Function And Molecular Mechanism Of SOX8 In Pancreatic Cancer Cell Stemness Maintenance And Chemotherapy Sensitivity

Background and Aim SOX8,combinated with SOX9 and SOX10,belongs to the SOXE family.It has been reported that the activation of Notch and Hedgehog pathway was correlated with the expression level of SOX8 in retinal cells.Recent studies have shown that SOX8 also has a close influence on the occurrence and development of tumors.The high expression of SOX8 which is regulated by Micro RNA 124 can promote the proliferation of tumor cells in non small cell lung cancer.Similarly,the high level of SOX8 which is positively correlated with the high expression of beta-catenin also promotes the proliferation of tumor cells in hepatocellular carcinoma cells [4].In the other hand,SOX8 also can be used as a molecular marker which evaluate the prognosis of malignant glioma.Our previous studies found that the expression of SOX8 was higher in pancreatic ductal adenocarcinoma tissues and cell lines than that in normal pancreatic tissues and cells,but the potential clinical significance is unclear.Therefore,in this study we aim to explore the expression and function of SOX8 from the level of clinical histology,cell biology,molecular biology and model animal,to analyze its correlation with prognosis,to elucidate the mechanism of SOX8 on stemness maintenance and chemo-resistance,and also its expression under hypoxia microenvironment.Method 1.The expression levels of SOX8 in normal pancreatic tissue and pancreatic ductal adenocarcinoma were analyzed by TCGA database.At the same time,paraffin specimens of pancreatic ductal adenocarcinoma tissues,normal pancreas tissues and clinical pathologic data were collected.Immunohistochemistry staining of paraffin-fixed sections was performed to detect the expression of SOX8 in pancreatic ductal adenocarcinoma tissues and normal pancreas tissues.The correlation between SOX8 expression and clinical and pathological parameters was analyzed by SPSS software.2.Investigating the basal expression of SOX8 in pancreatic cancer cell lines by Western blot.Stable transfected cell lines which were overexpression anddownexpression of SOX8 were established by lentiviral stable transfection system and confirmed by Western blot.Sphere-forming assay,soft agar cloning formation assay,CCK8 cell proliferation assay,cell apoptosis,cell cycle and stem cell surface molecular analysis and subcutaneous pancreatic tumor model in nude mice were used for the investigation of SOX8 direct function which maintain the stemness of pancreatic ductal adenocarcinoma cells.Western blot was used to detect the expression level of ?-catenin in different SOX8 stable cell lines.3.In order to investigate the effect of SOX8 expression on chemoresistance in vitro,CCK8 cell toxicity test was used to detect half-maximal inhibitory concentrations(IC50)of Gemcitabine in different SOX8 stable cell lines.In the other hand,apoptotic rate of Gemcitabine in different SOX8 stable cell lines were measured using flow cytometry assay.At the same time,subcutaneous pancreatic tumor model in nude mice was constructed with different SOX8 stable cell lines to investigate the effect of SOX8 expression on chemoresistance in vivo.Observing the antitumor effect of Gemcitabine intraperitoneal injection to these models of nude mice for the investigation of SOX8 function on chemoresistance of pancreatic ductal adenocarcinoma.4.In order to investigate the effect of hypoxia on pancreatic cancer cell lines,sphere-forming assay,CCK8 cell proliferation and toxicity assay,cell apoptosis analyse by flow cytometry were used.Futhermore,we repeated above experiments under hypoxic conditions in pancreatic cancer cell lines which were downexpression of SOX8.5.The correlation of SOX8 and HIF-1a in protein and m RNA level was examined in the consecutive paraffin-fixed sections of pancreatic ductal adenocarcinoma tissues by immunohistochemistry staining and in HIF-1a overexpression and downexpression pancreatic cancer cell lines by Western blot and RT-PCR experiment.Chromatin immunoprecipitation assay were used to investigate the regulation mechanism of SOX8 and HIF-1a.Results1.TCGA database analysis showed that not only the number of SOX8 gene copies in pancreatic cancer tissues was significantly higher than that of paracancerous tissues,but also that was also more significant than that of other members of SOXE family(SOX2 and SOX9).30 normal pancreatic tissues and 88 pancreatic ductal adenocarcinoma tissues were used to detect the expression of SOX8 by immunohistochemical staining.We found that SOX8 was not or extremely low expressed in the normal pancreatic ductal epithelium,but was significantly increased in some pancreatic ductal adenocarcinoma tissues.In 88 pancreatic ductal adenocarcinoma tissue specimens,SOX8 high expression accounted for 63.6%(56 cases)of all cases,low expression accounted for 36.4%(32 cases);Logistic regression analysis showed that the expression level of SOX8 was positively correlation with postoperative pathological stage,tumor size and lymphnode metastasis and negatively correlated with the degree of tissue differentiation.Survival analysis showed that the overall survival and relapse-free survival of patients with low SOX8 expression in pancreatic ductal adenocarcinoma were significantly longer than those in patients with high expression of SOX8(median overall survival: 22 vs 14 months,p <0.01;median relapse-free survival: 16 vs 10 months P<0.01).2.SOX8 was not or extremely low expressed in the normal pancreatic ductal epithelium cell line(HPDE6C7),but was significantly increased in some pancreatic ductal adenocarcinoma cell lines(Panc-1,CFPAC-1,MIA Pa Ca-2 and Bx PC-3).According to the constructive expression level of SOX8 in different pancreatic ductal adenocarcinoma cell lines,we constructed the SOX8 overexpressing cell lines(CFPAC-1 and Panc-1)and downexpression cell lines(CFPAC-1 and Bx PC-3)by lentivirus infection.Western blot was used to confirm the expression of SOX8 in constructed cell lins.Compare with control group,SOX8 overexprerssion stable transfected cell lines showed superior sphere-forming ability with larger spheres and more quantities,more colonies in soft agar and higher proliferation activity.In addition,the apoptotic rate of anoikis of SOX8 overexprerssion pancreatic ductal adenocarcinoma cell lines was lower than that of control group.Similarly,the expression of CD133 in SOX8overexprerssion pancreatic ductal adenocarcinoma cell lines was higher than that of control group.To explore effect of SOX8 on pancreatic ductal adenocarcinoma cell line in vivo,we transplanted CFPAC-1-plsi-sox8 or CFPAC-1-plsi-Control into the ventral of nude mice.When 1.0×103 cells were transplanted,four in ten mice were tumorigenesis in CFPAC-1-plsi-sox8 group,but ten mice were all tumorigenesis in CFPAC-1-plsi-Control group.3.The result of CCK8 cell toxicity test showed that the IC50 value of Gemcitabine in SOX8 downexprerssion pancreatic ductal adenocarcinoma cell lines was lower than that of control group.Similarly,the cell apoptosis rate was higher in SOX8 downexprerssion pancreatic ductal adenocarcinoma cell lines than that of control group.Constructing nude mice subcutaneously xenograft model to demonstrate the effection of SOX8 expression on pancreatic cancer cell by intraperitoneal injection Gemcitabine.The results suggest that the tumor-inhibiting rate was higher in mice which were transplanted SOX8 downexpression pancreatic ductal adenocarcinoma cell line than that of control group.The result show that downexpressing SOX8 can increase the sensitivity of Gemcitabine in vivo.4.Under hypoxic conditions,pancreatic cancer cell lines showed superior sphere-forming ability with larger spheres and more quantities,higher proliferation activity and lower apoptotic rate of anoikis than those of under normoxia.Meanwhile,the IC50 value of gemcitabine to pancreatic ductal adenocarcinoma cell lines was higher than that of under normoxia.The cell apoptosis of pancreatic ductal adenocarcinoma cell lines to gemcitabine was lower under hypoxic conditions by flow cytometry.Furthermore,downexpression SOX8 under hypoxic conditions,all the trends were reduced.5.The result of immunohistochemistry staining showed that expression level of SOX8 in pancreatic ductal adenocarcinoma cells was significantly positively correlated with HIF-1a.We detected the expression of SOX8 in HIF-1a overexpression and downexprerssion pancreatic ductal adenocarcinoma cell lines by Western blot and RT-PCR.The result suggested that SOX8 expression level increased in HIF-1a overexpression cell lines and decreased in HIF-1a downexpression cell lines.Bioinformatics analysis suggested that there werehypoxia response element(HRE)for HIF-1a transcriptional binding in SOX8 promoter.Furthermore chromatin immunoprecipitation assay were performed which confirmed that HIF-1a could directly bind the promoter region of SOX8.Conclusions 1.SOX8 expression level was elevated in pancreatic ductal adenocarcinoma tissue.The expression level of SOX8 was negatively correlation with poor prognosis of patients with pancreatic ductal adenocarcinoma.2.The overexpression of SOX8 can enhance the stem cell characteristics and reduce the chemotherapy sensitivity of pancreatic ductal adenocarcinoma cell which may be related to the activation of the ?-catenin.3.Hypoxia microenvironment can enhance the stem cell characteristics and reduce the chemotherapy sensitivity of pancreatic ductal adenocarcinoma cell which could be inhibited by downexpression SOX8.4.HIF-1a can directly bind to the specific sequence of SOX8 promoter region and then regulate the expression of SOX8 in pancreatic cancer.