1. Identification of SIP1-modulated genes during the epithelial-to-mesenchymal transition and interactions with KLF factors in EMT control Benjamin Koopmansch 1, Anne Brysse 2, Christine Gilles 2, Jean-Michel Foidart 2, Rosita Winkler 1 1 GIGA-Research, Molecular Oncology Unit, Université de Liège 2 GIGA-Research, Laboratory of Tumor & Development Biology Unit, Université de Liège In A431 cells, recruitment of KLF4 on the E-cadherin promoter is diminished more than 3 times following SIP1 forced expression. Prm1 promoter was used as a negative control for KLF4 binding. The E-cadherin promoter contains 2 E- boxes to which SIP1 is known to bind (Comijn et al., 2001). A GC-box (putative binding site for KLF4) is located between these two sites. To study the interplay between SIP1 and KLF4, we co-transfected reporter vectors mutated for theses binding sites and a SIP1- expression vector. CONCLUSION EMT (epithelial-to-mesenchymal transition) is a process characterized by the loss of epithelial properties and the gain of mesenchymal properties. Among the transcription factors involved in EMT, SIP1 is known as a transcriptional repressor of epithelial genes, including E-cadherin. It has been shown that SIP1 binds to DNA at 2 E-boxes separated by 40 to 50 bases. A repression model suggested that SIP1, by binding to theses 2 sites, is able to close a region of DNA and to impair the binding of an activating factor. KLF4, a Krüppel/Sp1-like family member, is down-regulated in some cancers (colon, oesophagus) but up-regulated in others (breast and pancreas). To date, KLF4 is described as a positive regulator of E-cadherin and has been shown to inhibit the EMT in MCF10A cells. Our project aims at understanding the implication of KLF4 in the EMT in breast cancer KLF4 is present on the E-cadherin promoter in MCF7 cells (not expressing SIP1), but not in SIP1- expressing cells (MDA-MB-231 and Hs578T). Study of the E-cadherin promoter for the role of SIP1 and KLF4 : RESULTS INTRODUCTION A431 and MCF7 cells show epithelial properties (E-cad+, Vim- and SIP1-) Hs578T and MDA-MB-231 show mesenchymal properties (Ecad-, Vim+ and SIP1+). Although KLF4 up-regulates E-cadherin and is more expressed in «mesenchymal » cells, these cells do not express E-cadherin. Mutation of the putative KLF4 binding site didn’t impair the SIP1 repression. Luciferase assay showed a potent activator effect of the GC-box on the promoter, but this site is not involved in the SIP1 repressive effect. Mutation of the E-box located at -75 is sufficient to impair SIP1 repression of E- cadherin promoter activity. Experiment conducted in triplicate in MCF7 cells, confirmed in other cell lines. This result is different from other published results. Although KLF4 is expressed at high levels in « mesenchymal » breast cancer cell lines, it doesn’t bind to the E-cadherin promoter. We postulated that SIP1 could be responsible for the absence of KLF4 on the E-cadherin promoter. Our first results didn’t allow us to confirm this or find the underlying mechanism. These results prompted us to test if SIP1 is responsible for the absence of KLF4 on the E-cadherin promoter Recruitment of KLF4 on the E-cadherin promoter in A431 and breast cancer cells: ChIP analysis