Investigation Of The Role Of Krupple-like Factor 12 On The Regulation Of PD-L1 In Non-small Cell Lung Cancer

ObjectiveTumor immune evasion plays an important role in tumorigenesis and cancer development.Tumor cells can escape from immune surveillance by expression of immunosuppression molecules,low immunogenicity,antigenic modulation,and producing physical barriers.In this way,the tumor further develops,recurs or even metastasizes.Researchs have shown that programmed death-1(PD-1)and programmed death ligand-1(PD-L1)play a key role in tumor immune evasion.PD-L1 is highly expressed in several kinds of cancers including melaloma,non-small cell lung cancer,ovary cancer and renal cell carcinoma.The binding of PD-L1 to the PD-1 on the surface of T cells affects the survival,proliferation and differentiation of T cells via several signaling pathways such as RAS/MEK/ERK and PI3K/Akt,thereby inhibits the normal function of the effector T cells and induces apoptosis.PD-L1 also promotes the differentiation and function of the immunosuppressive regulatory T cells(Tregs).Thus,PD-L1 mediates the tumor immune evasion.The significant effects of PD-1/PD-L1 in tumor immune evasion makes the targeted-drug research and development of PD-1/PD-L1 a hot spot.So far,five PD-1 or PD-L1 monoclonal antibodies have been approved by FDA,and domestic antibodies such as Toripalimab have also been approved or being reported for approval.The antibodies were initially used for treatment of melanoma and Hodgkin's lymphoma.Subsequently,they were applied to treat non-small cell lung cancer,bladder cancer and other solid tumors.Although progress has been made in treatment of cancer,PD-1/PD-L1 monoclonal antibodies still face problems such as low single drug response rate,adverse reaction,long half-life period,poor controllability.Thus,the PD-1/PDL1 small molecule inhibitors are also paied much attention in the new drug research and development,which mainly based on the regulatory mechanisms of PD-1/PD-L1.Therefore,fully understanding the PD-1/PD-L1 regulatory mechanisms is of great significance for the research and development of the small molecule inhibitors.Non-small cell lung cancer is the main type of lung cancer and is also one of the indications for PD-1/PD-L1 inhibitors.Deeply investigate the regulation mechanism of non-small cell lung cancer is of great value.In this study,we found KLF12,a transcription factor which may regulate the expression of PD-L1 in non-small cell lung cancer by gene chip screening.We try to explore the role and mechanism of KLF12 in PD-L1 regulation and provide theoretical foundation for PD-1/PD-L1 targeted therapy of non-small cell lung cancer.MethodsNon-small cell lung cancer cells NCI-H460 with high and low expression of PD-L1 were sorted by flow cytometry.The sorted cells were cultured to multiply and after verificating the expression of PD-L1,the cells were sent for gene chip analysis to screen genes correlate with up-regulation of PD-L1 expression,and the remaining cells were cultured and passaged.The transcription level of PD-L1 and the candidate genes in the passaged cells were detected by quantitative real time-PCR(q-RT-PCR).PD-L1 was knocked down by small interfering RNA(siRNA),KLF12 transcription level changes were detected by q-RT-PCR,KLF12 protein level changes were analyzed by western blot.KLF12 protein expression levels of different non-small cell lung cancer cell lines were examined by western blot.KLF12 was knocked down by KLF12 siRNA,the expression changes of PD-L1 were detected by q-RT-PCR and western blot.KLF12 plasmid was transfected to transient overexpress KLF12,the changes of PD-L1 expression were investigated by western blot.KLF12 was knocked down by KLF12 siRNA,followed by transient overexpression of KLF12 and the changes of PD-L1 protein level were investigated by western blot.Dual luciferase reporter assay was performed to detect the effect of KLF12 transient overexpression on the transcription activity of the PD-L1 promoter region.Protein synthesis was inhibited by Cycloheximide(CHX),the changes of PD-L1 protein stability after KLF12 knockdown were detected by western blot.NCI-H460 cells were treated with the classic PD-L1-inducing factor IFNy,the effect of KLF12 siRNA knockdown on induced PD-L1 expression was analyzed by western blot.PD-L1 emprical transcription regulation factor and/or KLF12 were knocked down by siRNA,western blot was performed to investigate the role of empirical transcription factor in KLF12 mediated modulation of PD-L1.Expression correlation of KLF12 and PD-L1 in tissue samples from non-small cell lung cancer patients was analyzed by immunohistochemistry.Investigation on the Cancer Genome Atlas(TCGA)database was performed to analyze correlation of KLF12 and PD-L1 in non-small cell lung cancer patients,as well as the relationship between KLF12 expression and prognosis.Results(1)Screening of genes correlate with PD-L1 up-regulation.NCI-H460 cells were labled with PD-L1 antibody,and two groups of cells with high and low expression of PD-L1 membrane surface protein were sorted by flow cytometry.The sorted cells were cultured to multiply.After verificating the expression of PD-L1,the cells were sent for gene chip analysis,and the remaining cells were cultured and passaged.Gene chip analysis was performed to investigate changes in target genes while PD-L1 expression level was changed.In the gene chip,400 protein-coding genes which up-regulated by more than 2-fold were screened,followed by investigation of protein location and function in Genecards,and 178 genes with certain location or function were found eventually.Our research focused on the transcription regulation related protein-coding genes which correlated with the up-regulation of PD-L1.Thus,q-RT-PCR was performed to detect the the transcription level of CD274 gene and the candidate genes of the passaged cells.The results showed that the CD274 gene of the PD-L1 high-expressed NCI-H460 up-regulated by 3.55 times(p = 0.0273),compared to PD-L1 low-expressed NCI-H460,and the KLF12 gene also up-regulated by 2.48 times(p = 0.0393).Knockdown of PD-L1 in NCI-H1299 by siCD274#1 and#2 showed no significant changes of KLF12 transcription levels(p =0.216 and 0.570)and protein expression(p = 0.829 and 0.767).The above results showed that KLF12 correlated with the up-regulation of PD-L1,PD-L1 had no effect on KLF12 expression,KLF12 might serve as an upstream factor modulating PD-L1 expression.(2)The regulation of PD-L1 by KLF12.Western blot was performed to dectect the KLF12 protein expression of different non-small cell lung cancer cell lines.The results showed that NCI-H1299 and NCI-H460 cells had a higher expression of KLF12,while A549 had a lower KLF12 expression.KLF12 in NCI-H1299 was knocked down by siKLF12#1 and#2,q-RT-PCR showed that the transcription level of PD-L1 was significantly down-regulated by 3.10 times(p = 0.0137)and 2.42 times(p-0.0318),respectively.Western blot showed that the PD-L1 protein expression level was also down-regulated by 1.46 times(p = 0.0220)and 1.98 times(p = 0.0107),respectively.Also,KLF12 in NCI-H460 and was knocked down by siKLF12#1 and#2,q-RT-PCR showed that the transcription level of PD-L1 was significantly down-regulated by 2.44 times(p = 0.0496)and 2.10 times(p = 0.0249),respectively.Western blot showed that the PD-L1 protein level was also down-regulated by 1.50 times(p = 0.0431)and 1.71 times(p = 0.0481),respectively.In A549 cells,knockdown of KLF12 also caused down-regulation of PD-L1 protein level.Plasmid transient overexpression of KLF12 caused 3.35 times(p = 0.000768)of up-regulation of PD-L1 protein level in A549 cells and 2.32 times(p = 0.0292)of up-regulation of PD-L1 in NCI-H1299 cells.Besides,in NCI-H1299,re-expression of KLF12 reversed the down-regulation of PD-L1 protein level caused by siRN A.The above experiments showed that KLF12 promoted PD-L1 expression of PD-L1.(3)The mechanism of PD-L1 transcription regulation by KLF12.The Luciferase plasmid x containing the KLF12 binding segment "CACCC-box" and the PD-L1 empirical transcriptional regulator(STAT1/3,STAT2/5 and IRF-1)binding segment were constructed,NCI-H1299 and 293T cells were co-transfected with the dual-luciferase plasmid and KLF12 transient expression plasmid.The results showed that transient overexpression of KLF12 in NCI-H1299 enhanced the PD-L1 transcription activities of"CACCC-box" containing segment and PD-L1 empirical transcriptional regulator binding segment by 1.99 times(p = 0.0394)and 2.43 times(p = 0.03410),and the overexpression KLF12 in 293T also enhanced the PD-L1 transcription activities of the two segments by 1.34 times(p = 0.00619)and 1.96 times(p = 0.0211).NCI-H1299 was transfected with KLF12 siRNA for 12 hrs and then treated with 10 ?g/ml CHX for different time(0,3,6,12,24 hrs).It was shown that knockdown of the KLF12 did not significantly affect the protein stability of PD-L1(p = 0.119).Knockdown of KLF12 in NCI-H460 before treatment of classical PD-L1-inducing factor IFNy inhibited the up-regulation of PD-L1 protein.The IFNy pathway related PD-L1 transcriptional regulators IRF-1 and STAT1 were knocked down by siRNA in NCI-H460 and NCI-H1299,respectively.The results showed that PD-L1 protein level was down-regulated after knockdown of STAT1 or IRF-1.Knockdown of both IRF-1 and KLF12 by siRNA further promoted the down-regulation of PD-L1 protein expression in NCI-H1299,and knockdown of both STAT1 and KLF12 had a similar effect,suggesting that KLF12 mediated PD-L1 regulation was independent of IRF-1 or STAT1.Besides,knockdown of cMyc had no significant effect on PD-L1 protein expression(p = 0.714 and 0.142),knockdown of STAT3 also had no apparent effect on PD-L1 expression(p = 0.121).The above experiments showed that KLF12 increased the PD-L1 expression by promoting its transcription,despite its role in IFNy-induced PD-L1 regulation,KLF12 mediated PD-L1 modulation was independent of empirical PD-L1 transcription regulatory factors.(4)Clinical correlation of KLF12 and PD-L1.Immunohistochemical analysis of 33 tumor tissue samples of non-small cell lung cancer patients showed positive correlation of KLF12 and PD-L1 expression(R = 0.432,p = 0.0120).The TCGA database showed that there was a positive correlation between KLF12 and PD-L1 expression in lung adenocarcinoma,lung squamous cell carcinoma and the merged samples(R = 0.4,0.22 and 0.27).KLF12 expression had no significant correlation with the overall survival of non-small cell lung cancer patients(Logrank p = 0.179,0.978 and 0.742),and it the high expression of KLF12 was only associated with poor prognosis in patients who had a long survival time.The above experiments showed that the expression of KLF12 in non-small cell lung cancer patient samples had clinical significance and might be used for indication of PD-L1 expression and evaluation of patient prognosis.ConclusionThis study found that KLF12 increased PD-L1 expression in non-small cell lung cancer by enhancing transcription rather than affecting its protein stability.Knockdown of KLF12 by siRNA caused down-regulation of PD-L1 expression.Plasmid transient expression of KLF12 promoted PD-L1 expression and enhanced the transcription activity of both "CACCC-box" binding segment and empirical transcriptional regulator binding segment on PD-L1 promoter region.Although KLF12 involved in IFN?-induced PD-L1 expression,the regulation of PD-L1 by KLF12 was independent of empirical PD-L1 transcription regulatory factors.The expression of KLF12 was positively correlated with PD-L1 both in non-small cell lung cancer patient samples and TCGA databases,and KLF12 expression was associated with poor prognosis in lung cancer patients who had long survival time.This study not only found a novel regulatory factor for PD-L1 in non-small cell lung cancer,but also suggested that inhibition of KLF12 expression might be a potential therapeutic strategy for non-small cell lung cancer.KLF12 might also be used for indication of PD-L1 expression and evaluation of patient prognosis.