Abstract 3864: Development of high-specific and sensitive reporter assays for the detection of Wnt/β-catenin signaling activity

Abstract

Abstract The Wnt/β-catenin signaling pathway is involved in development, tissue homeostasis, and regeneration. Since aberrant activation of this pathway plays a crucial role in human carcinogenesis, inhibition of this pathway has been shown to be a promising therapeutic strategy for cancers with de-regulated Wnt signaling. Although cell-based assays using synthetic TCF/LEF reporters (also known as TOPFLASH) have contributed to the discovery of small molecules that modulate Wnt signaling pathway, these assays often raise a problem of the specificity in the screening. In this study, we aimed to develop a specific reporter for the detection of Wnt signaling activity. We performed integrated transcriptome analysis and found that expression of histidine ammonia-lyase gene (HAL) is negatively regulated by β-catenin/TCF. In addition to TOPFLASH, we leveraged a promoter region of HAL as another transcriptional readout of Wnt signaling. Subsequently, HepG2 cells stably expressing both optimized HAL promoter reporter and TOPFLASH reporter were established. Since inhibition of the β-catenin/TCF transcriptional activity decreased TOPFLASH activity and simultaneously increased HAL reporter activity, we termed this assay as a bidirectional reporter assay. To verify the effectiveness of this assay, we compared the number of hits in the screening of a test library using the bidirectional assay with that using the conventional TOPFLASH/FOPFLASH assay. Among the 361 compounds in the library, the bidirectional reporter assay identified 9 hits, while the TOPFLASH/FOPFLASH assay did 79 hits. Four compounds were overlapped between these assays, suggesting that the bidirectional assay decreased false positives compared with the conventional assay. The concept of bidirectional assay might be useful for the screening of compounds that regulate other transcriptional factors and/or disease-associated signaling pathways. Citation Format: Kiyoshi Yamaguchi, Chi Zhu, Tomoyuki Ohsugi, Akari Saku, Tsuneo Ikenoue, Yoichi Furukawa. Development of high-specific and sensitive reporter assays for the detection of Wnt/β-catenin signaling activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3864.