Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells

Abstract

Purpose: G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84. Methods: Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 lM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined. Results: Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immuno-precipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth. Conclusions: Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.

Muscarinic Receptor Agonists and Antagonists: Effects on Cancer

“…Follow-up studies by Frucht and co-workers (Frucht et al 1999; Yang and Frucht 2000) demonstrated that the receptors were primarily M3, were increased approximately eightfold in tumor versus normal, and that carbamylcholine stimulated proliferation of colon carcinoma cell lines expressing M3 receptors. Raufman et al (2003) and Ukegawa et al (2003) confirmed those findings, again showing the importance of M3 receptors and also demonstrated that the proliferative action of M3 receptors depended in part on the transactivation of EGF receptors. The actual role of M3 receptors in colon cancer development was further confirmed by Raufman et al (2008) who showed that M3 receptor knockout mice were resistant to the development of colon tumors in the azoxymethane-induced colon neoplasia model.…”

Divergent effects of muscarinic receptor subtype gene ablation on murine colon tumorigenesis reveals association of M3R and zinc finger protein 277 expression in colon neoplasia

“…Whereas work from our [19] and other groups [2-4,7,8] provide strong evidence that M3R acts as a tumor promoter, the present observations newly suggest that in the colon M1R acts as a tumor suppressor. This putative role for M1R is unmasked by combined M1R and M3R deficiency in dual KO mice where tumor formation is similar to that observed in WT mice.…”

“…Activation of muscarinic receptors and downstream signaling was shown to stimulate proliferation of cells derived from lung [1,2], breast [3,4], prostate [5], colon [6,7], and skin [8] cancers, and muscarinic receptors are frequently over-expressed in these common cancers [9]. Hence, it is highly likely that activation of muscarinic receptor signaling plays a fundamentally important role in neoplastic transformation and progression.…”

Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia

“…Frucht et al (1999) demonstrated that the M3 expression was increased about eight-fold in tumor versus normal tissue, and that the cholinergic agonist carbamylcholine stimulated the proliferation of M3-expressing colon carcinoma cell lines (Yang and Frucht, 2000). These findings were confirmed by other research groups, who demonstrated that the proliferative action of the M3 receptors depended partially on the trans- activation of epithelial growth factor (EGF) receptors (Raufman et al, 2003; Ukegawa et al, 2003). Later, it was shown that the M3 receptor knockout mice were resistant to the development of colon tumors in the azoxymethane-induced colon neoplasia model (Raufman et al, 2008).…”