Klotho (KL) is a transmembrane protein that can be shed, and act as a circulating hormone and modulate several signaling pathways. There also exists a splice variant of Klotho mRNA, which encodes a putative secreted protein (Klotho-S, KL-S) in both human and mouse. The potential anti-senescence gene Klotho has been recently found to participate in the progression of several different human cancers. In the current study, we undertook to study the expression and activity of Klotho in lung cancer cell line A549. Klotho expression was studied by using RT-PCR and western blotting. Effects of Klotho on cell growth and motility were assessed using MTT and scratch motility assay, and the apoptosis was assessed by TUNEL. Wnt signaling pathway activity was measured by western blotting. We established that the Klotho was endogenous expressed in A549 cells, but the expression level is lower compared with normal lung tissues. The overexpression of KL or KL-S could inhibit the cell proliferation, motility, and induce apoptosis in a dose-dependent manner. Also, we report KL could inhibit activation of Wnt -TCF/β-catenin signaling pathway, and it is involved in KL-induced growth inhibition. These studies indicate Klotho works as a potential tumor suppressor in lung cancer, and suggest that the Klotho tumor suppressive activities could be mediated through its KL-S isoform. These results suggest the use of Klotho or KL-S as potential strategy for the development of novel therapeutic interventions for lung cancers.

Gastric cancer incidence demonstrates a strong etiologic association with smoking. Nicotine, the major component in tobacco, is a survival agonist that inhibits apoptosis induced by certain chemotherapeutic agents, but the precise mechanisms involved remain largely unknown. Recently studies have indicated that α5-nicotinic acetylcholine receptor (α5-nAChR) is highly associated with lung cancer risk and nicotine dependence. Nevertheless, no information has been available about whether nicotine also affects proliferation of human gastric cancer cells through regulation of α5-nAChR. To evaluate the hypothesis that α5-nAChR may play a role in gastric cancer, we investigated its expression in gastric cancer tissues and cell lines. The expression of α5-nAChR increased in gastric cancer tissue compared with para-carcinoma tissues. In view of the results, we proceeded to investigate whether nicotine inhibits cisplatin-induced apoptosis via regulating α5-nAChR in gastric cancer cell. The results showed that nicotine significantly promoted cell proliferation in a dose and time-dependent manner through α5-nAChR activation in human gastric cells. Furthermore, nicotine inhibited apoptosis induced by cisplatin. Silence of α5-nAChR ablated the protective effects of nicotine. However, when co-administrating LY294002, an inhibitor of PI3K/AKT pathway, an increased apoptosis was observed. This effect correlated with the induction of Bcl-2, Bax, Survivin and Caspase-3 by nicotine in gastric cell lines. These results suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that α5-nAChR/AKT signaling plays a key role in the anti-apoptotic activity of nicotine induced by cisplatin.

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 CHRNΑ5 encoding α5-nicotinic acetylcholine receptor (α5-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 α5-nAChR mediates lung adenocarcinoma (LUAD) epithelial-mesenchymal transition (EMT) and metastasis via STAT3/Jab1. However, the link between α5-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 α5-nAChR and PD-L1 were detected by immunohistochemistry in a tissue microarray. α5-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-γ, 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 α5-nAChR-mediated immune escape via PD-L1 pathway. Results: The expression of α5-nAChR was correlated with PD-L1 expression, smoking status and lower survival of LUAD in vivo. In vitro, the expression of α5-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 α5-nAChR suppressed the function of CD4⁺CD25⁺FOXP3⁺ Tregs, enhanced IFN-γ secretion, and increased T cell-mediated killing of LUAD cells. In the Jurkat T cells and LUAD cells coculture assay, inhibition of α5-nAChR increased IL-2 secretion. In tumour xenograft tissues, α5-nAChR expression was related to PD-L1, Jab1, pSTAT3, CD4 and granzyme B expression (GB). Conclusions: Our results suggest that the novel α5-nAChR/STAT3-Jab1-PD-L1 axis is involved in LUAD immune escape, which could lead to potential therapeutic strategies for cancer immunotherapy.

Nicotine and nicotinic acetylcholine receptors (nAChRs) are considered to be involved in lung cancer risk, onset and progression, but their precise physiological roles in these contexts remain unclear. Our previous studies suggested that α5-nAChR mediates nicotine-induced lung cancer cell proliferation, migration and invasion in vitro. In this study, we aimed to determine the role of α5-nAChR in the development and progression of non-small cell lung cancer (NSCLC). Our microarray results reveal that knockdown of the CHRNA5 gene encoding α5-nAChR significantly modulates key pathways including the cell cycle, DNA replication, pathway in cancer. α5-nAChR knockdown in cultured A549 cells affected cell cycle distribution, apoptosis, and cyclin expression. In vivo, α5-nAChR silencing inhibited the growth of lung tumors, especially in the context of nicotine exposure. Importantly, α5-nAChR expression in patient tumors correlated with the primary T stage, N stage and reduced survival time. These results reveal that α5-nAChR silencing inhibits the progression of nicotine-related NSCLC, making this receptor a potential pharmacological target for the treatment of nicotine-related lung carcinogenesis.