The Role Of M2 MAChR In The Malignant Phenotype Of NSCLC And The Underlying Signaling Pathway

Cholinergic receptors(AChRs) play crucial roles in mediating signaling of neurotransmitter acetylcholine(ACh). Recently, increasing evidence has shown that cholinergic components exist in various non-neuronal cell types. Non-neuronal ACh can act as an autocrine or paracrine growth factor to modulate basic biology of various cells through both muscarinic acetylcholine receptors(mAChRs) and nicotinic acetylcholine receptors(nAChRs). It’s of great interest whether and how ACh in tumor microenvironment regulates the malignant phenotype of lung cancer. Nicotinic AChRs belong to ligand-gated ion channels, while muscarinic AChRs(M1-M5) belong to the G-Protein Coupled Receptors(GPCR) superfamily. M1, 3, 5 mAChR subtypes are coupled to Gq-type G-proteins, while M2, 4 mAChR subtypes are linked to G-proteins of the Gi/Go family. It is very important to understand the ACh signaling network that regulates the biology of lung cancer.Cigarette smoking is the high risk factor for lung cancer. It has been demonstrated that nicotine,a main component of cigarettes, contributes directly to lung cancer malignant phenotype through activating nAChRs. There are some evidence that tumor cells growth is associated with stimulation of M3 receptors. However, little is known about the role of M2 mAChR in the malignant phenotype of NSCLC and the underlying signaling pathways. On the other side, targeted therapy make great progress in the treatment of lung cancer and meet a bottleneck of drug resistance as well. Cancer stem cells may have close association with the occurrence of drug resistance. The roles of ACh receptors in the development of drug-resistance and the regulation of cancer stem cells need to be elucidated.Firstly, we examined the expression of M1-M5 mAChR in NSCLC cells(A549 and PC9 cells) and evaluated the role of selective antagonists of M1/M2/ M3 mAChR on proliferation and the underlying mechanism. Blocking M2 muscarinic receptor(mAChR) signaling using selective M2 mAChR antagonist methoctramine or short hairpin RNA(shRNA) inhibited NSCLC PC9 and A549 cell proliferation and decreased the phosphorylation of MAPK and AKT. Furthermore, treatment with methoctramine(MT) inhibited A549 xenograft tumor growth in vivo and also decreased phosphorylation of MAPK and Akt in xenograft tumors in nude mice.Secondly,MT and M2 mAChR shRNA inhibited EMT, migration, invasion of NSCLC PC9 and A549 cells and decreased the phosphorylation of p65 NF-kB as well as expression of snail and zeb1. Moreover, MT inhibited EMT of A549 xenograft tumor and M2 mAChR shRNA reduced the metastasis nodules in lung.At last, MT reversed EMT of PC9-GR cells(PC9 cells with resistance to EGFR TKI gefitinib). M2 mAChR was significantly increased in accordance with EMT phenotype in CD221+-SPC-A1 cells(NSCLC stem cells).Our results show that blocking M2 mAChR can inhibit NSCLC cells growth, EMT, migration and invasion both in vitro and in vivo. The underling mechanisms include decreased phosphorylation of MAPK, Akt and NF-kB. We also demonstrate that M2 mAChR antagonist reverses EMT under basal and muscarinic agonist pilocarpine-induced conditions in gefitinib-resistant NSCLC cells. Moreover, we find that M2 mAChR may be associated in maintaining the stem-like phenotype of NSCLC stem cells.To our knowledge, this is the first study to investigate the regulatory effects of M2 mAChR on NSCLC cells, NSCLC TKI-resistant cells and NSCLC stem cells, as well as the underlying mechanisms. These findings provide important insights into developing M2 mAChR selective antagonists as potential approach of lung cancer treatment.