NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells - PubMed (original) (raw)

NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells

Bruno R B Pires et al. PLoS One. 2017.

Abstract

The metastatic process in breast cancer is related to the expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1 and TWIST1. EMT-TFs and nuclear factor-κB (NF-κB) activation have been associated with aggressiveness and metastatic potential in carcinomas. Here, we sought to examine the role of NF-κB in the aggressive properties and regulation of EMT-TFs in human breast cancer cells. Blocking NF-κB/p65 activity by reducing its transcript and protein levels (through siRNA-strategy and dehydroxymethylepoxyquinomicin [DHMEQ] treatment) in the aggressive MDA-MB-231 and HCC-1954 cell lines resulted in decreased invasiveness and migration, a downregulation of SLUG, SIP1, TWIST1, MMP11 and N-cadherin transcripts and an upregulation of E-cadherin transcripts. No significant changes were observed in the less aggressive cell line MCF-7. Bioinformatics tools identified several NF-κB binding sites along the promoters of SNAIL, SLUG, SIP1 and TWIST1 genes. Through chromatin immunoprecipitation and luciferase reporter assays, the NF-κB/p65 binding on TWIST1, SLUG and SIP1 promoter regions was confirmed. Thus, we suggest that NF-κB directly regulates the transcription of EMT-TF genes in breast cancer. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-κB as a potential target for breast cancer treatment.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Migration assay.

A representative wound healing assay evaluating cell migration at 24 h after DHMEQ treatment of MDA-MB-231 (A) HCC-1954 (B) and MCF-7 (C) cells is shown. The box plots represent migratory ability as indicated by the percent of wound closure. Magnification x100. The data were expressed as the mean ± SD. * = p<0.05, ns = not statistically significant.

Fig 2

Fig 2. Invasiveness assay.

A representative Matrigel transwell assay evaluating invasive potential at 24 h after DHMEQ treatment of MDA-MB-231 (A) HCC-1954 (B) and MCF-7 (C) cells is shown. The bar graph represents the relative invasive potential of MDA-MB-231, HCC-1954 and MCF-7 cells. The cells were stained with crystal violet. Magnification x200. The data were expressed as the mean ± SD. * = p<0.05, ** = p<0.01, ns = not statistically significant.

Fig 3

Fig 3. Relative expression of the EMT-inducing factors after NF-κB/p65 signaling inhibition.

The mRNA levels of SNAIL1, SLUG, TWIST1, and SIP1 were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. NF-κB/p65 inhibition was evaluated at protein levels by western blot assay at 16 and 24 h of DHMEQ treatment. Ponceau staining was used as a loading control. Ctrl: control. The data were expressed as the mean ± SD. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 4

Fig 4. Relative expression of EMT-phenotype markers after NF-κB/p65 signaling inhibition.

The mRNA levels of E-CADHERIN, N-CADHERIN and MMP11 were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells at 8, 16 and 24 h of DHMEQ treatment. The data were expressed as the mean ± SD. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 5

Fig 5. Relative expression of EMT-related genes after NF-κB/p65 genetic silencing using a siRNA approach.

The mRNA levels of the EMT-inducing factors SNAIL1, SLUG, TWIST1, and SIP1 and EMT-phenotype markers E-CADHERIN, N-CADHERIN and MMP11 together with NF-κB/p65 inhibition at the protein level were assessed in MDA-MB-231 (A) and HCC-1954 (B) cells (scramble and siNF-κB/p65). Moreover, Slug expression was evaluated at the protein level for MDA-MB-231 by western blot assay in scramble and siNF-κB cells. Ponceau staining was used as a loading control. The data were expressed as the mean ± SD. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 6

Fig 6

Representative scheme of putative NF-κB binding sites located in the SNAIL1 (A), SLUG (B), TWIST1 (C) and SIP1 (D) promoter regions predicted by Tfsitescan, TESS, TFBind, TFSearch and Transfac bioinformatics tools. An alignment of the DNA region showed evolutionarily conservation among metazoan species. Identical nucleotides are in bold. Gray lines indicate regions investigated by chromatin immunoprecipitation. +1: transcription start site.

Fig 7

Fig 7

ChIP-qPCR of predicted NF-κB/p65 binding sites in the SNAIL1, SLUG, TWIST1 and SIP1 promoter regions using MDA-MB-231 (A) and HCC-1954 (B) cells. The histograms set a fold-change of each site by comparing the IgG negative control to NF-κB/p65 antibodies with the natural and treated (DHMEQ) condition. The data were expressed as the mean ± SD. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 8

Fig 8

Relative luciferase activity in MDA-MB-231 cells transfected with pGL3-plasmid containing the SLUG (A), TWIST1 (B) and SIP1 (C) promoter regions. The firefly luciferase was normalized to the renilla vector, and the values are relative to the pGL3 (Mock) signal. The black boxes in the schematic representation plasmid constructs represent NF-κB binding sites. The bar graphs represent the relative luciferase activities of each construct in MDA-MB-231 cells, the white bars indicate natural NF-κB expression, and the black bars show NF-κB inhibition through DHMEQ treatment (10 μg/ml for 16 h). Each bar represents the mean ± SD.

Fig 9

Fig 9. Schematic representation of our findings.

A) Inhibition of NF-κB/p65 translocation reduces the expression of EMT-transcription factors that have been shown to regulate target genes of NF-κB. This inhibition increased E-cadherin expression, decreased N-cadherin and MMP11 expression and reduced cell motility and invasiveness potential. B) Then, NF-κB/p65 transcriptionally regulates the promoter regions of TWIST1, SLUG and SIP1, which in turn represses the epithelial marker E-cadherin and activates the mesenchymal markers N-cadherin and MMP11, resulting in induction of the EMT process.

References

    1. GLOBOCAN. Cancer Incidence and Mortality Worldwide: IARC Cancer Base [Internet]. Lyon, France: International Agency for Research on Cancer. 2012. Available: http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx. Accessed July 2015
    1. World Health Organization. Available: http://www.who.int/mediacentre/factsheets/fs297/en/ Accessed July 2015
    1. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol. 2005; 17(5):548–58. 10.1016/j.ceb.2005.08.001 - DOI - PubMed
    1. Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, et al. The transcription factor snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000; 2(2):76–83. 10.1038/35000025 - DOI - PubMed
    1. Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J, de Herreros AG. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000; 2(2):84–89. 10.1038/35000034 - DOI - PubMed

MeSH terms

Substances

LinkOut - more resources