Abstract B7: Cross-talk between Bach1, NF-кB and IL-6 in epithelial-mesenchymal transition
Authors: Andrey Brydun, Ayako Nakanome, Mitsuyo Matsumoto, Kazushige Ota, Yoshihiro Dohi, Kazuhiko Igarashi
Journal: Cancer Research (2009)

Abstract The epithelial-mesenchymal transition (EMT) is a complex biological program that allows cancer cells to aquire motility and invasiveness. Transcriptional repressor Bach1 (BTB and CNC homology 1) belongs to cap'n'collar basic leucin zipper transcription factors family and represses transcription of stress-responsive genes such as heme oxygenase-1 (Hmox-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) by binding to the MARE (Maf recognition element) sequenceses in the regulatory regions of these genes. We have recently reported that Bach1 also recruites histon deacetylase-1 (HDAC1) to a subset of p53 target genes (Perp1, p21, Noxa, Puma, PAI-1) and contributes to the inhibition of p53-dependent cellular senescence (Dohi Y. et al. Nat Struct Mol Biol. 2008;15(12):1246–54). In the present study we addressed whether Bach1 regulated cells motility and invasiveness, contributing to EMT. First, we analyzed the phenotype of Bach1-deficient (Bach1−/−) mice. We found increased spontaneous motility of immortalized embryonic fibroblasts (MEF) derived from Bach1−/− mice in the wound-healing assay. To identify differential gene expression which might explain enhanced motility of Bach−/− MEFs, we compared gene expression profiles of Bach1−/− and wild type (WT) MEFs. We found that NF-кB essential modulator, NEMO (IKBKG) was highly expressed in Bach1−/− MEFs. Mouse genome database search revealed three MARE sites located in 10 kb upstream of the first exon of NEMO gene. We confirmed that Bach1 bound to these sites in ChIP and EMSA assays and also repressed transcription of the reporter gene construct containing MARE sequences from the NEMO promoter. DNA binding activity of NF-кB, measured by EMSA, and expression of NF-кB-dependent reporter construct were enhanced in Bach1−/− MEFs. To identify Bach1 target genes downstream of NF-кB, we compared mRNA expression profiles of the Bach1−/− and WT MEFs, treated with TNF-α. Genes encoding cytokines, chemokines and matrix metalloproteinases (IL-6, CXCL-1, CXCL-2, MMP2, MMP3, MT2-MMP) were up-regulated in Bach1−/− cells. Interestingly, IL-6 mRNA level changed earlier and more intensively than other factors; moreover, we detected higher level of IL-6 in the culture supernatants from Bach1−/− MEFs. Phoshorylation of STAT3, a target of IL-6 and a regulator of many MMPs and chemokines, was enhanced in Bach1−/− MEFs. RNAi targeting of both NEMO and IL-6 significantly redused motility of Bach1−/− MEFs indicating causative role of the both factors. Thus, Bach1 may regulate cells motility by fine-tuning the expression of chemokines and matrix metaloproteinases via cross-talk with the NF-кB signal transduction pathway in IL-6 dependent manner. Next, we addressed whether the lost of Bach1 in epithelia or stroma would activate EMT program in cancer cells. RNAi targeting of Bach1 in breast cancer cell line MCF-7 resulted in lower E-cadherin expression, reduced number of cell-to-cell, cell-to-substrate contacts and increased spontaneous motility. Co-culture with Bach1−/− but not WT MEFs induced transwell migration and basement membrane matrix invasion of MCF-7 cells. In conclusion, our data indicate that Bach1 regulates cell autonomous and non-autonomous induction of chemokines and extracellular matrix degrading enzymes, inhibiting motility, invasiveness and EMT. Citation Information: Cancer Res 2009;69(23 Suppl):B7.