Acetylcholine-activated receptors are divided broadly into two major structurally distinct classes: ligand-gated ion channel nicotinic and G-protein-coupled muscarinic receptors. Each class encompasses several structurally related receptor subtypes with distinct patterns of tissue expression and post-receptor signal transduction mechanisms. The activation of both nicotinic and muscarinic cholinergic receptors has been associated with the induction and progression of gastrointestinal neoplasia. Herein, after briefly reviewing the classification of acetylcholine-activated receptors and the role that nicotinic and muscarinic cholinergic signaling plays in normal digestive function, we consider the mechanics of acetylcholine synthesis and release by neuronal and non-neuronal cells in the gastrointestinal microenvironment, and current methodology and challenges in measuring serum and tissue acetylcholine levels accurately. Then, we critically evaluate the evidence that constitutive and ligand-induced activation of acetylcholine-activated receptors plays a role in promoting gastrointestinal neoplasia. We focus primarily on adenocarcinomas of the stomach, pancreas, and colon, because these cancers are particularly common worldwide and, when diagnosed at an advanced stage, are associated with very high rates of morbidity and mortality. Throughout this comprehensive review, we concentrate on identifying novel ways to leverage these observations for prognostic and therapeutic purposes.

Studying individual data types in isolation provides only limited and incomplete answers to complex biological questions and particularly falls short in revealing sufficient mechanistic and kinetic details. In contrast, multi-omics approaches to studying health and disease permit the generation and integration of multiple data types on a much larger scale, offering a comprehensive picture of biological and disease processes. Gastroenterology and hepatobiliary research are particularly well-suited to such analyses, given the unique position of the luminal gastrointestinal (GI) tract at the nexus between the gut (mucosa and luminal contents), brain, immune and endocrine systems, and GI microbiome. The generation of ‘big data’ from multi-omic, multi-site studies can enhance investigations into the connections between these organ systems and organisms and more broadly and accurately appraise the effects of dietary, pharmacological, and other therapeutic interventions. In this review, we describe a variety of useful omics approaches and how they can be integrated to provide a holistic depiction of the human and microbial genetic and proteomic changes underlying physiological and pathophysiological phenomena. We highlight the potential pitfalls and alternatives to help avoid the common errors in study design, execution, and analysis. We focus on the application, integration, and analysis of big data in gastroenterology and hepatobiliary research.

M₃ muscarinic receptors (M₃R) modulate β-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M₃R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M₃R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M₃R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M₃R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M₃R/βPix/β-catenin axis may have therapeutic potential.

M₃ muscarinic receptor (M₃R) activation stimulates colon cancer cell proliferation, migration, and invasion; M₃R expression is augmented in colon cancer and ablating M₃R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M₃R, M₁R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M₁R versus M₃R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M₁R agonists. We detected divergent expression of M₁R and M₃R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M₁R agonists, we found that in contrast to the effects of M₃R activation, selective activation of M₁R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M₁R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M₁R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M₁R agonist-induced attenuated cell proliferation. Lastly, adding M₁R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M₁R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.

Small intestinal goblet cells robustly express both Chrm1 and Chrm3; mucosal immune cells express primarily Chrm3. Mice with intestinal epithelial cell (IEC) Chrm3/M ₃ R deletion have fewer small intestinal goblet cells but more tuft cells. The increase in tuft cells, which produce acetylcholine, may represent a feedback mechanism to compensate for reduced muscarinic receptor (MR) expression. IEC-selective deletion of MRs does not impact murine weight or enteroid development, suggesting that MRs beyond the intestinal epithelium regulate weight.