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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-mediated EMT in TNBC cells is dependent on AP-1–ZEB2 signaling(A) BT549 and Hs578T cells were serum-starved overnight and then treated with or without TNFα (10 ng/ml) for 72 hours. The cells were examined by phase contrast microscopy and Western blots analysis for epithelial (E-cadherin) and mesenchymal (N-cadherin and fibronectin) markers. β-actin was used as a loading control. Representative images from two independent studies. (B) BT549 cells transfected with control, Fra-1 or c-Jun siRNA were treated as above. Western blot confirms knockdown of Fra-1 or c-Jun levels. (C) BT549 cells transfected with control or ZEB2 siRNA were treated as above. Western blot confirms knockdown of ZEB2 levels. The same image for control siRNA is shown for Figure 1B and Figure 1C because all conditions were assessed in the same experiment.
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Figure 1: TNFα-mediated EMT in TNBC cells is dependent on AP-1–ZEB2 signaling(A) BT549 and Hs578T cells were serum-starved overnight and then treated with or without TNFα (10 ng/ml) for 72 hours. The cells were examined by phase contrast microscopy and Western blots analysis for epithelial (E-cadherin) and mesenchymal (N-cadherin and fibronectin) markers. β-actin was used as a loading control. Representative images from two independent studies. (B) BT549 cells transfected with control, Fra-1 or c-Jun siRNA were treated as above. Western blot confirms knockdown of Fra-1 or c-Jun levels. (C) BT549 cells transfected with control or ZEB2 siRNA were treated as above. Western blot confirms knockdown of ZEB2 levels. The same image for control siRNA is shown for Figure 1B and Figure 1C because all conditions were assessed in the same experiment.

Mentions: EMT is characterized by down-regulation of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers such as N-cadherin and fibronectin. Figure 1A shows that the TNBC cell lines BT549 and Hs578T acquired EMT-like morphological features, such as a spindle-shaped appearance, in response to TNFα treatment. In agreement with the change in cellular appearance, TNFα treatment led to significant reduction in E-cadherin protein expression as well as increases in N-cadherin and fibronectin protein expression (Figure 1A), all characteristics of EMT.


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-mediated EMT in TNBC cells is dependent on AP-1–ZEB2 signaling(A) BT549 and Hs578T cells were serum-starved overnight and then treated with or without TNFα (10 ng/ml) for 72 hours. The cells were examined by phase contrast microscopy and Western blots analysis for epithelial (E-cadherin) and mesenchymal (N-cadherin and fibronectin) markers. β-actin was used as a loading control. Representative images from two independent studies. (B) BT549 cells transfected with control, Fra-1 or c-Jun siRNA were treated as above. Western blot confirms knockdown of Fra-1 or c-Jun levels. (C) BT549 cells transfected with control or ZEB2 siRNA were treated as above. Western blot confirms knockdown of ZEB2 levels. The same image for control siRNA is shown for Figure 1B and Figure 1C because all conditions were assessed in the same experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: TNFα-mediated EMT in TNBC cells is dependent on AP-1–ZEB2 signaling(A) BT549 and Hs578T cells were serum-starved overnight and then treated with or without TNFα (10 ng/ml) for 72 hours. The cells were examined by phase contrast microscopy and Western blots analysis for epithelial (E-cadherin) and mesenchymal (N-cadherin and fibronectin) markers. β-actin was used as a loading control. Representative images from two independent studies. (B) BT549 cells transfected with control, Fra-1 or c-Jun siRNA were treated as above. Western blot confirms knockdown of Fra-1 or c-Jun levels. (C) BT549 cells transfected with control or ZEB2 siRNA were treated as above. Western blot confirms knockdown of ZEB2 levels. The same image for control siRNA is shown for Figure 1B and Figure 1C because all conditions were assessed in the same experiment.
Mentions: EMT is characterized by down-regulation of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers such as N-cadherin and fibronectin. Figure 1A shows that the TNBC cell lines BT549 and Hs578T acquired EMT-like morphological features, such as a spindle-shaped appearance, in response to TNFα treatment. In agreement with the change in cellular appearance, TNFα treatment led to significant reduction in E-cadherin protein expression as well as increases in N-cadherin and fibronectin protein expression (Figure 1A), all characteristics of EMT.

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