Cancers arising from gastrointestinal epithelial cells are common, aggressive, and difficult to treat. Progress in this area resulted from recognizing that the biological behavior of these cancers is highly dependent on bioactive molecules released by neurocrine, paracrine, and autocrine mechanisms within the tumor microenvironment. For many decades after its discovery as a neurotransmitter, acetylcholine was thought to be synthesized and released uniquely from neurons and considered the sole physiological ligand for muscarinic receptor subtypes, which were believed to have similar or redundant actions. In the intervening years, we learned this former dogma is not tenable. (1) Acetylcholine is not produced and released only by neurons. The cellular machinery required to synthesize and release acetylcholine is present in immune, cancer, and other cells, as well as in lower organisms (e.g., bacteria) that inhabit the gut. (2) Acetylcholine is not the sole physiological activator of muscarinic receptors. For example, selected bile acids can modulate muscarinic receptor function. (3) Muscarinic receptor subtypes anticipated to have overlapping functions based on similar G protein coupling and downstream signaling may have unexpectedly diverse actions. Here, we review the relevant research findings supporting these conclusions and discuss how the complexity of muscarinic receptor biology impacts health and disease, focusing on their role in the initiation and progression of gastric, pancreatic, and colon cancers.

Conflict of interest: Aspects of treating cancer with anticholinergic agents are the subject of a patent ("Hybrid cholinergic agents and compositions, methods of making, and methods of using to treat a cholinergic disorder," US 6,624,155) issued on September 23, 2003, to the University of Arkansas; JPR is an inventor on this patent. JPR owns equities in Agile Therapeutics, Gilead Sciences, and Procter & Gamble.

Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. βPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and post-wounding restitution of rat intestinal epithelial monolayers. We hypothesized βPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined βPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective βPix deletion [βPixflox/flox/Tg(Villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 CKO and control mice, we investigated the consequences of βPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear βPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile with normal gross intestinal architecture but reduced small intestinal villus height, villus/crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite adding Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared to littermate control mice, dextran sodium sulfate-treated βPix-deficient mice lost more weight, had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal βPix expression is important for enterocyte development, intestinal homeostasis and resistance to toxic injury.

Muscarinic receptors (MRs) in the G protein-coupled receptor superfamily are recipients and mediators of parasympathetic neural transmission within the central and enteric nervous systems. MR subtypes, M1R–M5R, encoded by CHRM1-CHRM5, expressed widely throughout the gastrointestinal (GI) tract, modulate a range of critical, highly regulated activities in healthy tissue, including secretion, motility, and cellular renewal. CHRM3/M3R overexpression in colon cancer is associated with increased cell proliferation, metastasis, and a worse outcome, but little is known about the role of the other four muscarinic receptor subtypes. To address this gap in knowledge, we queried the NCI Genomic Data Commons for publicly available TCGA-COAD samples collected from colon tissue. RNA-seq data were collected and processed for all available primary adenocarcinomas paired with adjacent normal colon. In this unbiased analysis, 78 paired samples were assessed using correlation coefficients and univariate linear regressions; gene ontologies were performed on a subset of correlated genes. We detected a consistent pattern of CHRM1 downregulation across colorectal adenocarcinomas. CHRM1 expression levels were positively associated with those for APC and SMAD4, and negatively associated with CTNNB1, the gene for β-catenin, and with coordinate changes in the expression of β-catenin target genes. These findings implicating CHRM1/M1R as an important deterrent of colon cancer development and progression warrant further exploration.

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.