The Role And Mechanism Of Tumor-derived IL-35 In Chemoresistance Of Pancreatic Cancer

BackgroundInterleukin-35(IL-35)is a new member of the interleukin 12 cytokine family and consists of EBI3 and P35 two subunits.We previously reported that IL-35 is highly expressed in pancreatic cancer and associated with poor prognosis.On the one hand,tumor-derived IL-35 promotes extravasation and metastasis of tumor cells by up-regulating the expression of ICAM1.On the other hand,tumor-derived IL-35 promotes angiogenesis and tumor growth by up-regulating CCL5 which can recruit monocytes.Current research of IL-35 mainly focused on inflammatory diseases,autoimmune diseases,infectious diseases,and a few tumors.However,there is no study on the relationship between tumor-derived IL-35 and chemotherapy effect.In this study we found that the high expression of tumor-derived IL-35 was negatively correlated with progression-free survival and gemcitabine chemotherapy effect.Therefore,we designed this project to further explore the role and mechanism of IL-35 in pancreatic cancer chemotherapy from the clinical level,cell level and animal experiment level,and further explore whether tumor-derived IL-35 can be a biomarker for guiding the choice of chemotherapy for pancreatic cancer?Method1.Paraffin specimens of pancreatic cancer receiving gemcitabine monotherapy and liver metastasis specimens of pancreatic cancer were collected,and the expression of EBI3 and p35 were detected by immunohistochemical staining.The collected case was followed up for prognosis.Kaplan-Meier curve and chi-square test was used to analysis the relationship between IL-35 expression level and patient recurrence-free survival and gemcitabine chemotherapy effect.2.Construction and validation of stable pancreatic cell lines with decreased expression/overexpression of IL-35 in human pancreatic cancer cells.Flow cytometry and CCK8 assays were used to detect the apoptosis rate of tumor cells with different IL-35 expression levels under the killing of gemcitabine.The subcutaneous tumor formation model of SCID immunodeficient mice was established.Gemcitabine chemotherapeutic drugs were administered intraperitoneally to observe the effects of different IL-35 expression levels on the growth curve of transplanted tumors,survival curves of mice and apoptosis rate of tumor cells.In vivo and in vitro experiments to explore whether IL-35 affects the killing effect of gemcitabine on pancreatic cancer cells.3.The levels of reactive oxygen species(ROS)and cell membrane potential of pancreatic cancer cells with different IL-35 expression levels after gemcitabine stimulation were detected by DHE staining and JC-1 staining.Caspase3 and BAX,which activated in ROS-related apoptotic signaling pathways were detected by Western blot.The role of ROS in IL-35-mediated chemoresistance was identified by ROS clearance assays and ROS induction assays.4.The target of IL-35 downstream which affect ROS metabolism was screened by transcriptome sequencing;the sequencing results were verified by Western blot and RT-PCR;the molecular mechanism of IL-35 regulatory target was determined by Western blot,RT-PCR,CHIP and dual luciferase assay.The role of the target in IL-35-mediated chemoresistance was further determined by blocking assays.5.The expression of IL-35 in gemcitabine treated pancreatic cancer cells was detected by RT-PCR,Western-blot and ELISA.Bioinformatics analysis was used to screening of signaling pathways which affect IL-35 expression,ChIP and dual luciferase assays were used to explore the molecular mechanism of the up-regulation of IL-35 by gemcitabine.6.The role of IL-35 neutralizing antibody in chemotherapy sensitization of pancreatic cancer was explored by subcutaneous tumor model in mice.The results1.Immunohistochemical staining was performed on 92 tissue specimens of pancreatic cancer and 49 biopsy specimens of liver metastasis of pancreatic cancer receiving gemcitabine chemotherapy,and it was found that the relapse-free survival period of patients with high IL-35 expression was significantly lower than that of patients with low IL-35 expression(median survival: 375.0 vs 456.7 days).At the same time,chemotherapy effect of gemcitabine was poor in patients with high IL-35 expression.2.In vitro gemcitabine killing experiments confirmed that overexpression of IL-35 resulted in decreased sensitivity of pancreatic cancer cells to gemcitabine,while decreased expression of IL-35 resulted in increased sensitivity of pancreatic cancer to gemcitabine.In vivo experiments,overexpression of IL-35 resulted in a larger tumor volume,shorter mouse survival,and decreased apoptotic rate;whereas decreased expression of IL-35 had opposite results.3.In vitro experiments,we revealed that IL-35 overexpression significantly reduced gemcitabine-induced ROS levels,whereas IL-35 down-regulation significantly increased gemcitabine-induced ROS levels.In vitro recovery experiments and blocking experiments confirmed that IL-35 reduces the killing effect of gemcitabine by reducing the level of cellular reactive oxygen species.4.Transcriptome sequencing revealed that the expression of superoxide dismutase 2(SOD2)was up-regulated after IL-35 overexpression,and the regulation of IL-35 on SOD2 was confirmed by RT-PCR and Western blot.We found that when over-expressing IL-35 cells down-regulated SOD2 expression,the cell line no longer had a significant increase in sensitivity to gemcitabine.In mechanism,IL-35 activates STAT1 phosphorylation via the receptor gp130,and the expression of SOD2 is activated by p-STAT1 homodimer into the nucleus.5.The expression of IL-35 protein and mRNA in tumor cells was significantly increased after treatment with gemcitabine.Mechanism,gemcitabine can activate the NFκB signaling pathway,and it was confirmed by ChIP assay and dual luciferase assay that p65 can directly bind to the promoter regions of EBI3 and P35 and regulate its transcriptional activity.6.In the subcutaneous tumor model of mouse-derived Pan 02 cell line,IL-35 neutralizing antibody can significantly enhance the chemotherapy effect of gemcitabine,which is characterized by prolonged survival and decreased tumor burden in mice,and increased apoptosis rate in tumor tissues,decreased ROS level.Conclusions1.Patients with high expression of IL-35 in pancreatic cancer have poor recurrence-free survival.Gemcitabine chemotherapy effect is poor in patients with high expression of IL-35 in liver metastases from pancreatic cancer.2.IL-35 activates the JAK-STAT signaling pathway through the receptor gp130,and p-STAT1 homodimers enter the nucleus to promote the expression of SOD2,further reducing the level of cellular reactive oxygen species and reducing the killing effect of gemcitabine.3.Gemcitabine promotes the transcription of two subunits of IL-35 by activating NFκB signaling pathway,thereby promoting the expression of IL35,further promoting the ROS clearance ability of pancreatic cancer cells and enhancing the drug resistance of cancer cells.4.IL-35 neutralizing antibody can enhance the killing effect of gemcitabine,so IL-35 may be a potential target for the treatment of pancreatic cancer.