Blockage of Cholinergic Signaling via Muscarinic Acetylcholine Receptor 3 Inhibits Tumor Growth in Human Colorectal Adenocarcinoma

Abstract

Cholinergic signaling via the muscarinic M3 acetylcholine receptor (M3R) is involved in the development and progression of colorectal cancer (CRC). The present study aimed to analyze the blocking of M3R signaling in CRC using darifenacin, a selective M3R antagonist. Darifenacin effects were studied on HT-29 and SW480 CRC cells using MTT and BrdU assays, Western blotting and real time RT-PCR. In vivo, blocking of M3R was assessed in an orthotopic CRC xenograft BALB/cnu/nu mouse model. M3R expression in clinical tumor specimens was studied by immunohistochemistry on a tissue microarray of 585 CRC patients. In vitro, darifenacin decreased tumor cell survival and proliferation in a dose-dependent manner. Acetylcholine-induced p38, ERK1/2 and Akt signaling, and MMP-1 mRNA expression were decreased by darifenacin, as well as matrigel invasion of tumor cells. In mice, darifenacin reduced primary tumor volume and weight (p < 0.05), as well as liver metastases, compared to controls. High expression scores of M3R were found on 89.2% of clinical CRC samples and correlated with infiltrative tumor border and non-mucinous histology (p < 0.05). In conclusion, darifenacin inhibited components of tumor growth and progression in vitro and reduced tumor growth in vivo. Its target, M3R, was expressed on the majority of CRC. Thus, repurposing darifenacin may be an attractive addition to systemic tumor therapy in CRC patients expressing M3R.

Potential Role for Combined Subtype-Selective Targeting of M1 and M3 Muscarinic Receptors in Gastrointestinal and Liver Diseases

“…Such agents with potential for oncotherapy continue to be developed. For example, the M 3 R-specific antagonist darifenacin which is approved to treat bladder dysfunction ( Yamada et al, 2006 ) reportedly inhibits tumor progression and invasiveness in human-derived cell lines, most recently in colorectal cancer cell lines ( Hering et al, 2021 ). As darifenacin is in clinical use with a known safety profile, it is an attractive candidate for adjunctive therapy, especially for cancers already shown to overexpress M 3 R, like colon cancer cells ( Frucht et al, 1999 ; Cheng et al, 2014 ), PDAC ( Zhang et al, 2016 ), and non-small cell lung cancer ( Lin et al, 2014 ).…”

Differential Actions of Muscarinic Receptor Subtypes in Gastric, Pancreatic, and Colon Cancer

“…M 3 R activation and post-receptor signaling is instrumental to colon cancer initiation, invasion, and metastasis. M 3 R is overexpressed in colon cancer, a finding that correlates with poor prognostic features including increased tumor burden, invasion, and metastasis [ 62 , 101 , 102 ]. M 3 R signal transduction, via the EGFR/ERK and PKC/p38 MAPK pathways, results in the induction and release of selected matrix metalloproteinases (MMP1, MMP7, and MMP10), collagenases that facilitate cell invasion by breaking down the extracellular matrix [ 103 , 104 ].…”

“…Notably, this may open the door to a therapeutic strategy that blocks M 3 R expression/activation while enhancing M 1 R expression/activation to prevent or treat colon neoplasia. In this respect, initial findings in an orthotopic xenograft mouse model suggest that darifenacin, a selective M 3 R antagonist approved in the United States, Canada, and the European Union to treat urinary incontinence, could be repurposed to attenuate the growth of M 3 R-expressing colon cancers [ 62 ]. In vitro, darifenacin also appeared to suppress ACh-stimulated colon cancer cell invasion, with a corresponding suppression of MMP1 mRNA expression, presumably via inhibition of p38, ERK1/2, and Akt signaling [ 62 ].…”

“…In this respect, initial findings in an orthotopic xenograft mouse model suggest that darifenacin, a selective M 3 R antagonist approved in the United States, Canada, and the European Union to treat urinary incontinence, could be repurposed to attenuate the growth of M 3 R-expressing colon cancers [ 62 ]. In vitro, darifenacin also appeared to suppress ACh-stimulated colon cancer cell invasion, with a corresponding suppression of MMP1 mRNA expression, presumably via inhibition of p38, ERK1/2, and Akt signaling [ 62 ]. Whether this therapeutic approach prevents or attenuates metastatic disease in mice remains to be determined; current studies were underpowered to answer these questions [ 62 ].…”

“…In vitro, darifenacin also appeared to suppress ACh-stimulated colon cancer cell invasion, with a corresponding suppression of MMP1 mRNA expression, presumably via inhibition of p38, ERK1/2, and Akt signaling [ 62 ]. Whether this therapeutic approach prevents or attenuates metastatic disease in mice remains to be determined; current studies were underpowered to answer these questions [ 62 ]. Nonetheless, the results of these exploratory preclinical studies appear promising and suggest that clinical trials of FDA-approved M 3 R-selective antagonists are warranted.…”