Abstract 1423: Locus-specific proteomics to define transcriptional regulation of GUCY2C hormone expression in colorectal cancer

Abstract The GUCY2C intestinal receptor regulates fluid secretion and epithelial transformation through its hormone ligand guanylin (GUCA2A). GUCY2C supports intestinal homeostasis and has been characterized as a tumor suppressor in colorectal cancer. In early tumorigenesis, GUCY2C signaling is silenced through transcriptional repression of guanylin. Guanylin repression is mediated by Wnt/β-catenin/TCF4, a critical signaling pathway aberrantly activated in ∼90% of colorectal cancers. Specifically, Wnt signaling regulates expression at a recently characterized 2, 683-base pair locus control region (LCR) upstream of the GUCA2A gene. This regulation is conferred indirectly, and TCF4, the transcription factor effector of the Wnt cascade, does not interact with the LCR. However, many downstream TCF4 targets are transcription factors canonically implicated in tumorigenesis. Therefore, our goal is to identify factors specifically interacting with the LCR and regulating guanylin expression. Colorectal cancer cell lines expressing inducible Wnt-silencing constructs were used to toggle the expression of guanylin. Using these models, we sought to interrogate Wnt regulation at the LCR by employing C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging), an unbiased proteomics-based approach to profile locus-specific protein interactions. This system relies on peroxidase-mediated biotinylation of proteins by a dCas9-APEX2 fusion protein, driven to a locus of interest by a targeting gRNA. Following localized biotinylation, proteins are enriched by streptavidin pulldown and identified by mass spectrometry analysis. Assessing proteins in the presence and absence of Wnt signaling can reveal Wnt-regulated factors controlling target gene expression. By ChIP-qPCR, we have captured the GUCA2A gene locus using targeted gRNAs. Further, dCas9-APEX2 binding does not interfere with the expression of guanylin or its Wnt regulation. Finally, streptavidin-based pulldowns reveal protein interactions at the promoter that can be further characterized by mass spectrometry to elucidate the Wnt regulatory machinery at the GUCA2A locus. Identifying the mechanism of Wnt-mediated guanylin silencing, and the transcription factors controlling that regulation, will provide novel insights to inform therapeutic targeting to reconstitute the GUCY2C signaling axis to prevent colorectal cancer. Citation Format: Adi Caspi, Ariana A. Entezari, Jasmine R. Alvarez, Jeffrey A. Rappaport, Adam E. Snook, Scott A. Waldman. Locus-specific proteomics to define transcriptional regulation of GUCY2C hormone expression in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1423.