Prognostic Value And Regulatory Mechanism Of Gamma Interferon-ineucible Lysosomal Thiol Reductase Expression In Breast Cancer

BackgroundBreast cancer is the most common malignancy among women worldwide.In the past three decades,breast cancer mortality has been substantially reduced attributed to increased screening and the development of personalized systematic therapy.However,approximately 200%-30%of breast cancer patients still experience relapse or die years after the initial treatment.Finding new biomarkers can more accurately predict the prognosis of patients and provide evidence for treatment,which has always been an important field of breast cancer research.In recent years,with the application of multiomics technology in the diagnosis and treatment of breast cancer,the prognosis prediction of breast cancer patients has become more accurate.However,the low popularity of detection platform limits the wide application of these technologies,especially in relatively undeveloped countries and regions.Simple and classical methods,such as immunohistochemistry,are more convenient and efficient to evaluate the expression of prognostic markers for prognostic prediction.Our previous study identified significant heterogeneity of gamma-interferon-inducible lysosomal thiol reductase(GILT,also known as IFI30)expression levels in breast cancer tissues compared with normal breast tissues.GILT expression in breast cancer cells was significantly associated with the 3-year disease-free survival(DFS)of breast cancer patients.GILT is a reductase that catalyzes the reduction of disulfide bonds in the endocytic compartments.Studies have shown that GILT is involved in several key biological processes,including immune response,autophagy,cellular redox state and proliferation,which are processes involved in carcinogenesis and cancer progression.The aberrant expression of GILT has been associated with the survival of melanoma,diffuse large B cell lymphoma(DLBCL)and glioma patients.However,the mechanism of how GILT affect the prognosis of cancer patients are still unclear.Early studies about GILT had been focusing on the immunity roles in antigen processing,suggesting that GILT may involve in antigen presenting process of tumor cells.While with the recently in-depth researching,GILT was found to play roles in many physiological and pathological processes besides immune.In order to further verify the prognostic value of GILT expression for breast cancer patients and conduct external data verification,we conducted this study to explore the influence and mechanism of GILT expression on biological behavior and sensitivity to treatment of breast cancer cells.Therefore,here we used data of breast cancer patients from multiple centers with a longer follow-up period as cohort 1 and cohort 2.We validated the prognostic value of GILT expression in breast cancer and investigated the potential biological mechanism of GILT underlying its influence on the prognosis of breast cancer patients.Method1.For the cohort 1,patients diagnosed with primary breast cancer at The Second Hospital,Cheeloo College of Medicine,Shandong University between 2006 and 2014 were assessed for eligibility.A final study population of 416 breast cancer patients was included as cohort 1.We recruited another 210 breast cancer patients between 2004 and 2009 from Linyi People’s Hospital and Linyi Tumor Hospital as cohort 2.This study was approved by the respective ethics review boards.GILT immunohistochemical(IHC)staining was performed on breast cancer tissue sections,and correlation analysis was conducted with clinical information of patients.The differences of disease free survival(DFS)and breast cancer-specific survival(BCSS)between patients with high and low GILT expression were compared.Key prognostic indicators related to breast cancer were included in the multivariate survival analysis for correction to determine whether GILT expression was an independent prognostic indicator of breast cancer patients.The baseline characteristics of patients were compared between high and low GILT expression groups.Spearman correlation analysis was used to analyze the bivariate correlation between the total GILT expression score(0-8 points)and the proportion of ER positive rate,PR positive rate,tumor size,lymph node metastasis status,histological grade,Ki67 level and other clinical factors,2.Human breast cancer cell lines were transfected with GILT-overexpressing and GILT RNAi lentivirus.Biological behaviors of transfected cells were assessed.3.Proteomics analysis was performed to analyze the changes of protein expression after GILT overexpression.Effect of GILT on reactive oxygen species(ROS)was assayed.Changes of proteins in autophagy pathway were assayed.Cells were treated with ROS and autophagy inhibitors to observe whether GILT could affect the proliferation of breast cancer cells by affecting ROS and autophagy.Results1.Patients were categorized into groups of "high GILT expression"(total score>4)and low GILT expression"(total score ≤4)based on IHC staining score.Correlation analysis showed that total GILT expression was positively correlated with the proportion of ER positive rate and PR positive rate,and negatively correlated with tumor size,lymph node metastasis status and Ki67 level.Patients with high GILT expression in breast cancer cells showed higher BCSS and DFS rates than their counterparts.After further adjustment for age at diagnosis,clinical characteristics and treatment,the differences in BCSS and DFS between two groups persisted.The results were consistent in cohort 1 and cohort 2.2.High expression of GILT in breast cancer cells suppressed cell proliferation rate,cell cycle progression,migration,invasion and subcutaneous tumorigenesis ability in nude mice.GILT can also increase the sensitivity of triple negative breast cancer cell MDA-MB-231 and Luminal A cell MCF-7 to docetaxel and epirubicin,improve the sensitivity of HER2-positive breast cancer cell BT-474 to trastuzumab,improve the sensitivity of MDA-MB-231 and BT-474 cells to radiotherapy.3.GILT overexpressing inhibited the protein expression of autophagy pathway and the ROS level.After GILT silencing,the cell proliferation ability was enhanced,while after culturing with autophagy and ROS inhibitors,the proliferation ability returned to the normal level,suggesting that GILT may inhibit the proliferation of breast cancer cells by inhibiting autophagy and ROS.ConclusionsIn this study,we confirmed that high GILT expression was an independent predictor of better prognosis for breast cancer patients,both in cohort 1 and cohort 2.GILT was positively correlated with the proportion of ER positive rate and PR positive rate,and negatively correlated with tumor size,lymph node metastasis status and Ki67 level,which was consistent with clinical analysis results.GILT overexpression inhibits multiple biological activities of breast cancer cells and enhances sensitivity to treatment.Mechanism studies have shown that GILT inhibits cell proliferation by inhibiting autophagy and ROS levels in breast cancer.