Proteomics at the guanylin gene locus reveals Wnt/beta-catenin regulatory factors underlying GUCY2C hormone silencing in colorectal cancer

Guanylin (GUCA2A) is a paracrine hormone expressed in the colonic epithelium that activates the transmembrane receptor guanylyl cyclase C (GUCY2C). Activated GUCY2C promotes fluid secretion, genome stability, barrier integrity, and homeostasis through cGMP signaling. In early colorectal cancer (CRC), guanylin expression is universally suppressed, silencing the GUCY2C signaling cascade and contributing to tumorigenesis. Repression of guanylin is mediated transcriptionally by aberrant Wnt/beta-catenin/TCF4 signaling at a Wnt-sensitive enhancer upstream of the GUCA2A gene. Using unbiased proteomics in Wnt-inducible CRC cell lines, we sought to directly identify regulators that mediate Wnt sensitivity to guanylin expression and control its transcriptional silencing in the context of tumorigenesis. sgRNAs targeting the GUCA2A regulatory element, consisting of the promoter and enhancer, were coupled to a dCas9-APEX2 fusion protein to capture the desired DNA locus. ChIP-qPCR and ChIP-seq confirmed sgRNA effectiveness and specificity in targeting GUCA2A . APEX2-mediated proximity biotinylation was performed to label proteins associated with the targeted site. Streptavidin affinity pulldown followed by LC-MS/MS-based proteomics revealed candidate regulatory proteins differentially enriched at the GUCA2A promoter under Wnt-on and Wnt-off conditions, including the previously identified Wnt-modulated guanylin activator p38alpha (MAPK14). Additionally, other candidates were enriched at the promoter irrespective of Wnt status, suggesting regulation of guanylin that is either Wnt-independent or requires post-translational control downstream of Wnt signaling. Potential regulators of guanylin expression identified in this analysis are undergoing validation using a knockdown screen in combination with a GUCA2A luciferase reporter with the goal of identifying the regulatory machinery underlying guanylin loss in CRC. As GUCY2C functions as a tumor suppressor, new insights into the mechanism of guanylin transcriptional silencing will provide an opportunity to inform prevention and treatment strategies to repair the signaling pathway and oppose disease.