Limits...
AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

Qiao Y, Shiue CN, Zhu J, Zhuang T, Jonsson P, Wright AP, Zhao C, Dahlman-Wright K - Oncotarget (2015)

Bottom Line: We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1.Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions.Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden.

ABSTRACT
The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

No MeSH data available.


Related in: MedlinePlus

TNFα induces AP-1 activation and ZEB2 expression in TNBC cells(A) Western blot analysis of p-Fra-1, Fra-1, p-c-Jun, c-Jun, and β-actin (as a loading control) in BT549 cells treated with or without TNFα (10 ng/ml) for 2, 4 and 6 hours. (B) TNFα mediates Fra-1 and c-Jun expression via the PI3K/Akt and MAPK/ERK pathways. Western blot analysis of pAkt, Akt, pERK1/2, ERK1/2, Fra-1, c-Jun and β-actin (as a loading control) in BT549 cells pretreated with LY294002 (25 μM) or PD98059 (25 μM) for 6 hours, followed by stimulation with TNFα (10 ng/ml) for 6 hours. (C) TNFα induces ZEB2 expression at both the mRNA and protein levels in BT549 and Hs578T cells. ZEB2 mRNA levels were determined by qPCR after treatment with or without TNFα (10 ng/ml) for the indicated times. Data are shown as means with SD. **p < 0.01, ***p < 0.001 compared with TNFα minus (n = 3). The protein levels of ZEB2 after 6 hours of treatment with TNFα were analyzed by Western blot. β-actin was used as a loading control. (D) Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) ChIP-qPCR analysis showing increased binding of Fra-1 and c-Jun to ZEB2 following TNFα treatment. Columns, mean fold enrichment of Fra-1 or c-Jun relative to IgG; bars, SD (n = 3). *p < 0.05, **p < 0.01 compared with TNFα minus. (F) ChIP-qPCR analysis showing increased binding of phospho-Fra-1 to ZEB2 following TNFα treatment. TFF1 is used as a negative control. ChIP was performed with antibody against phospho-Fra-1. Columns, mean fold enrichment of phospho-Fra-1 relative to IgG; bars, SD (n = 3). **p < 0.01 compared with TNFα minus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480717&req=5

Figure 2: TNFα induces AP-1 activation and ZEB2 expression in TNBC cells(A) Western blot analysis of p-Fra-1, Fra-1, p-c-Jun, c-Jun, and β-actin (as a loading control) in BT549 cells treated with or without TNFα (10 ng/ml) for 2, 4 and 6 hours. (B) TNFα mediates Fra-1 and c-Jun expression via the PI3K/Akt and MAPK/ERK pathways. Western blot analysis of pAkt, Akt, pERK1/2, ERK1/2, Fra-1, c-Jun and β-actin (as a loading control) in BT549 cells pretreated with LY294002 (25 μM) or PD98059 (25 μM) for 6 hours, followed by stimulation with TNFα (10 ng/ml) for 6 hours. (C) TNFα induces ZEB2 expression at both the mRNA and protein levels in BT549 and Hs578T cells. ZEB2 mRNA levels were determined by qPCR after treatment with or without TNFα (10 ng/ml) for the indicated times. Data are shown as means with SD. **p < 0.01, ***p < 0.001 compared with TNFα minus (n = 3). The protein levels of ZEB2 after 6 hours of treatment with TNFα were analyzed by Western blot. β-actin was used as a loading control. (D) Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) ChIP-qPCR analysis showing increased binding of Fra-1 and c-Jun to ZEB2 following TNFα treatment. Columns, mean fold enrichment of Fra-1 or c-Jun relative to IgG; bars, SD (n = 3). *p < 0.05, **p < 0.01 compared with TNFα minus. (F) ChIP-qPCR analysis showing increased binding of phospho-Fra-1 to ZEB2 following TNFα treatment. TFF1 is used as a negative control. ChIP was performed with antibody against phospho-Fra-1. Columns, mean fold enrichment of phospho-Fra-1 relative to IgG; bars, SD (n = 3). **p < 0.01 compared with TNFα minus.

Mentions: As shown in Figure 2A, TNFα treatment markedly up-regulated the levels of phospho-Fra-1, Fra-1, phospho-c-Jun, and c-Jun proteins at 2, 4 and 6 hours. To determine whether the PI3K/Akt and MAPK/ERK signaling pathways mediate TNFα up-regulation of AP-1 levels and activity, we used two pharmacological inhibitors, LY294002 and PD98059, to block the PI3K/Akt and MAPK/ERK pathways, respectively. We found that LY294002 and PD98059 in TNFα-stimulated cells inhibited TNFα-induced expression of Fra-1 and c-Jun proteins (Figure 2B), suggesting that activation of the PI3K/Akt and MAPK/ERK pathways is responsible for TNFα-induced AP-1 protein expression. The effect of inhibitor treatment on these pathways was confirmed by assessing the level of Akt and ERK1/2 phosphorylation (Figure 2B). We next examined ZEB2 expression after TNFα stimulation. Compared to untreated cells, TNFα significantly increased ZEB2 mRNA levels after 2 and 4 hours of treatment in the BT549 and Hs578T cell lines with a subsequent increase in ZEB2 protein levels (Figure 2C). Finally, a role of AP-1 signaling in mediating TNFα-induced up-regulation of ZEB2 was provided by depleting Fra-1 or c-Jun in BT549 cells prior to TNFα stimulation. Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα (Figure 2D). Furthermore, TNFα treatment increased binding of Fra-1 and c-Jun to an AP-1 binding region in an intron of the ZEB2 gene that we have previously described [13] (Figure 2E). To further determine whether phospho-Fra-1 is binding to DNA, we assessed the ability of phospho-Fra-1 to bind to the ZEB2 gene. Consistent with the markedly elevated levels of phospho-Fra-1 upon TNFα treatment, phospho-Fra-1 binding was increased by TNFα (Figure 2F). Collectively, these results show that TNFα-induced AP-1 activation, via distinct pathways, is a critical mediator of ZEB2 up-regulation in TNBC cells.


AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

Qiao Y, Shiue CN, Zhu J, Zhuang T, Jonsson P, Wright AP, Zhao C, Dahlman-Wright K - Oncotarget (2015)

TNFα induces AP-1 activation and ZEB2 expression in TNBC cells(A) Western blot analysis of p-Fra-1, Fra-1, p-c-Jun, c-Jun, and β-actin (as a loading control) in BT549 cells treated with or without TNFα (10 ng/ml) for 2, 4 and 6 hours. (B) TNFα mediates Fra-1 and c-Jun expression via the PI3K/Akt and MAPK/ERK pathways. Western blot analysis of pAkt, Akt, pERK1/2, ERK1/2, Fra-1, c-Jun and β-actin (as a loading control) in BT549 cells pretreated with LY294002 (25 μM) or PD98059 (25 μM) for 6 hours, followed by stimulation with TNFα (10 ng/ml) for 6 hours. (C) TNFα induces ZEB2 expression at both the mRNA and protein levels in BT549 and Hs578T cells. ZEB2 mRNA levels were determined by qPCR after treatment with or without TNFα (10 ng/ml) for the indicated times. Data are shown as means with SD. **p < 0.01, ***p < 0.001 compared with TNFα minus (n = 3). The protein levels of ZEB2 after 6 hours of treatment with TNFα were analyzed by Western blot. β-actin was used as a loading control. (D) Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) ChIP-qPCR analysis showing increased binding of Fra-1 and c-Jun to ZEB2 following TNFα treatment. Columns, mean fold enrichment of Fra-1 or c-Jun relative to IgG; bars, SD (n = 3). *p < 0.05, **p < 0.01 compared with TNFα minus. (F) ChIP-qPCR analysis showing increased binding of phospho-Fra-1 to ZEB2 following TNFα treatment. TFF1 is used as a negative control. ChIP was performed with antibody against phospho-Fra-1. Columns, mean fold enrichment of phospho-Fra-1 relative to IgG; bars, SD (n = 3). **p < 0.01 compared with TNFα minus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480717&req=5

Figure 2: TNFα induces AP-1 activation and ZEB2 expression in TNBC cells(A) Western blot analysis of p-Fra-1, Fra-1, p-c-Jun, c-Jun, and β-actin (as a loading control) in BT549 cells treated with or without TNFα (10 ng/ml) for 2, 4 and 6 hours. (B) TNFα mediates Fra-1 and c-Jun expression via the PI3K/Akt and MAPK/ERK pathways. Western blot analysis of pAkt, Akt, pERK1/2, ERK1/2, Fra-1, c-Jun and β-actin (as a loading control) in BT549 cells pretreated with LY294002 (25 μM) or PD98059 (25 μM) for 6 hours, followed by stimulation with TNFα (10 ng/ml) for 6 hours. (C) TNFα induces ZEB2 expression at both the mRNA and protein levels in BT549 and Hs578T cells. ZEB2 mRNA levels were determined by qPCR after treatment with or without TNFα (10 ng/ml) for the indicated times. Data are shown as means with SD. **p < 0.01, ***p < 0.001 compared with TNFα minus (n = 3). The protein levels of ZEB2 after 6 hours of treatment with TNFα were analyzed by Western blot. β-actin was used as a loading control. (D) Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) ChIP-qPCR analysis showing increased binding of Fra-1 and c-Jun to ZEB2 following TNFα treatment. Columns, mean fold enrichment of Fra-1 or c-Jun relative to IgG; bars, SD (n = 3). *p < 0.05, **p < 0.01 compared with TNFα minus. (F) ChIP-qPCR analysis showing increased binding of phospho-Fra-1 to ZEB2 following TNFα treatment. TFF1 is used as a negative control. ChIP was performed with antibody against phospho-Fra-1. Columns, mean fold enrichment of phospho-Fra-1 relative to IgG; bars, SD (n = 3). **p < 0.01 compared with TNFα minus.
Mentions: As shown in Figure 2A, TNFα treatment markedly up-regulated the levels of phospho-Fra-1, Fra-1, phospho-c-Jun, and c-Jun proteins at 2, 4 and 6 hours. To determine whether the PI3K/Akt and MAPK/ERK signaling pathways mediate TNFα up-regulation of AP-1 levels and activity, we used two pharmacological inhibitors, LY294002 and PD98059, to block the PI3K/Akt and MAPK/ERK pathways, respectively. We found that LY294002 and PD98059 in TNFα-stimulated cells inhibited TNFα-induced expression of Fra-1 and c-Jun proteins (Figure 2B), suggesting that activation of the PI3K/Akt and MAPK/ERK pathways is responsible for TNFα-induced AP-1 protein expression. The effect of inhibitor treatment on these pathways was confirmed by assessing the level of Akt and ERK1/2 phosphorylation (Figure 2B). We next examined ZEB2 expression after TNFα stimulation. Compared to untreated cells, TNFα significantly increased ZEB2 mRNA levels after 2 and 4 hours of treatment in the BT549 and Hs578T cell lines with a subsequent increase in ZEB2 protein levels (Figure 2C). Finally, a role of AP-1 signaling in mediating TNFα-induced up-regulation of ZEB2 was provided by depleting Fra-1 or c-Jun in BT549 cells prior to TNFα stimulation. Depletion of Fra-1 or c-Jun markedly impaired the induction of ZEB2 gene expression by TNFα (Figure 2D). Furthermore, TNFα treatment increased binding of Fra-1 and c-Jun to an AP-1 binding region in an intron of the ZEB2 gene that we have previously described [13] (Figure 2E). To further determine whether phospho-Fra-1 is binding to DNA, we assessed the ability of phospho-Fra-1 to bind to the ZEB2 gene. Consistent with the markedly elevated levels of phospho-Fra-1 upon TNFα treatment, phospho-Fra-1 binding was increased by TNFα (Figure 2F). Collectively, these results show that TNFα-induced AP-1 activation, via distinct pathways, is a critical mediator of ZEB2 up-regulation in TNBC cells.

Bottom Line: We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1.Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions.Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden.

ABSTRACT
The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

No MeSH data available.


Related in: MedlinePlus