Abstract 1988: Role of N- ZEB1 in epithelial-mesenchymal transition (EMT)

Abstract

Background: ZEB1 (Zn Finger E-box Binding Homeobox) transcription factor is important in both development and disease, including the TGFβ-induced epithelial-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 contains 2 Zn finger domains (ZD1-ZD2) which bind DNA independently. ZEB1 expresses as two phosphorylated forms important for its biological role. We had characterized the role of phosphorylated C-term ZEB1. Aim: Our goal is to uncover the role of signaling pathway/s in the phosphorylation on N-term ZD1 (N-ZEB1) and characterize the functional role of N-ZEB1 in the process of EMT in mammary epithelial cells NMuMG. Results: A 728 aa fragment of N-ZEB1 was overexpressed in HEK293T cells and Immunoprecipitation with N-ZEB1 antibodies followed by western blot analysis with anti phospho kinases (AKT, MEK/ERK, PKC or PKA) substrate Antibodies. The results revealed that N-ZEB1 is solely phosphorylated by PI3K (AKT), MAPK and PKC pathways. IGF1-mediated phosphorylation can regulate nuclear localization of C-term ZEB1. Two N-ZEB1-GFP clones harboring first 490aa (eGFPZ1) and a 2nd clone of 130 aa, eGFPZ2 (included in the former) were transfected to CHO-K1 cells and treated with inhibitors and activators of kinase pathways for 15-60 minutes in serum free medium. Fluorescence microscopy techniques showed that IGF-1 induced relocation of a nuclear eGFPZ1 into the cytosol of CHO-K1 cells; on the contrary, eGFPZ2 was unresponsive to IGF-1. PMA/IONO (activator of PKC) treatment relocated both clones in the cytosol. Later on, NMuMG cells were used to investigate the role of N-ZEB1 in EMT. These epithelial cells, that can turn into mesenchymal type under TGFβ treatment, were stably transfected with eGFPZ2 or empty vector (control). The epithelial markers E-cadherin and actin assessed by Western blot and confocal microscopy were diminished or absent in eGFPZ2 cells. The activity of Matrix Metallo Proteases 2 was also increased in NMuMG-GFPZ2 cells. Also, proliferation, migration and invasion were significantly increased (P<0.001, P<0.001 and P<0.05, respectively) in NMuMG-GFPZ2 cells vs. controls. Taking together, these results show that the cells were induced to EMT by N-ZEB1. Conclusions: We have identified a small fragment in N-ZEB1 that is able to elicit the EMT program in a TGFβ independent manner. In addition, changes in ZEB1 cellular localization induced by either PKC or IGF-1 could regulate this transcription factor biological activity. Citation Format: M Candelaria Llorens, Ana M. Cabanillas. Role of N- ZEB1 in epithelial-mesenchymal transition (EMT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2015-1988