| BackgroundPancreatic ductal adenocarcinoma(PDAC)is a highly lethal tumour.Despite recent advances in combination chemotherapy regimens,the prognosis remains poor.Checkpoint blockage has been established as novel pillar of cancer therapy for several tumour types;however,its efficacy in PDAC remains poor when used as a single agent.EHF(ETS homologous factor /epithelium-specific ETS factor family member 3,ESE3)is a member of the E26 transformation-specific(ETS)superfamily.Our previous investigation identified that EHF inhibits PDAC epithelial-mesenchymal transition and metastasis by transcriptionally upregulating E-cadherin.Here,we report a novel function of EHF in tumour immune microenvironment editing and checkpointblockage therapy efficacy prediction.Method 1.In vivo efficacy studies were conducted to evaluate the anti-tumour efficacy of PD1 blockage therapy and Treg plus MDSC depletion therapy in the PANC02-vector group and PANC02-EHF group.2.In vitro coculture studies,expression analyses,chromatin immunoprecipitation(ChIP)assays,luciferase analyses and in vivo studies were designed to explore the mechanism.3.Some of the observations were further confirmed in PDAC patient samples.The resultsTumour volumes in PANC02-vector tumour-bearing mice were not significantly different from those in PANC02-EHF tumour-bearing mice in nude hosts(P=0.576).However,tumour volume in PANC02-vector group was significantly larger than in the PANC02-EHF group in immunocompetent C57BL/6 mice(P=0.009),indicating that EHF play a role in the tumour immune response.A notable decrease in Tregs and MDSCs and an increased infiltration of CD8+T cells were observed in PANC02-EHF tumours.PANC02-EHF tumour tissues exhibited decreased CD8+T cell apoptosis rates,higher percentages of IFN-γ+CD8+T cells and lower percentages of exhaustion.PDAC tissues with low tumoural EHF showed increased accumulation of Tregs and MDSCs but decreased infiltration of CD8+T cells.The median survival time for lowEHF group was 15.00 months,which was significantly shorter than that in the highEHF group(45.00 months,P<0.001).Multivariate analysis identified EHF and pTNM as independent prognostic factors of PDAC patients.Tumoural EHF deficiency induced Treg/MDSCs conversion,expansion and increase the function of Treg/MDSCs.Q-PCR was used and identified that TGFβ1 and GM-CSF were the most significantly downregulated cytokines.Western blot,ELISA,and IHC demonstrated that TGFβ1 and GM-CSF decreased in the EHF-overexpressing cell lines and increased in the EHFknockdown cell lines in protein level.To evaluate whether EHF directly binds to the promoters of TGFB1 and CSF2,a Ch-IP assay was performed using the PANC-1 and HKE293 T cell line.In chromatin fractions pulled down by anti-EHF antibody,we detected EBS1 and EBS2 in both the TGFB1 promoter and the CSF2 promoter by PCR.Luciferase analysis was performed and found that EHF directly controlled TGFB1(mainly through EBS1)and CSF2(mainly through both EBS1 and EBS2)transcription and maintained the gene under a repressive status in PDAC.In vitro and in vivo TGFβ1 and GM-CSF blocking study indicated tumour EHF deficiency induces immune suppression through TGFβ1 and GM-CSF.EHF-deficient tumour can benefit from Treg and MDSCs depletion treatment.Subcutaneous and orthotopic C57BL/6 tumour mouse model were used and found that tumour with high EHF expression exhibit good response to single anti-PD1 therapy.ConclusionsOur study is the first to identify that EHF,as a tumour suppressor in PDAC,significantly transcriptionally inhibited the expressions of TGFβ1 and GM-CSF,which are the two suppressive cytokines involved in the induction of Treg and MDSCs accumulation.Tumoural EHF can be used as a promising biomarker to evaluate the immune microenvironment status of PDAC and screen for patient response to single anti-PD1/PD-L1 therapy. |
