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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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Effects of NFI family members on the expression of KLF4 and E-cadherin. (A) Expression of HA, KLF4, and E-cadherin proteins was analyzed by western blotting (left panel) and quantified (right panel) in MCF7 cells transfected with HA-tagged-NFI-A, −NFI-B, −NFI-C, or -NFI-X expressing constructs. (B) The transcriptional activity of the E-cadherin promoters was evaluated by luciferase assay in MCF7 cells transfected with NFI-A-, NFI-B-, NFI-C-, or NFI-X-expressing constructs. The data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01).
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Fig4: Effects of NFI family members on the expression of KLF4 and E-cadherin. (A) Expression of HA, KLF4, and E-cadherin proteins was analyzed by western blotting (left panel) and quantified (right panel) in MCF7 cells transfected with HA-tagged-NFI-A, −NFI-B, −NFI-C, or -NFI-X expressing constructs. (B) The transcriptional activity of the E-cadherin promoters was evaluated by luciferase assay in MCF7 cells transfected with NFI-A-, NFI-B-, NFI-C-, or NFI-X-expressing constructs. The data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01).

Mentions: NFI family members inhibit tumorigenesis by regulating oncogenic transcriptional factors such as brain fatty acid-binding protein (B-FABP) and JUN proteins [29-31]. To investigate the function of the four NFI factors on EMT/MET regulation, we examined the effects of NFI family member overexpression on KLF4 and E-cadherin promoter activity. Expression of each of the NFI isoforms was confirmed by western blotting with the anti-HA antibody after transfection. Overexpression of NFI-C and NFI-X resulted in a statistically significant increase in KLF4 and E-cadherin transcriptional activity in MCF7 breast cancer cells, while NFI-A and NFI-B overexpression did not (Figure 4).Figure 4


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 family members on the expression of KLF4 and E-cadherin. (A) Expression of HA, KLF4, and E-cadherin proteins was analyzed by western blotting (left panel) and quantified (right panel) in MCF7 cells transfected with HA-tagged-NFI-A, −NFI-B, −NFI-C, or -NFI-X expressing constructs. (B) The transcriptional activity of the E-cadherin promoters was evaluated by luciferase assay in MCF7 cells transfected with NFI-A-, NFI-B-, NFI-C-, or NFI-X-expressing constructs. The data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359555&req=5

Fig4: Effects of NFI family members on the expression of KLF4 and E-cadherin. (A) Expression of HA, KLF4, and E-cadherin proteins was analyzed by western blotting (left panel) and quantified (right panel) in MCF7 cells transfected with HA-tagged-NFI-A, −NFI-B, −NFI-C, or -NFI-X expressing constructs. (B) The transcriptional activity of the E-cadherin promoters was evaluated by luciferase assay in MCF7 cells transfected with NFI-A-, NFI-B-, NFI-C-, or NFI-X-expressing constructs. The data are presented as the mean ± SD of triplicate experiments. * denotes values significantly different from the control (P < 0.01).
Mentions: NFI family members inhibit tumorigenesis by regulating oncogenic transcriptional factors such as brain fatty acid-binding protein (B-FABP) and JUN proteins [29-31]. To investigate the function of the four NFI factors on EMT/MET regulation, we examined the effects of NFI family member overexpression on KLF4 and E-cadherin promoter activity. Expression of each of the NFI isoforms was confirmed by western blotting with the anti-HA antibody after transfection. Overexpression of NFI-C and NFI-X resulted in a statistically significant increase in KLF4 and E-cadherin transcriptional activity in MCF7 breast cancer cells, while NFI-A and NFI-B overexpression did not (Figure 4).Figure 4

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