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Nuclear factor I-C regulates E-cadherin via control of KLF4 in breast cancer.

Lee HK, Lee DS, Park JC - BMC Cancer (2015)

Bottom Line: We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT.NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis.The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Histology-Developmental Biology & Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehagro, Chongro-gu, Seoul, 110-749, South Korea. h35071221@snu.ac.kr.

ABSTRACT

Background: Progression to metastasis is the leading cause of most cancer-related mortality; however, much remains to be understood about what facilitates the spread of tumor cells. In the present study, we describe a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness.

Methods: We examined nuclear factor I-C (NFI-C) expression in MCF10A human breast epithelial cells, MCF7 non-invasive breast cancer cells, and MDA-MB231 invasive breast cancer cells by real-time PCR and western blotting. To investigate the loss- and gain-function of NFI-C, we determined whether NFI-C regulated KLF4 expression by real-time PCR, western blotting, and promoter assay. To understand the biological functions of NFI-C, we observed cell invasion, migration, adhesion in human tumor cells by transwell assay, wound healing assay, quantitative RT-PCR, cell adhesion assay, western blotting, and immunohistochemistry.

Results: We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT. NFI-C is expressed in normal mammary gland or noninvasive breast cancer cells with epithelial characteristics. NFI-C overexpression induced expression of KLF4 and E-cadherin, but not Slug, in breast cancer cells. NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis. Cells overexpressing NFI-C maintained their epithelial differentiation status, which could drive mesenchymal-epithelial transition (MET) via the NFI-C-KLF4-E-cadherin axis in breast cancer cells. Consequently, NFI-C suppressed EMT, migration, and invasion in breast cancer cells.

Conclusions: Our study reveals a novel signaling pathway that is important during breast cancer tumorigenesis: the NFI-C-KLF4-E-cadherin pathway. The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.

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Related in: MedlinePlus

Effects of NFI-C overexpression and inactivation on the KLF4-E-cadherin signaling pathway in MCF7 breast cancer cells. (A, B) Expression of NFI-C, KLF4, Slug, E-cadherin, Vimentin, N-cadherin, and P-cadherin was analyzed by real-time PCR (A) and western blotting (B) in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. (C, D) Transcriptional activity of the KLF4 promoter and the E-cadherin promoter were evaluated by luciferase assays using NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. Control samples were transfected with only E-cadherin promoter constructs. Data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01). (E) NFI-C binding to the KLF4 promoter was investigated using ChIP assays after transfection with NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. (F) The NFI-C binding motif in the KLF4 promoter was confirmed by DNAP assay using extracts from MCF7 cells that had been transfected with NFI-C-expressing or NFI-C-siRNA constructs. Biotinylated oligonucleotides corresponding to the KLF4 promoter (WT or mutated) were used as probes. WT, wild type; MT, mutant type.
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Fig2: Effects of NFI-C overexpression and inactivation on the KLF4-E-cadherin signaling pathway in MCF7 breast cancer cells. (A, B) Expression of NFI-C, KLF4, Slug, E-cadherin, Vimentin, N-cadherin, and P-cadherin was analyzed by real-time PCR (A) and western blotting (B) in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. (C, D) Transcriptional activity of the KLF4 promoter and the E-cadherin promoter were evaluated by luciferase assays using NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. Control samples were transfected with only E-cadherin promoter constructs. Data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01). (E) NFI-C binding to the KLF4 promoter was investigated using ChIP assays after transfection with NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. (F) The NFI-C binding motif in the KLF4 promoter was confirmed by DNAP assay using extracts from MCF7 cells that had been transfected with NFI-C-expressing or NFI-C-siRNA constructs. Biotinylated oligonucleotides corresponding to the KLF4 promoter (WT or mutated) were used as probes. WT, wild type; MT, mutant type.

Mentions: MET initiates and completes the invasion-metastasis cascade of cancer cells [24]. Our previous results showed that NFI-C induces MET during normal odontoblast differentiation [17]. Therefore, we speculated that NFI-C also regulates the balance between KLF4 and E-cadherin in breast cancer cells, which is important for MET. To address this, we examined whether NFI-C could regulate the transcription of KLF4 and E-cadherin and subsequently alter the expression of marker genes in MCF7 breast cancer cells. MCF7 cells were transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. NFI-C overexpression increased the transcription of KLF4 and E-cadherin mRNAs, which play an important role in the MET of cancer cells. In contrast, siRNA or TGF-β-mediated silencing of NFI-C decreased transcription of KLF4 and E-cadherin. However, NFI-C expression levels did not significantly affect the expression of N-cadherin compared with other mRNA (Figure 2A). Slug (Snail2), a Snail family member, is the dominant regulator of EMT initiation in vitro and in vivo, as EMT is inhibited following Slug depletion [25]. KLF4 and another epithelial determinant, FoxA1, are direct transcriptional inhibitors of Slug expression in mouse and human prostate cancer cells [26]. Interestingly, similar with microarray data with pulp cells of Nfic knockout mice [17], Slug mRNA was increased in NFI-C downregulated cells (Figure 2A). Also, Vimentin and P-cadherin mRNA was slightly increased in NFI-C downregulated cells by siRNA or TGF-β (Figure 2A). In western blot analyses, although NFI-C overexpression did not alter the expression levels of Slug and N-cadherin, it enhanced the expression of KLF4 and E-cadherin. Conversely, NFI-C inactivation by siRNA suppressed E-cadherin expression and induced Slug expression (Figure 2B). In addition, increasing the concentration of NFI-C significantly enhanced the expression of luciferase reporter genes under the control of the mouse KLF4 promoter. In contrast, depletion of NFI-C using a specific siRNA suppressed promoter activity of the KLF4 reporter construct in MCF7 cells (Figure 2C). NFI-C overexpression augmented E-cadherin promoter activity; whereas, siRNA-mediated NFI-C inactivation decreased E-cadherin promoter activity (Figure 2D). Furthermore, overexpression of NFI-C and KLF4 showed a synergistic effect on E-cadherin transcription levels (Figure 2D).Figure 2


Nuclear factor I-C regulates E-cadherin via control of KLF4 in breast cancer.

Lee HK, Lee DS, Park JC - BMC Cancer (2015)

Effects of NFI-C overexpression and inactivation on the KLF4-E-cadherin signaling pathway in MCF7 breast cancer cells. (A, B) Expression of NFI-C, KLF4, Slug, E-cadherin, Vimentin, N-cadherin, and P-cadherin was analyzed by real-time PCR (A) and western blotting (B) in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. (C, D) Transcriptional activity of the KLF4 promoter and the E-cadherin promoter were evaluated by luciferase assays using NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. Control samples were transfected with only E-cadherin promoter constructs. Data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01). (E) NFI-C binding to the KLF4 promoter was investigated using ChIP assays after transfection with NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. (F) The NFI-C binding motif in the KLF4 promoter was confirmed by DNAP assay using extracts from MCF7 cells that had been transfected with NFI-C-expressing or NFI-C-siRNA constructs. Biotinylated oligonucleotides corresponding to the KLF4 promoter (WT or mutated) were used as probes. WT, wild type; MT, mutant type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Effects of NFI-C overexpression and inactivation on the KLF4-E-cadherin signaling pathway in MCF7 breast cancer cells. (A, B) Expression of NFI-C, KLF4, Slug, E-cadherin, Vimentin, N-cadherin, and P-cadherin was analyzed by real-time PCR (A) and western blotting (B) in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. (C, D) Transcriptional activity of the KLF4 promoter and the E-cadherin promoter were evaluated by luciferase assays using NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. Control samples were transfected with only E-cadherin promoter constructs. Data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01). (E) NFI-C binding to the KLF4 promoter was investigated using ChIP assays after transfection with NFI-C-expressing or NFI-C-siRNA constructs in MCF7 cells. (F) The NFI-C binding motif in the KLF4 promoter was confirmed by DNAP assay using extracts from MCF7 cells that had been transfected with NFI-C-expressing or NFI-C-siRNA constructs. Biotinylated oligonucleotides corresponding to the KLF4 promoter (WT or mutated) were used as probes. WT, wild type; MT, mutant type.
Mentions: MET initiates and completes the invasion-metastasis cascade of cancer cells [24]. Our previous results showed that NFI-C induces MET during normal odontoblast differentiation [17]. Therefore, we speculated that NFI-C also regulates the balance between KLF4 and E-cadherin in breast cancer cells, which is important for MET. To address this, we examined whether NFI-C could regulate the transcription of KLF4 and E-cadherin and subsequently alter the expression of marker genes in MCF7 breast cancer cells. MCF7 cells were transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β. NFI-C overexpression increased the transcription of KLF4 and E-cadherin mRNAs, which play an important role in the MET of cancer cells. In contrast, siRNA or TGF-β-mediated silencing of NFI-C decreased transcription of KLF4 and E-cadherin. However, NFI-C expression levels did not significantly affect the expression of N-cadherin compared with other mRNA (Figure 2A). Slug (Snail2), a Snail family member, is the dominant regulator of EMT initiation in vitro and in vivo, as EMT is inhibited following Slug depletion [25]. KLF4 and another epithelial determinant, FoxA1, are direct transcriptional inhibitors of Slug expression in mouse and human prostate cancer cells [26]. Interestingly, similar with microarray data with pulp cells of Nfic knockout mice [17], Slug mRNA was increased in NFI-C downregulated cells (Figure 2A). Also, Vimentin and P-cadherin mRNA was slightly increased in NFI-C downregulated cells by siRNA or TGF-β (Figure 2A). In western blot analyses, although NFI-C overexpression did not alter the expression levels of Slug and N-cadherin, it enhanced the expression of KLF4 and E-cadherin. Conversely, NFI-C inactivation by siRNA suppressed E-cadherin expression and induced Slug expression (Figure 2B). In addition, increasing the concentration of NFI-C significantly enhanced the expression of luciferase reporter genes under the control of the mouse KLF4 promoter. In contrast, depletion of NFI-C using a specific siRNA suppressed promoter activity of the KLF4 reporter construct in MCF7 cells (Figure 2C). NFI-C overexpression augmented E-cadherin promoter activity; whereas, siRNA-mediated NFI-C inactivation decreased E-cadherin promoter activity (Figure 2D). Furthermore, overexpression of NFI-C and KLF4 showed a synergistic effect on E-cadherin transcription levels (Figure 2D).Figure 2

Bottom Line: We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT.NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis.The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Histology-Developmental Biology & Dental Research Institute, School of Dentistry, Seoul National University, 101 Daehagro, Chongro-gu, Seoul, 110-749, South Korea. h35071221@snu.ac.kr.

ABSTRACT

Background: Progression to metastasis is the leading cause of most cancer-related mortality; however, much remains to be understood about what facilitates the spread of tumor cells. In the present study, we describe a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness.

Methods: We examined nuclear factor I-C (NFI-C) expression in MCF10A human breast epithelial cells, MCF7 non-invasive breast cancer cells, and MDA-MB231 invasive breast cancer cells by real-time PCR and western blotting. To investigate the loss- and gain-function of NFI-C, we determined whether NFI-C regulated KLF4 expression by real-time PCR, western blotting, and promoter assay. To understand the biological functions of NFI-C, we observed cell invasion, migration, adhesion in human tumor cells by transwell assay, wound healing assay, quantitative RT-PCR, cell adhesion assay, western blotting, and immunohistochemistry.

Results: We identified the downstream factors of NFI-C, such as KLF4 and E-cadherin, which play roles in EMT. NFI-C is expressed in normal mammary gland or noninvasive breast cancer cells with epithelial characteristics. NFI-C overexpression induced expression of KLF4 and E-cadherin, but not Slug, in breast cancer cells. NFI-C bound directly to the KLF4 promoter and stimulated KLF4 transcriptional activity, thereby regulating E-cadherin expression during tumorigenesis. Cells overexpressing NFI-C maintained their epithelial differentiation status, which could drive mesenchymal-epithelial transition (MET) via the NFI-C-KLF4-E-cadherin axis in breast cancer cells. Consequently, NFI-C suppressed EMT, migration, and invasion in breast cancer cells.

Conclusions: Our study reveals a novel signaling pathway that is important during breast cancer tumorigenesis: the NFI-C-KLF4-E-cadherin pathway. The results indicate the important role of NFI-C in regulating KLF4 during tumorigenesis.

Show MeSH
Related in: MedlinePlus