Digoxin And Angiotensin Ⅱ Contribute To The Formation Of NSCLC Immunosuppressive Microenvironment And The Underlying Mechanisms

In recent years,immunotherapies targeting immune checkpoints PD-1/PD-L1 have made breakthrough progress in the clinical application of advanced non-small cell lung cancer(NSCLC)treatment.It has been approved by the FDA for first-line treatment of advanced lung cancer.However,only about 20%of patients with advanced NSCLC have an effective response to PD-1/PD-L1 inhibitors.The main cause of this phenomenon is the formation of tumor immunosuppressive microenvironment.Therefore,exploring new factors and mechanisms that promote the formation of tumor immunosuppressive microenvironment will help to better improve the body’s immune recognition and killing ability.Digoxin and AngiotensinⅡ(AngⅡ)both play important roles in the cardiovascular system.Digoxin is a strong cardiac glycoside drug,which has a variety of biological functions such as enhancing myocardial contractility and inhibiting Na+/K+-ATPase.AngⅡ is the main active substance of renin-angiotensin system(RAS)and has important physiological functions such as regulating the balance of water and salt metabolism and blood pressure.The role of digoxin and AngⅡ in cardiovascular diseases has been extensively studied,but the role in tumor immunity has been reported relatively little.Therefore,studying the role of digoxin and AngⅡ in tumor immunity has important clinical significance for discovering new biological functions of digoxin and AngⅡand expanding the beneficiary population of immunotherapy.In this study,a mouse model of lung adenocarcinoma with LLC was established.Digoxin or AngⅡ were injected intraperitoneally for 13 and 15 days,respectively.It was found that digoxin and AngⅡ could significantly promote the growth of tumor and increase the weight of tumor in LLC-bearing lung adenocarcinoma mice.Interestingly,both digoxin and AngⅡ could reduce the infiltration of intratumoral CD4+T and CD8+T lymphocytes,increase the accumulation of tumor-associated macrophages(TAMs)cells,and AngⅡ could also increase the aggregation of immunosuppressive granulocytes.In addition,both digoxin and AngⅡ could up-regulate mRNA levels of tumor immunosuppression microenvironment associated genes.The above results indicated that both digoxin and AngⅡ induced the formation of immunosuppressive microenvironment in non-small cell lung cancer.Programmed death ligand 1(PD-L1)is highly expressed in the tumor microenvironment.It is one of the main molecular mechanisms of tumor immune escape.It has been previously demonstrated that digoxin and AngⅡ can promote the formation of immunosuppressive microenvironment in non-small cell lung cancer,so it is worth studying whether PD-L1 is involved in this process.In this study,digoxin and AngⅡ not only induced up-regulation of PD-L1 protein expression in mouse lung adenocarcinoma tumor tissues in vivo,but also increased PD-L1 expression in non-small cell lung cancer cells in vitro,including LLC,H1975 and human lung adenocarcinoma primary cells LAC275.The above results suggested that PD-L1 may participate in digoxin and AngⅡ to promote the formation of immunosuppressive microenvironment in non-small cell lung cancer.In order to further study the molecular mechanism of PD-L1 expression induced by digoxin and AngⅡ,experimental methods such as mRNA half-life and luciferase reporter gene were used.This result showed that digoxin and AngⅡ extended the half-life of PD-L1 mRNA and enhanced activity of the PD-L1 3’-untranslated region(3’-UTR)reporter gene.Meanwhile,this result suggested that the expression of PD-L1 induced by digoxin and AngⅡ may occur at the post-transcriptional level,and this process may be related to the stability of mRNA.Since the AU-rich element(ARE)interacts with the corresponding RNA-binding protein,it can regulate the stability of mRNA,thereby achieving the control of gene expression at the post-transcriptional level.The results of this study found that the RNA binding protein human antigen R(HuR)binded to the ARE sequence AUUUA in PD-L1 3’-UTR,and digoxin and AngⅡ could up-regulate HuR mRNA expression.The above results suggested that the induction of PD-L1 mRNA expression by digoxin and AngⅡ may be related to the promotion of the binding of ARE in PD-L1 3’-UTR and HuR and thus increasing the stability of PD-L1 mRNA.Full text summary:digoxin and AngⅡ promoted the formation of immunosuppressive microenvironment in non-small cell lung cancer by inducing the expression of PD-L1.The molecular mechanism may be caused by the enhanced binding of HuR and PD-L1 3’-UTR,thus mediating the stability of mRNA.Here are the innovations of this study.Firstly,It was clear that digoxin and AngⅡpromoted the formation of tumor immunosuppressive microenvironment.Secondly,digoxin and AngⅡ regulated the mRNA expression of PD-L1 through ARE.Thirdly,HuR positively regulated the mRNA expression of PD-L1.Collectively,this study confirms the role of digoxin and AngⅡ in the immune escape of NSCLC,which has important clinical significance for discovering new biological functions of digoxin and AngⅡ and expanding the benefit of immunotherapy.