Cholinergic Mechanisms in Gastrointestinal Neoplasia

Abstract

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.

Keywords: acetylcholine; brain–gut axis; cancer; cellular signaling; gastrointestinal cancer; muscarinic receptors; nicotinic receptors; protein kinases.

Figure 1

Overview of acetylcholine (ACh) synthesis. Choline enters cells via the sodium-coupled choline transporter (1a), whereas acetyl-coenzyme A (CoA) is produced in mitochondria from glycolysis or the metabolism of fatty acids (1b). Choline acetyltransferase (ChAT) catalyzes the synthesis of ACh from acetyl-CoA and choline (2); free CoA is a byproduct. ACh is loaded into vesicles by the vesicular ACh transporter (VAChT), a proton antiporter (3), and released by exocytosis (4) into either the synaptic cleft for neuronal ACh synthesis or the extracellular space for non-neuronal ACh synthesis. Prior to interacting with receptors, ACh may be hydrolyzed by acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) (5a), forming acetate and choline; the resulting choline may then undergo reuptake (6a). ACh may bind to nicotinic (nAChR) (5b), muscarinic (mAChR) (5c), or other receptors, in a paracrine (5b/c) or autocrine (6b) fashion. The binding of two ACh molecules activates nAChR and opens a non-selective cation channel. Activated mAChRs can recruit either G proteins, which initiate either Gq/11 or Gi/o signaling, or G-protein-coupled receptor kinases, which recruit β-arrestins, leading to receptor desensitization via their internalization. Created with BioRender.com.

Figure 2

Overview of post-receptor acetylcholine (ACh) signaling. Odd-numbered muscarinic receptors (M_odd: M₁R, M₃R, and M₅R) are coupled to G_q/11 signaling, which effects cellular change via phospholipid metabolism and increasing intracellular calcium concentrations, while even-numbered muscarinic receptors (M_even: M₂R and M₄R) are coupled to G_i/o, which inhibits the formation of cAMP by membrane-bound adenylyl cyclase (AC). As a result of M_even signaling, protein kinase C (PKC) is activated, which further activates mitogen-activated protein kinases (MAPK), such as p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase-1/2 (ERK1/2), to alter gene expression. ACh-bound M_odd also transactivates epidermal growth factor receptors (EGFR) via activation of matrix metalloproteinases (MMP), offering another mechanism whereby ERK1/2 can be activated. M_even activation is not known to directly transactivate EGFR, and instead leads to the inhibition of the activation of EGFR by protein kinase A (PKA). Nicotinic signaling is diverse and subtype-dependent. α7nAChR activates AC, leading to upregulation of cAMP production and PKA activation, both directly and through interplay with β-adrenergic receptors (β-AR). Muscarinic and nicotinic cholinergic receptors can also recruit β-arrestins, which mediate receptor internalization. Created with BioRender.com.

Figure 3

Roles of muscarinic signaling in GI cancer. Gastric and colon cancer cells produce non-neuronal ACh, which is implicated in autocrine and paracrine signaling. In gastric adenocarcinoma, M₃R is overexpressed relative to normal gastric epithelial cells, and M₃R activation stimulates gastric cancer progression and metastasis via an EGFR/MAP/ERK-dependent process. In pancreatic ductal adenocarcinoma (PDAC), M₃R is likewise overexpressed and associated with both cancer initiation and progression. Conversely, M₁R expression is downregulated in PDAC, and opposes the carcinogenic roles of M₃R both directly via inhibition of EGFR transactivation, and indirectly via inhibition of the cancer stem cell compartment. In colon cancer, activation of M₃R, either by ACh or bile acids, promotes expansion of the cancer stem cell compartment via Wnt signaling and promotes tumor invasion and growth by upregulating matrix metalloproteinase (MMP) expression. M₁R expression and activity are downregulated in colon cancer and M₁R signaling is associated with reduced colon cancer cell proliferation by currently unknown mechanisms. Created with BioRender.com.