Liting Lan1, Hua Wang2, Rui Yang3, Fengqi Liu4, Qingshang Bi4, Shiqi Wang5, Xiaoli Wei1, Haitao Yan6, Ruibin Su7. 1. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China. 2. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China; 69242 Force Health Center, No. 1, Hongxing Road, Turpan, Xinjiang 838000, China. 3. National Institutes for Food and Drug Control, No. 31, Huatuo Road, Daxing District, Beijing 102629, China. 4. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China; Department of Medical Laboratory, Changzhi Medical College, No.161 Jiefang East Street, Changzhi, Shanxi, 046000, China. 5. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China; Center for Drug Evaluation, NMPA. No. 128, Jianguo Road, Chaoyang District, Beijing 100022, China. 6. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China. Electronic address: yanht7809@aliyun.com. 7. State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, 27th Taiping Road, Beijing 100850, China. Electronic address: ruibinsu@126.com.
Abstract
AIMS: The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor that is expressed in cases of non-small cell lung cancer (NSCLC). Previous studies demonstrated that M3R antagonists reduce the proliferation of NSCLC. However, how antagonists inhibit the NSCLC proliferation and migration is still little known. This study aims to investigate the mechanism of M3R involved in the growth of NSCLC. MAIN METHODS: The CRISPR/Cas9 was used to knock out (KO) the M3R gene. A real-time cell analyzer (RTCA) was used to record the proliferation of NSCLC cells. The migration and cell cycle of NSCLC cells were evaluated with scratch test and flow cytometry (FCM), respectively. Antibody microarray analysis was performed to detect the expression of proteins after antagonizing M3R and knocking out of M3R, subsequently some of these important proteins were verified by western blot. KEY FINDINGS: The proliferation and migration of NSCLC cells were inhibited by M3R antagonist R2-8018 and knocking out of M3R. Antagonism or knocking out of M3R reduced the phosphorylation of EGFR. Moreover, c-Src and β-arrestin-1 are involved in the mechanism of how the inhibition of M3R affects EGFR in NSCLC. Further study demonstrated that PI3K/AKT and MEK/ERK signal pathways are involved in M3R-induced EGFR transactivation in NSCLC, and the molecules involved in the cell cycle progression and migration of NSCLC cells were identified. SIGNIFICANCE: This further understanding of the relationship between M3R and NSCLC facilitates the design of therapeutic strategy with M3R antagonist as an adjuvant drug for NSCLC treatment.
AIMS: The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor that is expressed in cases of non-small cell lung cancer (NSCLC). Previous studies demonstrated that M3R antagonists reduce the proliferation of NSCLC. However, how antagonists inhibit the NSCLC proliferation and migration is still little known. This study aims to investigate the mechanism of M3R involved in the growth of NSCLC. MAIN METHODS: The CRISPR/Cas9 was used to knock out (KO) the M3R gene. A real-time cell analyzer (RTCA) was used to record the proliferation of NSCLC cells. The migration and cell cycle of NSCLC cells were evaluated with scratch test and flow cytometry (FCM), respectively. Antibody microarray analysis was performed to detect the expression of proteins after antagonizing M3R and knocking out of M3R, subsequently some of these important proteins were verified by western blot. KEY FINDINGS: The proliferation and migration of NSCLC cells were inhibited by M3R antagonist R2-8018 and knocking out of M3R. Antagonism or knocking out of M3R reduced the phosphorylation of EGFR. Moreover, c-Src and β-arrestin-1 are involved in the mechanism of how the inhibition of M3R affects EGFR in NSCLC. Further study demonstrated that PI3K/AKT and MEK/ERK signal pathways are involved in M3R-induced EGFR transactivation in NSCLC, and the molecules involved in the cell cycle progression and migration of NSCLC cells were identified. SIGNIFICANCE: This further understanding of the relationship between M3R and NSCLC facilitates the design of therapeutic strategy with M3R antagonist as an adjuvant drug for NSCLC treatment.
Authors: Gloria M Calaf; Leodan A Crispin; Juan P Muñoz; Francisco Aguayo; Tammy C Bleak Journal: Cancers (Basel) Date: 2022-05-07 Impact factor: 6.575