News and views on EMT Fra-1 controls EMT in mammary epithelial cells

Epithelial-to-mesenchymal transition (EMT) is a complex biological program occurring in physiological and pathological conditions. The acquisition of a mesenchymal state by cancer cells is associated with loss of epithelial features, decreased cell adhesion and increased migration and invasion, which together are crucial for metastasis. Down-regulation of the adherens junction protein E-cadherin is a hallmark of EMT. E-cadherin expression and epithelial plasticity are regulated by a number of molecular signaling pathways and regulators operating within dynamic circuits. Transcription factors (TFs) of the Snail, Twist and Zinc finger E-box binding homeobox (Zeb) families (collectively EMT-TFs) repress E-cadherin transcription and modulate additional epithelial and mesenchymal genes. EMT-TFs are controlled by growth factors such as Transforming growth factor b (TGFb) and microRNAs, but also by a number of TFs. Fos-related antigen 1 (Fra-1) dimerize with Jun proteins to form the Activator Protein 1 (AP-1) transcription factor complex. Several studies, using human tumor material and cell lines correlated FRA-1 expression with malignancy. Compelling evidence implicates FRA-1 in EMT of human breast cancer cells and reports have connected FRA-1 as a dimer with c-JUN, with the EMT-TFs SNAI2/SLUG, ZEB1 and ZEB2. In our recent publication, we set out to define how Fra-1 contributes to EMT and assess the evolutionary conservation of EMT-TF regulation by Fra-1. We used the EpH4 murine non-tumourigenic mammary epithelial cells, which display a well documented phenotypic plasticity in response to oncogenes or growth factors. Ectopic expression of Fra-1 in EpH4 cells (EpFra1) was sufficient to trigger a proliferative, mesenchymal and invasive phenotype. Importantly, EpFra1 cells formed tumors in orthotopic and lung colonisation assays. Mechanistically, Fra-1 induced critical changes in the mRNA and protein expression of Tgfb1 and 2 EMT-TFs: Zeb1 and Zeb2. Gene expression profiling confirmed that Fra-1 induced gene expression programmes characteristic of EMT, transformation and invasiveness. In addition, we identified a Zeb1/2 molecular signature in Fra1-expressing cells, which was further validated by mRNA and protein analyses. Moreover, and in line with the recently emerging relationship between EMT, stemness and Fra-1/AP-1 in breast cancer, EpFra1 cells displayed a mammary stem cell signature. Finally, distinct changes in the expression of genes such as Secreted acidic cysteine rich glycoprotein (Sparc), Matrix metallopeptidase 2 (Mmp2), Interleukin 13 receptor, a 2 (Il13ra2), and Angiopoietin-like 4 (Angptl4), previously associated with lung tropism of human breast cancer cells, were measured. We next determined how Fra-1 modulates Tgfb1 and Zeb1/2 expression and how these genes contribute to the phenotypes observed in EpFra1 cells. Chromatin immunoprecipitation demonstrated that Fra-1 bound the mouse Tgfb1 promoter. However, inhibiting TGFb signaling in EpFra1 cells only modestly increased epithelial marker expression, indicating that TGFb signaling contributes, but is likely not essential for Fra-1-induced EMT. We next tested whether Fra-1 directly regulates Zeb1 and Zeb2 transcription, focusing on genomic sequences with