhibitor. The red scale bar represents 200 μm.
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Figure 4: NF-κB activation and upregulation of genes involved in inflammation-induced tumor progression by EI24 knockdown(A) The transcriptional activity of NF-κB in control and EI24-overexpressing MDA-MB-231 cells was evaluated using a NF-κB luciferase reporter assay. Data shown are mean ± S.D. n = 6, **p <0.0001. (B) Immunoblot analysis of p65 expression levels in nuclear extracts from control and EI24-overexpressing MDA-MB-231 cells. (C) and (D) GSEA for gene signatures in EI24 knockdown cells indicating genes that contain p65-binding sites in their promoter regions (p <0.001; C) and genes that are targets of NF-κB and are associated with tumor invasiveness (p <0.001; D). NES, normalized enrichment score. (E) Relative mRNA expression levels of the indicated genes in control and ZR-shEI24 cells analyzed by real-time qPCR. Data shown are the mean ± S.D. of two independent experiments with each sample assayed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.005, **p <0.0001, ***p <0.0005. (F) Cell migratory potential was measured after treatment of ZR-shEI24 cells with an NF-κB inhibitor. The red scale bar represents 200 μm.

Mentions: To elucidate the molecular mechanism governing EI24-mediated regulation of EMT, we treated ZR-shEI24 cells with specific inhibitors of several pathways. Although most of the inhibitors had no effect, the NF-κB inhibitor BAY 11-7082 restored the epithelial morphology (Supplemental Figure 3A). From these data, we surmised that EI24 suppresses EMT by negatively regulating NF-κB signaling. Consistent with this, overexpression of EI24 suppressed NF-κB reporter activity in MDA-MB-231 cells (Figure 4A). These observations were also confirmed using EI24 overexpression and knockdown systems in HeLa cells (Supplemental Figure 3B, 3C). EI24 overexpression in MDA-MD-231 cells significantly decreased nuclear localization of the p65 subunit of NF-κB (Figure 4B), whereas F10-shEI24 cells exhibited an increase in p65 in the nucleus (Supplemental Figure 3D). Furthermore, when GSEA was performed using a gene set from the Molecular Signatures Database (MSigDB) that contained the GGGRATTTCC motif, the consensus binding sequence for p65 transcription factors, in their promoter regions [20] this gene signature was enriched in ZR-shEI24 cells (Figure 4C). Notably, the expression levels of NF-κB target genes that are associated with tumor invasiveness [21] were also significantly enhanced in ZR-shEI24 cells (Figure 4D). For example, ZR-shEI24 cells exhibited upregulation of IL6 and IL8, NF-κB target genes that are involved in inflammation-induced tumor metastasis (Figure 4E). Consistent with these data, IL6 and IL8 were downregulated in MDA-MB-231 cells upon overexpression of EI24 (Supplemental Figure 3E). Moreover, treatment of ZR-shEI24 cells with the NF-κB inhibitor BAY 11-7082 significantly decreased cell migration compared with the control treatment (Figure 4F). Taken together, these data demonstrate that EI24 attenuates NF-κB-mediated tumor malignancy.


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)

NF-κB activation and upregulation of genes involved in inflammation-induced tumor progression by EI24 knockdown(A) The transcriptional activity of NF-κB in control and EI24-overexpressing MDA-MB-231 cells was evaluated using a NF-κB luciferase reporter assay. Data shown are mean ± S.D. n = 6, **p <0.0001. (B) Immunoblot analysis of p65 expression levels in nuclear extracts from control and EI24-overexpressing MDA-MB-231 cells. (C) and (D) GSEA for gene signatures in EI24 knockdown cells indicating genes that contain p65-binding sites in their promoter regions (p <0.001; C) and genes that are targets of NF-κB and are associated with tumor invasiveness (p <0.001; D). NES, normalized enrichment score. (E) Relative mRNA expression levels of the indicated genes in control and ZR-shEI24 cells analyzed by real-time qPCR. Data shown are the mean ± S.D. of two independent experiments with each sample assayed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.005, **p <0.0001, ***p <0.0005. (F) Cell migratory potential was measured after treatment of ZR-shEI24 cells with an NF-κB inhibitor. The red scale bar represents 200 μm.
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Figure 4: NF-κB activation and upregulation of genes involved in inflammation-induced tumor progression by EI24 knockdown(A) The transcriptional activity of NF-κB in control and EI24-overexpressing MDA-MB-231 cells was evaluated using a NF-κB luciferase reporter assay. Data shown are mean ± S.D. n = 6, **p <0.0001. (B) Immunoblot analysis of p65 expression levels in nuclear extracts from control and EI24-overexpressing MDA-MB-231 cells. (C) and (D) GSEA for gene signatures in EI24 knockdown cells indicating genes that contain p65-binding sites in their promoter regions (p <0.001; C) and genes that are targets of NF-κB and are associated with tumor invasiveness (p <0.001; D). NES, normalized enrichment score. (E) Relative mRNA expression levels of the indicated genes in control and ZR-shEI24 cells analyzed by real-time qPCR. Data shown are the mean ± S.D. of two independent experiments with each sample assayed in triplicate; P values were calculated by unpaired t-test using GraphPad Prism software. *p <0.005, **p <0.0001, ***p <0.0005. (F) Cell migratory potential was measured after treatment of ZR-shEI24 cells with an NF-κB inhibitor. The red scale bar represents 200 μm.
Mentions: To elucidate the molecular mechanism governing EI24-mediated regulation of EMT, we treated ZR-shEI24 cells with specific inhibitors of several pathways. Although most of the inhibitors had no effect, the NF-κB inhibitor BAY 11-7082 restored the epithelial morphology (Supplemental Figure 3A). From these data, we surmised that EI24 suppresses EMT by negatively regulating NF-κB signaling. Consistent with this, overexpression of EI24 suppressed NF-κB reporter activity in MDA-MB-231 cells (Figure 4A). These observations were also confirmed using EI24 overexpression and knockdown systems in HeLa cells (Supplemental Figure 3B, 3C). EI24 overexpression in MDA-MD-231 cells significantly decreased nuclear localization of the p65 subunit of NF-κB (Figure 4B), whereas F10-shEI24 cells exhibited an increase in p65 in the nucleus (Supplemental Figure 3D). Furthermore, when GSEA was performed using a gene set from the Molecular Signatures Database (MSigDB) that contained the GGGRATTTCC motif, the consensus binding sequence for p65 transcription factors, in their promoter regions [20] this gene signature was enriched in ZR-shEI24 cells (Figure 4C). Notably, the expression levels of NF-κB target genes that are associated with tumor invasiveness [21] were also significantly enhanced in ZR-shEI24 cells (Figure 4D). For example, ZR-shEI24 cells exhibited upregulation of IL6 and IL8, NF-κB target genes that are involved in inflammation-induced tumor metastasis (Figure 4E). Consistent with these data, IL6 and IL8 were downregulated in MDA-MB-231 cells upon overexpression of EI24 (Supplemental Figure 3E). Moreover, treatment of ZR-shEI24 cells with the NF-κB inhibitor BAY 11-7082 significantly decreased cell migration compared with the control treatment (Figure 4F). Taken together, these data demonstrate that EI24 attenuates NF-κB-mediated tumor malignancy.

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