Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

Abstract

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.

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

“…Such cross talk may limit the benefits of currently approved EGFR-targeted biological (3,19,39). For example, in mouse models of colon cancer and several human colon cancer cell lines including H508 cells used in this study, we showed that cholinergic muscarinic receptors cross talk with EGFR to stimulate cell proliferation, tumor growth, and invasion (2,7,9,44).…”

“…(34), we hypothesized that similar interactions occur in intestinal epi- thelial cells. We used two human colon cancer epithelial cell lines, H508 and SNU-C4, that express high levels of EGFR and exhibit EGFR-mediated interactions with other signaling mechanisms (2,7) to test the effects of a variety of AhR agonists on colon cancer cell proliferation. AhR ligands included environmental contaminants (MC and TCDD) and natural ligands (indirubin, I3C, and DIM).…”

HT29 Cell Line

“…It is also remarkable that when using cell cultures, the expression of a particular receptor may depend on the degree of cell differentiation and the growing conditions. Recently, the presence of opioid, serotonin, muscarinic, PPARβ/δ receptors has been reported in this cell line (Zoghbi et al 2006 ;Ataee et al 2010 ;Belo et al 2011 ;Foreman et al 2011 ). Information on the studies on receptors carried out in this cell line can be found at Zweibaum et al ( 2011 ).…”

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells

“…It was previously described that mAChR activation stimulates migration and invasion of human colonic cancer cells 25,67 . In these cases, the transactivation of EGFR mediates the effect of M3 mAChR.…”

“…ACh binding to M3 mAChR results in MMP activation (MMP1 or MMP7) that releases EGFR ligand named HBEGF 43,68 . The downstream signaling involves ERK and PI3K dependent rapid RhoA activation as well as FAK activation 67,69 . We also found that…”