A New Fluorescence-Based Reporter Gene Vector as a Tool for Analyzing and Fishing Cells with Activated Wnt Signaling Pathway

The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β-galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. 1. Introduction The dysregulated Wnt signaling pathway is linked with cancer diseases and is also one of the most mutated signaling pathways in colorectal cancer carcinomas？？[1–3]. In a normal cell, the key protein β-catenin is permanently expressed, bound to the APC protein in the destruction complex, and marked for degradation by GSK3β and β-TrCP. As a consequence the level of free β-catenin is low？？[4]. With activation of the canonical Wnt signaling pathway by extracellular ligands like the Wnt proteins or by mutation of one of the destruction complex proteins and due to the following misregulation β-catenin is translocated into the nucleus and activates expression of target genes, resulting, for example, in increased cell proliferation, reduced apoptosis, or decreased cell differentiation？？[5, 6]. This is supposed to be one of the major steps in carcinogenesis from a normal cell to a tumor cell？？[7]. Specific drugs which interrupt this misregulation might have high potential in targeted cancer therapy with low side effects especially in the aggressive chemotherapies？？[8, 9]. The searching for specific Wnt pathway inhibitors as possible cancer drugs forged ahead during the last years although none of them finished clinical trials studies until now？？[10]. Most assays which are used to screen for Wnt modulating drugs are based on Korinek’s TOPFLASH reporter