The regulatory role of NF-êB and Twist1 in breast cancer cells

Abstract

The metastasis of breast cancer (BC) is related to the expression of epithelial-to-mesenchymal transition (EMT)-inducing genes. We sought to examine the role of NF-κB in the regulation of EMT in BC. NF-κB/p65 knockdown resulted in decreased invasiveness and migration, downregulated SLUG, SIP1, TWIST1 and N-CADHERIN transcripts, and upregulated E-CADHERIN transcripts in BC cells. Bioinformatics tools indicated NF-κB binding sites on promoters of EMT genes. Chromatin immunoprecipitation and luciferase reporter assays confirmed this binding on TWIST1, SLUG and SIP1 promoter regions. Thus, NF-κB directly regulates the transcription of EMT-inducing genes in BC. Furthermore, we investigated the role of NF-κB in redox alterations in gene profiles of BC subtypes by microarray assays. The NF-κB knockdown in BC cells led to differential expression of relevant factors involved in glutathione metabolism, cytochrome P450 and cyclooxygenase, suggesting a relationship between the redox balance and NF-κB. Biochemical analyses validated the microarray dataset focused on oxidative stress, which showed a distinct pattern for each of the three studied BC subtype models. We also investigated the role of Twist1, the master regulator of EMT, in BC subtypes. In HER2 patients, TWIST1 was overexpressed. When it was silenced in HER2 BC cells, TWIST1 modified hundreds of genes compared to control cells in microarray assays. In silico analysis revealed correlations between Twist1 and Th17-mediated immune response. IL-17 signaling was examined, and it was observed that TWIST1 knockdown caused the downregulation of leading members of this signaling. In addition, RORγt, the main regulator of Th17 differentiation, was overexpressed in HER2 BC, similar to TWIST1 expression, supporting our hypothesis proposing a link between Twist1 and Th17-like phenotype/IL-17 signaling in HER2 BC. Thus, demonstrated the role of NF-κB in metastatic and redox processes of BC and Twist1 in IL-17 signaling in HER2 BC and a possible relationship between EMT and immune/inflammatory signaling.