Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling

Background: Immunotherapy has proven to be an emerging treatment for non-small-cell lung cancer in recent years. Notably, smokers show higher programmed cell death ligand-1 (PD-L1) expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Genome-wide association studies show that the CHRNA5 encoding alpha 5-nicotinic acetylcholine receptor (alpha 5-nAChR) is especially relevant to lung cancer and nicotine dependence. Jab1 is a key regulatory factor and promotes the stabilization of PD-L1. Our previous study reported that alpha 5-nAChR mediates lung adenocarcinoma (LUAD) epithelial-mesenchymal transition (EMT) and metastasis via STAT3/Jab1. However, the link between alpha 5-nAChR and PD-L1 is unclear in LUAD. Methods: We used various bioinformatics databases to analyze the expression of related genes and their correlations. Expression and clinicopathologic significance of alpha 5-nAChR and PD-L1 were detected by immunohistochemistry in a tissue microarray. alpha 5-nAChR regulated LUAD cell immune escape by targeting the STAT3/Jab1-PD-L1 signalling by Western-blotting and ChIP in vitro. We used T cell coculture, flow cytometry, ELISA, CCK8 assay and crystal violet staining to detect the expression of regulatory T cell (Tregs), IFN-gamma, IL-2 and the ability of T cell-mediated tumour cell killing respectively. IF assays were performed in both cancer cells and tumour xenograft paraffin sections to analyze the protein expression. The in vivo experiments in mouse model were performed to show the alpha 5-nAChR-mediated immune escape via PD-L1 pathway. Results: The expression of alpha 5-nAChR was correlated with PD-L1 expression, smoking status and lower survival of LUAD in vivo. In vitro, the expression of alpha 5-nAChR mediated phosphorylated STAT3 (pSTAT3), Jab1 and PD-L1 expression. STAT3 bound to the Jab1 or PD-L1 promoter and mediated PD-L1 expression. Jab1 stabilized PD-L1 expression in LUAD cells. Furthermore, in primary T cell cocultured system, downregulation of alpha 5-nAChR suppressed the function of CD4(+)CD25(+)FOXP3(+) Tregs, enhanced IFN-gamma secretion, and increased T cell-mediated killing of LUAD cells. In the Jurkat T cells and LUAD cells coculture assay, inhibition of alpha 5-nAChR increased IL-2 secretion. In tumour xenograft tissues, alpha 5-nAChR expression was related to PD-L1, Jab1, pSTAT3, CD4 and granzyme B expression (GB). Conclusions: Our results suggest that the novel alpha 5-nAChR/STAT3-Jab1-PD-L1 axis is involved in LUAD immune escape, which could lead to potential therapeutic strategies for cancer immunotherapy.

Automated summary

This study reveals that the alpha 5-nAChR/STAT3-Jab1-PD-L1 axis is involved in LUAD immune escape, which could provide potential therapeutic strategies for cancer immunotherapy.