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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 regulates ZEB2 via two promoters dependent on AP-1 in TNBC cells(A) Structure of the 5′ region of the ZEB2 gene and schematic of mRNA initiated from two distinct first exons, named exon 1a and exon 1b, splicing to downstream exon 2. Origins of sequences: exon 1a was from ENST00000558170, and exon 1b from ENST00000409487. The size of each exon and distances between exons are indicated. ‘▼’ at exon 2 indicates ATG, ‘▄’ indicates AP-1 binding site and bent arrows indicate transcriptional start sites. RT-PCR primer sets to amplify exon 1a and exon 1b transcripts are also indicated. Expected sizes of the products are shown in the parentheses. (B) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in RNA isolated from breast cancer cell lines. Expression of β-actin mRNA was used as an internal control. (C) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in breast tumor RNA. (D) qPCR data showing decreased exon 1a and exon 1b transcript levels in BT549 cells upon Fra-1 or c-Jun depletion. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) qPCR data showing that depletion of Fra-1 or c-Jun markedly impaired the induction of exon 1a and exon 1b transcript expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared with control siRNA.
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Figure 3: TNFα regulates ZEB2 via two promoters dependent on AP-1 in TNBC cells(A) Structure of the 5′ region of the ZEB2 gene and schematic of mRNA initiated from two distinct first exons, named exon 1a and exon 1b, splicing to downstream exon 2. Origins of sequences: exon 1a was from ENST00000558170, and exon 1b from ENST00000409487. The size of each exon and distances between exons are indicated. ‘▼’ at exon 2 indicates ATG, ‘▄’ indicates AP-1 binding site and bent arrows indicate transcriptional start sites. RT-PCR primer sets to amplify exon 1a and exon 1b transcripts are also indicated. Expected sizes of the products are shown in the parentheses. (B) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in RNA isolated from breast cancer cell lines. Expression of β-actin mRNA was used as an internal control. (C) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in breast tumor RNA. (D) qPCR data showing decreased exon 1a and exon 1b transcript levels in BT549 cells upon Fra-1 or c-Jun depletion. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) qPCR data showing that depletion of Fra-1 or c-Jun markedly impaired the induction of exon 1a and exon 1b transcript expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared with control siRNA.

Mentions: As ZEB2 participates in regulation of EMT, a detailed knowledge of mechanisms for its expression is of fundamental importance. Human ZEB2 mRNAs initiate from two distinct first exons, referred to as exon 1a and exon 1b, respectively, which are located 2.2 kb apart and are spliced to the common exons 2–10 (Figure 3A). We examined the expression of exon 1a and exon 1b transcripts in a panel of breast cancer cell lines and primary human breast tumors. As shown in Figure 3B, among 11 breast cancer cell lines, expression of the two transcripts was detected in 5 cell lines. In primary breast tumors, the exon 1a transcript was detected in all analyzed samples, whilst the exon 1b transcript displayed a more restricted expression (Figure 3C). Together, these results reveal that two alternatively spliced 5′UTR isoforms of the ZEB2 gene can be expressed in breast cancer cell lines and breast cancer tissues, indicating that the two promoters, driving the respective transcripts, are functional.


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 regulates ZEB2 via two promoters dependent on AP-1 in TNBC cells(A) Structure of the 5′ region of the ZEB2 gene and schematic of mRNA initiated from two distinct first exons, named exon 1a and exon 1b, splicing to downstream exon 2. Origins of sequences: exon 1a was from ENST00000558170, and exon 1b from ENST00000409487. The size of each exon and distances between exons are indicated. ‘▼’ at exon 2 indicates ATG, ‘▄’ indicates AP-1 binding site and bent arrows indicate transcriptional start sites. RT-PCR primer sets to amplify exon 1a and exon 1b transcripts are also indicated. Expected sizes of the products are shown in the parentheses. (B) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in RNA isolated from breast cancer cell lines. Expression of β-actin mRNA was used as an internal control. (C) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in breast tumor RNA. (D) qPCR data showing decreased exon 1a and exon 1b transcript levels in BT549 cells upon Fra-1 or c-Jun depletion. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) qPCR data showing that depletion of Fra-1 or c-Jun markedly impaired the induction of exon 1a and exon 1b transcript expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared with control siRNA.
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Figure 3: TNFα regulates ZEB2 via two promoters dependent on AP-1 in TNBC cells(A) Structure of the 5′ region of the ZEB2 gene and schematic of mRNA initiated from two distinct first exons, named exon 1a and exon 1b, splicing to downstream exon 2. Origins of sequences: exon 1a was from ENST00000558170, and exon 1b from ENST00000409487. The size of each exon and distances between exons are indicated. ‘▼’ at exon 2 indicates ATG, ‘▄’ indicates AP-1 binding site and bent arrows indicate transcriptional start sites. RT-PCR primer sets to amplify exon 1a and exon 1b transcripts are also indicated. Expected sizes of the products are shown in the parentheses. (B) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in RNA isolated from breast cancer cell lines. Expression of β-actin mRNA was used as an internal control. (C) RT-PCR detection of the expression of exon 1a and exon 1b transcripts in breast tumor RNA. (D) qPCR data showing decreased exon 1a and exon 1b transcript levels in BT549 cells upon Fra-1 or c-Jun depletion. Values are mean ± SD (n = 3). *p < 0.05 compared with control siRNA. (E) qPCR data showing that depletion of Fra-1 or c-Jun markedly impaired the induction of exon 1a and exon 1b transcript expression by TNFα. Values are mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared with control siRNA.
Mentions: As ZEB2 participates in regulation of EMT, a detailed knowledge of mechanisms for its expression is of fundamental importance. Human ZEB2 mRNAs initiate from two distinct first exons, referred to as exon 1a and exon 1b, respectively, which are located 2.2 kb apart and are spliced to the common exons 2–10 (Figure 3A). We examined the expression of exon 1a and exon 1b transcripts in a panel of breast cancer cell lines and primary human breast tumors. As shown in Figure 3B, among 11 breast cancer cell lines, expression of the two transcripts was detected in 5 cell lines. In primary breast tumors, the exon 1a transcript was detected in all analyzed samples, whilst the exon 1b transcript displayed a more restricted expression (Figure 3C). Together, these results reveal that two alternatively spliced 5′UTR isoforms of the ZEB2 gene can be expressed in breast cancer cell lines and breast cancer tissues, indicating that the two promoters, driving the respective transcripts, are functional.

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