Cholinergic signals promote esophageal squamous cell carcinoma metastasis by activating EGFR/AKT signaling pathway

Abstract Purpose Increasing evidence has shown that the nervous system plays a vital role in the regulation of tumorigenesis. However, the regulatory mechanism of the parasympathetic nervous system in the progression of esophageal squamous cell carcinoma (ESCC) has not been elucidated. Methods Vesicular acetylcholine transporter (VAChT) protein expression was detected by immunohistochemistry (IHC) and parasympathetic nerve density was analyzed in human ESCC tissues. The co-culture model of ESCC cells and dorsal root ganglia (DRG) was established in vitro. The effects of cholinergic stimulation on cell growth, migration and invasion were investigated through CCK-8, Transwell and wound healing assays using acetylcholine (ACh). PCR and western blotting were utilized respectively for the purpose of detecting Epithelial-to-mesenchymal transition (EMT) related transcripts proteins. M3 muscarinic receptor (CHRM3) was knocked down using small interfering RNA (siRNA). RNA sequencing explored the possible altered gene transcriptome and signal pathways, which were further verified by Western blotting and CO-IP analysis. Results The intensity of parasympathetic nerve in ESCC was significantly higher than that in adjacent tissues, and was positively correlated with higher clinical staging and poorer prognosis. Co-incubation with DRG enhanced the migration and invasion ability of ESCC cells in vitro. Moreover, exogenous choline stimulation enhanced the migration, invasion ability together with EMT of ESCC cells, while CHRM3 knockdown were opposite. Mechanistically, cholinergic activation enhanced the phosphorylation levels of EGFR and AKT through CHRM3, which bound directly to EGFR.Conclusion We revealed a critical role of cholinergic signals ACh/CHRM3 in mediating the effects of parasympathetic nerve activity on ESCC through the EGFR/AKT signaling pathway, which might provide a novel therapeutic target for ESCC.