Most colon cancers overexpress M 3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M 3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by ϳ40% (P Ͻ 0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5-and 2-fold, respectively (P Ͻ 0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n ϭ 25) whereas half of colon cancer specimens (n ϭ 24) exhibited moderate to strong staining (P Ͻ 0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation. autocrine signaling; choline acetyltransferase; muscarinic receptors FOR BOTH MEN AND WOMEN IN the United States colon cancer is a common, frequently lethal disease. Although endoscopic or surgical cancer resection is highly successful in treating early disease, advanced colon cancer responds poorly to surgery, chemotherapy, and radiation, thereby accounting for 30% mortality. A fundamental tenet of cancer biology is that elucidating mechanisms underlying neoplastic cell proliferation will identify therapeutic targets. Hence, identifying colon cancer growth factors and growth factor receptors is a prominent research goal. Nonetheless, although it was recognized more than 15 years ago that muscarinic receptor signaling stimulates colon cancer cell proliferation (9) and that most colon cancers overexpress M 3 muscarinic receptors (M 3 R) (29), muscarinic receptor ligands and receptors have been neglected as potential therapeutic targets.Postmuscarinic receptor signaling has long been recognized as integral to gastrointestinal physiology. Cholinergic nerve endings are present in colonic mucosa and muscarinic signaling, particularly via M 3 R, plays a prominent role in mediating intestinal epithelial fluid and electrolyte transport (10,26,28,30). Muscarinic receptor ...

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ously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose-and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-B activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation. epidermal growth factor receptor; gene transcription; heparin-binding epidermal growth factor-like growth factor COLON CANCER, A COMMON and frequently lethal disease, progresses in a multistep process that involves tumor initiation and promotion. Therapeutic strategies directed at interfering with tumor promotion are of particular interest, because this is generally a reversible, long-term process (13). Hence, there is great interest in identifying key colon cancer growth factors and their receptors. Increasing evidence indicates that muscarinic receptors and ligands play key roles in intestinal neoplasia (8,9,14,15,17,27). The muscarinic cholinergic family of G proteincoupled receptors (GPCRs) consists of five muscarinic receptor subtypes designated M 1 R, M 2 R, M 3 R, M 4 R, and M 5 R (for reviews see Refs. 34 and 35). Using RT-PCR with primers specific for muscarinic receptor subtypes, Frucht and colleagues (14, 15) reported that 60% of colon cancer cell lines tested express M 3 R. Follow-up studies revealed that normal colon epithelial cells uniformly express M 3 R, and in 62% of colon cancers, M 3 R expression was increased up to eightfold compared with adjacent normal colon epithelium (37).Work from our laboratory extended these observations by showing that cholinergic ligand-induced proliferation of H508 human colon cancer cells, which exp...

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.