Mechanism Of Metformin Downregulates PD-L1 Expression In Esophageal Squamous Cell Carcinoma By Inhibiting IL-6 Signaling Pathway

Background and ObjectiveAccording to the latest national cancer statistics released by the national cancer center,in 2015,there were about 3.929 million new malignant tumor cases in China,with an incidence of 285.83 per 100,000,and a cumulative incidence of 21.44%between the ages of 0-74 years old,and a mortality rate of 170.09 per 100,000.Among them,the top five frequent tumors in China are lung cancer,breast cancer,gastric cancer,colorectal cancer and hepatocellular carcinoma.And the top five in terms of mortality are lung cancer,hepatocellular carcinoma,gastric cancer,esophageal cancer and colorectal cancer,respectively.About half of all esophageal cancer cases occur in China,and squamous cell carcinoma is the main pathological type of esophageal cancer patients in China.Most patients will experience recurrence or metastasis,and systemic chemotherapy is the first choice for patients with advanced esophageal cancer.However,because of drug resistance to chemotherapy and dose-limiting toxic adverse effects,a large number of patients in China could not been well treated.Therefore,it is urgent to improve the existing treatment measures,especially finding a new treatment strategy,to improve the survival and prognosis of esophageal cancer patients in our country,and it has important practical significance.In recent years,tumor immunotherapy has become the focus of the field of tumor therapy.American immunologist James Allison and Japanese biologist TasukuHonjo won the 2018 Nobel Prize in physiology or medicine for their discovery of negative immunoregulation therapy for cancer cells.Immunotherapy is to use the body’s own immune system to kill the tumor cells.PD-1/PD-L1 monoclonal antibodies,representing many kinds of immune checkpoint inhibitors,have been proven safety and efficacy in solid tumors,like lung cancer,colon cancer,malignant melanoma and other solid tumor.There were also a number of clinical trials confirmed that anti-PD-1 antibodies in patients with esophageal cancer has a significant therapeutic effect.MethodsThe Cancer Genome Atlas(TCGA)database was used to analyze the correlation between IL-6 and PD-L1 gene expression in esophageal Cancer.Esophageal squamous cell lines KYSE-450 and TE-7 were selected for subsequent experimental study.Enzyme linked immunosorbent assay(ELISA)was used to detect the effect of metformin on IL-6 secretion in esophageal squamous cell carcinoma cells.The effects of metformin on the phosphorylation level of the downstream signaling molecule JAK2/STAT3 and the transcription level and expression level of PD-L1 were detected by fluorescence quantitative PCR,western blot assay and immunofluorescence assay.At the same time,an IL-6 positive control group was set up to further confirm that metformin regulates PD-L1 level in esophageal squamous cell carcinoma and is related to the IL-6-JAK2-STAT3 signaling pathway.It was confirmed that metformin regulated the expression of PD-L1 in esophageal squamous cell carcinoma cells by knocking out AMPK molecules or choosing AMPK-specific blocker Compound-C to block the AMPK-ACC signaling pathway,which was independent of the classical pathway AMPK of metformin.Peripheral blood mononuclear lymphocytes cells(PBMCs)were isolated and co-cultured with esophageal squamous cell lines pretreated with metformin or IL-6 to simulate the tumor immune microenvironment.IL-2 secretion level of T cells was detected by ELISA,and ERK protein phosphorylation level of T cells was detected by western-blot,confirming the changes in activation level of T cells in each pretreated group.At the same time,the T-cell killing experiment confirmed that after metformin down-regulated the expression of PD-L1 in esophageal squamous cell carcinoma,the local T-cell killing function of tumor cells was enhanced.PBMCs were injected into the tail vein of male NPI mice for humanized immune reconstruction.At the same time,a cell line containing luciferase transfected with TE7-luc was constructed,and a model of esophageal squamous cell carcinoma was constructed by subcutaneous inoculation.The experiment was divided into 4 groups,and 5 mice in each group were given normal saline,metformin,PD-1 antibody and combined drugs,respectively.In vivo fluorescence imaging system was used to observe the growth of the transplanted tumor,and the expression of pd-11 in the transplanted tumor was observed by immunohistochemistry.At the same time,immunohistochemical staining of paraffin sections was used to detect the esophageal cancer sections.Combined with the medical history,the sections were divided into the metformin group and the control group,and the expression levels of PD-L1 in the tissue samples of the two groups were compared.Results1.TCGA data showed that IL-6 expression was positively correlated with PD-L1 expression in esophageal cancer tissues.The results showed that metformin was associated with low expression of PD-L1 in tissues.2.It showed that metformin reduced the transcription and cytokine secretion of IL-6 in esophageal squamous cell carcinoma cells and the phosphorylation of JAK2/STAT3 protein.3.Metformin could reduce the expression of PD-L1 in esophageal squamous cell carcinoma.4.Esophageal squamous cell cells were co-cultured with PBMCs.The T cell activation level was higher in the metformin pretreatment group,and the T cell killing function was the strongest.5.Animal experiments confirmed that metformin down-regulated the expression of PD-L1,and combined with metformin and PD-1 antibody,the treatment effect was the best.ConclusionMetformin down-regulated the expression of PD-L1 by blocking the IL6-JAK2-STAT3 signaling pathway of esophageal squamous cell carcinoma,which enhanced the activation level and killing tumor cells function of T cells in esophageal squamous cell carcinoma,and enhanced the anti-tumor immune response.Animal experiments confirmed that the combination of metformin and PD-1 antibody could enhance the anti-tumor efficacy,which enhances the effect of immunotherapy,improves the prognosis of patients,and provides a new theoretical basis for the innovation of esophageal squamous cell carcinoma treatment strategies.