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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 morphology, adhesion, migration, and invasion of breast cancer cells. (A) Morphology of MCF10A cells when transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β. Images obtained using phase-contrast microscopy (Magnification: 100×). (B) Cell adhesion was assessed in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs. Data are presented as the mean ± SD of triplicate experiments. (C) Migration was analyzed by wound healing assays in MCF7 cell transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β (Magnification: 400×). (D) The invasion capacity of MCF7 cells, which were transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β was determined by matrigel-coated transwell assays. Average cell counts from representative fields for each condition are given as mean ± S.D. (E) The effect of NFI-C on EMT of breast cancer cells was analyzed using gene expression data collected from atypical ductal hyperplasia with or without breast cancer in the Gene Expression Omnibus (GEO) database (GSE 2429). The mean and standard variants were calculated from four biological replicates for both activity and mRNA levels of NFI-C, KLF4, E-cadherin, TIAM1 (invasion activator), and SCAI (invasion suppressor). * denotes values significantly different from the control (P < 0.01). ADH: Atypical ductal hyperplasia.
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Fig3: Effects of NFI-C overexpression and inactivation on morphology, adhesion, migration, and invasion of breast cancer cells. (A) Morphology of MCF10A cells when transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β. Images obtained using phase-contrast microscopy (Magnification: 100×). (B) Cell adhesion was assessed in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs. Data are presented as the mean ± SD of triplicate experiments. (C) Migration was analyzed by wound healing assays in MCF7 cell transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β (Magnification: 400×). (D) The invasion capacity of MCF7 cells, which were transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β was determined by matrigel-coated transwell assays. Average cell counts from representative fields for each condition are given as mean ± S.D. (E) The effect of NFI-C on EMT of breast cancer cells was analyzed using gene expression data collected from atypical ductal hyperplasia with or without breast cancer in the Gene Expression Omnibus (GEO) database (GSE 2429). The mean and standard variants were calculated from four biological replicates for both activity and mRNA levels of NFI-C, KLF4, E-cadherin, TIAM1 (invasion activator), and SCAI (invasion suppressor). * denotes values significantly different from the control (P < 0.01). ADH: Atypical ductal hyperplasia.

Mentions: It is well-known that forced expression of TGF-β induces EMT [27]. Breast cancer cell lines range from epithelial-like with low invasiveness, to mesenchymal-like, which exhibit high invasive capacity [28]. Therefore, we first investigated whether NFI-C expression levels correlate with cellular phenotype in natural isolation. NFI-C-expressing MCF10A cells grew as tightly packed patches of epithelial sheets like normal MCF10A cells. In contrast, NFI-C-siRNA-transfected or TGF-β-treated MCF10A cells were longer, like mesenchymal cells (Figure 3A). In cell binding assays, NFI-C-expressing MCF7 cells showed significantly greater attachment than normal MCF7 cells on plates (Figure 3B). Next, we asked whether NFI-C controlled migration and invasion of breast cancer cells by MET. In wound healing assays, NFI-C enhanced MCF7 breast cancer cell migration. Conversely, ectopic NFI-C-siRNA or TGF-β treatment reduced MCF7 cell motility (Figure 3C). Transwell invasion assays also demonstrated a significantly decreased number of invasive MCF7 cells when transfected with NFI-C-expressing constructs compared with control cells, NFI-C-siRNA-transfected cells, or TGF-β-treated cells (Figure 3D). These results indicated that NFI-C is crucial for the inhibition of breast cancer cell migration and invasion in vitro.Figure 3


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 morphology, adhesion, migration, and invasion of breast cancer cells. (A) Morphology of MCF10A cells when transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β. Images obtained using phase-contrast microscopy (Magnification: 100×). (B) Cell adhesion was assessed in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs. Data are presented as the mean ± SD of triplicate experiments. (C) Migration was analyzed by wound healing assays in MCF7 cell transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β (Magnification: 400×). (D) The invasion capacity of MCF7 cells, which were transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β was determined by matrigel-coated transwell assays. Average cell counts from representative fields for each condition are given as mean ± S.D. (E) The effect of NFI-C on EMT of breast cancer cells was analyzed using gene expression data collected from atypical ductal hyperplasia with or without breast cancer in the Gene Expression Omnibus (GEO) database (GSE 2429). The mean and standard variants were calculated from four biological replicates for both activity and mRNA levels of NFI-C, KLF4, E-cadherin, TIAM1 (invasion activator), and SCAI (invasion suppressor). * denotes values significantly different from the control (P < 0.01). ADH: Atypical ductal hyperplasia.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359555&req=5

Fig3: Effects of NFI-C overexpression and inactivation on morphology, adhesion, migration, and invasion of breast cancer cells. (A) Morphology of MCF10A cells when transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β. Images obtained using phase-contrast microscopy (Magnification: 100×). (B) Cell adhesion was assessed in MCF7 cells transfected with NFI-C-expressing or NFI-C-siRNA constructs. Data are presented as the mean ± SD of triplicate experiments. (C) Migration was analyzed by wound healing assays in MCF7 cell transfected with NFI-C-expressing or NFI-C-siRNA constructs or treated with TGF-β (Magnification: 400×). (D) The invasion capacity of MCF7 cells, which were transfected with NFI-C-expressing or NFI-C-siRNA constructs, or treated with TGF-β was determined by matrigel-coated transwell assays. Average cell counts from representative fields for each condition are given as mean ± S.D. (E) The effect of NFI-C on EMT of breast cancer cells was analyzed using gene expression data collected from atypical ductal hyperplasia with or without breast cancer in the Gene Expression Omnibus (GEO) database (GSE 2429). The mean and standard variants were calculated from four biological replicates for both activity and mRNA levels of NFI-C, KLF4, E-cadherin, TIAM1 (invasion activator), and SCAI (invasion suppressor). * denotes values significantly different from the control (P < 0.01). ADH: Atypical ductal hyperplasia.
Mentions: It is well-known that forced expression of TGF-β induces EMT [27]. Breast cancer cell lines range from epithelial-like with low invasiveness, to mesenchymal-like, which exhibit high invasive capacity [28]. Therefore, we first investigated whether NFI-C expression levels correlate with cellular phenotype in natural isolation. NFI-C-expressing MCF10A cells grew as tightly packed patches of epithelial sheets like normal MCF10A cells. In contrast, NFI-C-siRNA-transfected or TGF-β-treated MCF10A cells were longer, like mesenchymal cells (Figure 3A). In cell binding assays, NFI-C-expressing MCF7 cells showed significantly greater attachment than normal MCF7 cells on plates (Figure 3B). Next, we asked whether NFI-C controlled migration and invasion of breast cancer cells by MET. In wound healing assays, NFI-C enhanced MCF7 breast cancer cell migration. Conversely, ectopic NFI-C-siRNA or TGF-β treatment reduced MCF7 cell motility (Figure 3C). Transwell invasion assays also demonstrated a significantly decreased number of invasive MCF7 cells when transfected with NFI-C-expressing constructs compared with control cells, NFI-C-siRNA-transfected cells, or TGF-β-treated cells (Figure 3D). These results indicated that NFI-C is crucial for the inhibition of breast cancer cell migration and invasion in vitro.Figure 3

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