Abstract
Background: Vagal innervation plays a pivotal role in gastric tumorigenesis and tumor progression. However, the upstream signaling regulating acetylcholine receptors (AChRs) and its contribution to carcinogenesis remains largely elusive. Methods: We constructed Trp53-/-; Cdh1-/- mouse gastric organoids to recapitulate the morphological and functional characteristics of diffuse-type gastric cancer (DGC) for FDA-approved drug (1464 compounds) screening. We investigated the effects of AChR inhibitors in diffuse-type patient-derived organoids (PDOs) by examining IC50 and xenograft tumorigenesis. Mass spectrometry and Co-immunoprecipitation was used to identify the interaction between cholinergic receptor muscarinic 3 (CHRM3) and PJA2. Ubiquitination and degradation assays were used to explore the regulation of CHRM3 by PJA2. 120 human gastric cancer specimens and GEO database were used to explore the clinical relevance of PJA2-CHRM3 signaling. Results: mAChR inhibitors were identified as the most effective at suppressing Trp53-/-; Cdh1-/- organoids. Consistently, the viability of PDOs with aberrant expression of CHRM3 can be significantly inhibited by oxybutynin hydrochloride (OXY) and nortriptyline hydrochloride (NOR). Mechanistically, the RING E3 ligase PJA2 ubiquitinates and degrades CHRM3, subsequently suppressing downstream TGFβ-pSMAD3 signaling and tumor cell progression. Clinically, low PJA2 expression was correlated with high CHRM3, p-SMAD3 and choline acetyltransferase (ChAT) expression and predicted poor outcomes. Biologically, PJA2 but not its catalytically dead ΔRING mutant could suppress PDOs with aberrant CHRM3 signaling. PJA2 depletion enhanced tumor metastasis of gastric cancer cells, subsequently reversed by OXY and NOR treatment. Conclusions: This previously unknown PJA2-CHRM3 signaling axis provides further understanding of cholinergic innervation as well as identifies a new therapeutic vulnerability in DGC.