Abstract 5289: In human endothelial cell line Wnt-beta-catenin pathway controls transcription of lymphoid enhancer-binding factor 1, a representative of vertebrate-specific Lef1/Tcf1 sub-family enhancing matrix metalloproteinase-2 transcription and endothelial cell invasion

Background: Wnt signaling is activated in many types of cancer and normal physiological processes. Various Wnt-related secreted factors may influence angiogenesis both in the tumor microenvironment and in normal tissues by direct action on endothelial cells. The mechanism of this Wnt action in angiogenesis is not well defined. Gradl D, Konig A, and Wedlich D (2002) show that members of Tcf1/Lef1 sub-family generate stronger Wnt throughput and Xenopus’ secondary axis induction per protein unit than members of Tcf3/Tcf4 sub-family. This stronger activity might require different, may be, stronger chemotherapy tools for angiogenesis control than the tools necessary to prevent colon cancer where Tcf4 signaling is prominent. We hypothesize that endothelial cells are responsive to Wnt signals and that Lef1, a member of the vertebrate-specific Wnt/beta-catenin throughput-inducing transcription factors’ sub-family Lef1/Tcf1, mediates this responsiveness and promotes endothelial cell invasion. Methods: A human endothelial cell line, EAhy926 was exposed to Wnt3a or directly transfected with Lef1. Readouts included assessment of nuclear β-catenin, Wnt throughput with a SuperTOPflash reporter assay, induction of Lef1 transcription, induction of matrix metalloproteinase (MMP)-2 transcription, cell proliferation and cell invasion through a matrix in vitro. The effects on MMP2 were also evaluated in the presence of Lef1 silencing siRNA. Results: Wnt3a increased nuclear β-catenin staining identified by DNA staining of nucleus with DAPI and up-regulated Wnt/β-catenin throughput without changing the activity of negative control reporter with mutated Lef/Tcf binding sites (FOP). Wnt3a increased Lef1 transcription and activity of the Lef1 promoter. Both Wnt3a treatment and Lef1 overexpression induced MMP2 transcription but this effect was completely abrogated in the presence of Lef1 siRNA. Inhibition of Lef1 also reduced basal MMP2 levels suggesting that Lef1 regulates MMP2 expression even in the absence of exogenous Wnt pathway activation. Lef1 slightly enhanced proliferation of EAhy926 cells after prolonged periods (96 and 120 hours post-transfection) and increased invasion by more than two-fold. Conclusions: EAhy926 cell line activates canonical Wnt signaling in response to Wnt3a ligand. A representative of vertebrate-specific Lef1/Tcf1 sub-family Lef1 specifically regulates MMP2 transcription in these cells and promotes endothelial cell invasion. This cell line provides a convenient alternative to primary human umbilical vein endothelial cells (HUVEC) in the study of angiogenesis and the role of Wnt signaling on endothelial cell function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5289. doi:1538-7445.AM2012-5289