Selective Activation of M1 Muscarinic Receptors Attenuates Human Colon Cancer Cell Proliferation

Abstract

M3 muscarinic receptor (M3R) activation stimulates colon cancer cell proliferation, migration, and invasion; M3R expression is augmented in colon cancer and ablating M3R expression in mice attenuates colon neoplasia. Several lines of investigation suggest that in contrast to these pro-neoplastic effects of M3R, M1R plays an opposite role, protecting colon epithelial cells against neoplastic transformation. To pursue these intriguing findings, we examined the relative expression of M1R versus M3R in progressive stages of colon neoplasia and the effect of treating colon cancer cells with selective M1R agonists. We detected divergent expression of M1R and M3R in progressive colon neoplasia, from aberrant crypt foci to adenomas, primary colon cancers, and colon cancer metastases. Treating three human colon cancer cell lines with two selective M1R agonists, we found that in contrast to the effects of M3R activation, selective activation of M1R reversibly inhibited cell proliferation. Moreover, these effects were diminished by pre-incubating cells with a selective M1R inhibitor. Mechanistic insights were gained using selective chemical inhibitors of post-muscarinic receptor signaling molecules and immunoblotting to demonstrate M1R-dependent changes in the activation (phosphorylation) of key downstream kinases, EGFR, ERK1/2, and p38 MAPK. We did not detect a role for drug toxicity, cellular senescence, or apoptosis in mediating M1R agonist-induced attenuated cell proliferation. Lastly, adding M1R-selective agonists to colon cancer cells augmented the anti-proliferative effects of conventional chemotherapeutic agents. Collectively, these results suggest that selective M1R agonism for advanced colon cancer, alone or in combination with conventional chemotherapy, is a therapeutic strategy worth exploring.

Cholinergic Mechanisms in Gastrointestinal Neoplasia

“…In vitro, McN-A-343 and xanomeline dose-dependently inhibited colon cancer cell proliferation, an effect reversed by pre-treating cells with a selective M 1 R inhibitor [163]. Moreover, adding McN-A-343 to standard colon cancer chemotherapy, namely 5-fluorouracil and oxaliplatin, potentiated their anti-proliferative effects on colon cancer cells [163].…”

“…M 3 R blockade in human colon cancer cell lines counteracted ACh-induced ERK1/2, Akt, and MMP-1 signaling, consequently attenuating cell proliferation and invasion [149]. Further, M 1 R expression is downregulated in human CRC tissue and may protect colon cells against neoplastic transformation [163]. In human colon cancer cell lines, Sundel et al demonstrated that M 1 R agonism inhibits cell proliferation and augments the effects of conventional chemotherapeutic agents [163].…”

“…Further, M 1 R expression is downregulated in human CRC tissue and may protect colon cells against neoplastic transformation [163]. In human colon cancer cell lines, Sundel et al demonstrated that M 1 R agonism inhibits cell proliferation and augments the effects of conventional chemotherapeutic agents [163]. Likewise, α7nAchR antagonism reverses the stimulatory actions of nicotine on colon cancer cell proliferation [156].…”

“…Drugs targeting M 1 R and M 3 R were shown to impact GI cancer progression (Table 2). Whereas non-subtype-selective muscarinic agonists such as carbamylcholine (carbachol) and bethanechol stimulate cancer cell proliferation [138,141,148,163], M 3 R antagonists attenuate cancer proliferation in vitro and in vivo.…”

“…McN-A-343 and xanomeline, selective M 1 R agonists studied in the context of GI neoplasia, were developed decades ago, McN-A-343 in 1961 [170] and xanomeline in 1997 [171]. In vitro, McN-A-343 and xanomeline dose-dependently inhibited colon cancer cell proliferation, an effect reversed by pre-treating cells with a selective M 1 R inhibitor [163]. Moreover, adding McN-A-343 to standard colon cancer chemotherapy, namely 5-fluorouracil and oxaliplatin, potentiated their anti-proliferative effects on colon cancer cells [163].…”