Role Of Hypoxia-inducible Factor-1α In The Regulation Of Programmed Death Ligand 1 Expression In Lung Adenocarcinoma

Background and ObjectiveNon-small-cell lung cancer(NSCLC)is a common malignancy worldwide,accounting for about 85%of all lung cancer cases[1].The incidence of lung adenocarcinoma is increasing[2].Programmed death-1(PD-1)/programmed death ligand 1(PD-L1),an important immune checkpoint involved in tumor immune escape[3],has become the hotspot of NSCLC therapy research.Recently,monoclonal antibodies targeting PD-1/PD-L1 immune checkpoint,such as nivolumab,pembrolizumab and atezolizumab,have been demonstrated in a serial of clinical trials to have promising clinical effect in a fraction of patients with advanced NSCLC C[4-6],and have been approved by FDA as the first line therapy for NSCLC patients recently.However,there are still many problems need to be resolved:the majority of patients did not respond to the immune checkpoint inhibitor treatment;and the mechanism affecting curative effect has not been fully elucidated[7-8].Therefore,it is of great important clinical significance to study the PD-L1 expression characteristics and underlying regulatory mechanism,and their prognostic and predicative value in the PD-1/PD-L1 pathway blockade treatment in tumor[9].Hypoxic microenvironment is one of the important biological characteristics in solid tumor[10].Hypoxia-inducible factor-1(HIF-1)is a transcriptional factor existing widely in most solid tumors,including an oxygen-regulated hypoxia-inducible factor-la(HIF-1α)subunit and a constitutively expressed hypoxia-inducible factor-1β(HIF-1β)subunit.Thus,under hypoxic conditions,HIF-1 a is an important factor controlling a variety of gene expression to make the tumor cells adapt hypoxic conditions.In the past two years,some research have reported that HIF-la can upregulate the expression of PD-L1 in hypoxic conditions[11-14].However,whether PD-L1 expression is regulated by the hypoxic conditions and HIF-1α in lung adenocarcinoma remains unknown.This study is to investigate the expression correlation of HIF-1α and PD-L1 in lung adenocarcinoma,and to investigate the potential role of HIF-1α in the regulation of PD-L1 expression,thereby revealing an important mechanism of tumor immune escape induced by hypoxic conditions.Methods99 samples of lung adenocarcinoma cases in our hospital were examined for the expressions of HIF-la and PD-L1 by immunohistochemical method;Two NSCLC datasets:one consists of 230 patients with lung adenocarcinoma[15]and another of 178 patients with lung squamous cell carcinoma[16]from TCGA database were analyzed for the correlation between HIF-la and PD-L1 mRNA respectively;The lung adenocarcinoma cell line NCI-H1975 and HCC827 were treated with hypoxia mimic CoCl2 respectively and divided into Normal group and CoCl2 group.The mRNA and protein expressions of HIF-1α and PD-L1 were detected with qRT-PCR and Western blotting;The lung adenocarcinoma cell line NCI-H 1975 and HCC827 were transfected with siRNA-HIF-1α respectively and divided into NC group and siR-HIF-1α group.The transfection efficiency of siRNAs and the expression of PD-L1 were detected by qRT-PCR and Western blotting.Results1.From the results of immunohistochemisty,HIF-1α positive expression was located in nucleus stained with brown fine particles,while PD-L1 positive expression was located in cell membranes and(or)cytoplasm stained with claybank granule in lung adenocarcinoma samples.The positive expression rates of HIF-la and PD-L1 were 47.48%and 34.34%in 99 cases of lung adenocarcinoma respectively.2.The expression of HIF-la was correlated with age and clinical stage(P<0.05)and was not correlated with gender,smoking status,tumor size and lymphatic matastasis(P>0.05)in 99 cases of lung adenocarcinoma.3.The expression of PD-L1 was correlated with clinical stage(P<0.05)and was not correlated with age,gender,smoking status,tumor size and lymphatic matastasis(P>0.05)in 99 cases of lung adenocarcinoma.4.Spearman correlation analysis of the immunohistochemistry results in lung adenocarcinoma demonstrated expressions of HIF-la and PD-L1 protein were positively correlated(r=0.292,P<0.01).We also found a statistically significant positive correlation of HIF-la and PD-L1 mRNA in TCGA dataset of 230 patients with lung adenocarcinoma(r=0.467,P<0.001),further confirming the results of immunohistochemistry analysis;In contrast,in another TCGA dataset of 178 patients with lung squamous cell carcinomas there revealed no correlation of HIF-1α and PD-L1 mRNA(r=0.108,P>0.05).5.The lung adenocarcinoma cell line NCI-H1975 were treated with hypoxia mimic CoCl2 for 24 hours,compared with the Normal group,the protein expression of HIF-la significantly increased(P<0.05),and the mRNA and protein expressions of PD-L1 increased subsequently in the CoCl2 group(P<0.01);The lung adenocarcinoma cell line HCC827 were treated with hypoxia mimic CoCl2 for 24 hours,compared with the Normal group,the protein expression of HIF-1α significantly increased(P<0.05),and the mRNA and protein expressions of PD-L1 increased subsequently in the CoCl2 group(P<0.05).6.The lung adenocarcinoma cell line NCI-H1975 were transfected with siRNA-HIF-1αfor 48 hours,then detected transfection efficiency to validate down-regulate HIF-la expression.Further detected PD-L1 expression demonstrated that the mRNA and protein expressions of PD-L1 decreased dramatically in the siR-HIF-la group compared with the NC group(P<0.05);The lung adenocarcinoma cell line HCC827 were transfected with siRNA-HIF-1α for 48 hours,then detected transfection efficiency to validate down-regulate HIF-1α expression.Further detected PD-L1 expression demonstrated that the mRNA and protein expressions of PD-L1 decreased dramatically in the siR-HIF-1αgroup compared with the NC group(P<0.05).ConclusionThe HIF-la and PD-L1 expressions are highly co-expressed and HIF-la can up-regulate the expression of PD-L1 in lung adenocarcinoma,revealing an important mechanism of tumor immune escape induced by hypoxic conditions.