The Mechanism And Prognostic Significance Of SOX9 In The Proliferation Of Cholangiocarcinoma

Background:Cholangiocarcinoma(CCA)is a highly malignant tumor originating from the epithelium of the bile ducts.Depending on its anatomical location,CCA can be divided into three subtypes,namely intrahepatic cholangiocarcinoma(iCCA),perihilar cholangiocarcinoma(pCCA)and distal cholangiocarcinoma(dCCA).The different subtypes of CCA have their own characteristics(such as risk factors,pathogenesis,treatment and prognosis,etc.).The diagnosis and treatment level in China is far behind.Surgery is still the first choice for the treatment of bile duct cancer;however,radical surgery is very difficult due to the rapid progression of CCA and the complex anatomy involved in surgery.Moreover,most patients are found late,which predicts little possibility of radical resection.Generally speaking,for patients with advanced CCA or CCA lost to surgery,their median overall survival is mostly no more than 1 year.Even if some patients undergo radical resection for cholangiocarcinoma,their 5-year overall survival rate generally does not exceed 30%.In addition to surgery,radiotherapy and chemotherapy have limited therapeutic effect on patients with bile duct cancer.Currently,in this era of precise treatment,research related to bile duct cancer lags far behind other common tumors such as gastric cancer and lung cancer.The main reasons for this phenomenon may include:(1)the overall prevalence of bile duct cancer is relatively low compared to other tumors,making it difficult to establish a relatively large study cohort;(2)the lack of relatively clear surgical guidelines for the surgical treatment of CCA,coupled with the complex anatomical locations involved in the surgical doing of bile duct cancer and the difficulty in obtaining patient specimens,making it difficult to conduct CCA-related trials in a sustainable manner;(3)most CCA patients have missed the best treatment period when diagnosed,which often leads to a poor prognosis for CCA patients with short survival time and difficulties in carrying out experimental treatment.Therefore,it is important to carry out clinical as well as basic research related to CCA in China.It was not until April 2020 that the first targeted drug for cholangiocarcinoma,pemigtinib(INCB054828),became available for the treatment of adult patients with treated advanced cholangiocarcinoma in the presence of an FGFR2 fusion gene or mutation.Since pemetinib was approved for the treatment of patients with FGFR2-fused cholangiocarcinoma,pemetinib-resistant cholangiocarcinoma patients have been consistently reported,but the mechanism of resistance is not fully understood.Based on the limited targeted therapies for cholangiocarcinoma,unclear resistance mechanisms,and lack of biomarkers,we formed three groups of patients with CCA undergoing radical resection,including 58 patients with intrahepatic cholangiocarcinoma(iCCA),123 patients with portohepatic cholangiocarcinoma(pCCA),and 101 patients with distal cholangiocarcinoma(dCCA),in order to find more biomarkers and resistance mechanisms related to the prognosis of CCA and therapeutic targets for CCA,in order to accurately predict the postoperative risk of CCA patients and to guide individualized patient treatment.SOX9(SRY(sex-determining region Y)box 9)plays an important role in the embryonic development of many tissues and organs,and is also a biomarker for various tissue progenitor cells.In adult liver,SOX9 is significantly expressed in bile duct cells and is often considered to be a regulator of biliary cell fate.Although SOX9 has been shown to drive bile duct cell proliferation and differentiation,the role of SOX9 in CCA tumorigenesis and progression remains unclear.It was previously reported in the literature that SOX9 was shown to be associated with chemotherapy resistance and poor prognosis in a small sample size iCCA cohort.Considering that SOX9 is a target gene of the Wnt-TCF/LEF signaling pathway and an important regulator of bile duct cell fate,we further investigated the mechanism of SOX9’s role in bile duct cancer proliferation through bioinformatics and experimental validation.Objectives:1.Based on high-throughput sequencing of 8 pairs of clinical samples,we analyzed the differential expression of SOX9 in CCA tissues and paracancerous tissues in the SOX family,and clarified the most significant differential genes in the SOX family.2.Based on TCGA cancer database,we analyzed the differential expression of SOX9 in 36 CCA tissues and 9 normal bile duct epithelial tissues to clarify the potential value of SOX9 as a tumor marker.3.The expression of SOX9 in different subtypes of CCA was further investigated by Western blot using surgically resected tissue specimens from CCA patients.4.Immunohistochemical staining(IHC)was performed on tissue mieroarray(TMA)of CCA patients,and the patients were divided into subgroups with low or high SOX9 expression by IHC score,and the expression in different subtypes of CCA tissues and paraneoplastic tissues was analyzed.The relationship between SOX9 and the prognosis of CCA patients was clarified by combining the postoperative follow-up of patients.5.To clarify the effect of SOX9 on the proliferation of CCA cells by qPCR,Western blot and other molecular biology techniques and in vivo and in vitro experiments.Methods:1.High-throughput sequencing was performed on 8 pairs of pCCA and paracancer clinical samples to calculate the FPKM values of the SOX family in pCCA and adjacent normal tissues,and the differences in SOX family expression were reflected by heat maps.2.With the help of TCGA database,mRNA expression data related to CCA(including 36 CCA tissues and 9 normal bile duct epithelial tissues)were downloaded from TCGA database,and the differences in SOX9 mRNA expression levels in CCA tissues and normal bile duct epithelial tissues were statistically analyzed by unpaired t-test analysis(unpaired t tests).3.Fresh surgically resected specimens from 4 pairs of iCCA,4 pairs of pCCA and 4 pairs of dCCA patients were selected to further clarify the differences of SOX9 mRNA expression in CCA and paracancerous tissues by qRT-PCR.4.Fresh surgical resection specimens from 2 pairs of iCCA,2 pairs of pCCA and 2 pairs of dCCA patients were selected to compare the protein expression differences of SOX9 between different subtypes of cholangiocarcinoma,cancerous and paraneoplastic tissues by western blot assay.5.A total of 282 paraffin tissue specimens(including 58 cases of iCCA,123 cases of pCCA and 101 cases of dCCA)were collected from patients who underwent radical bile duct cancer surgery at Qilu Hospital of Shandong University between January 2010 and December 2020.The collected tissue specimens were made into tissue microarrays,and the tissue microarrays were stained with IHC and scored for SOX9 expression.6.The ROC curve(receiver operating characteristic curve)was used to calculate the cut-off values of iCCA,pCCA and dCCA,and the patients were divided into the low and high SOX9 expression groups using the cut-off values.One-way survival analysis was performed by Kaplan-Meier method and long-rank test to clarify the clinicopathological characteristics of bile duct cancer and the influence of SOX9 expression on patients’ prognosis.Multi-factor analysis was performed by Cox regression model to clarify whether SOX9 was an independent risk factor affecting the prognosis of cholangiocarcinoma.7.We constructed lentiviral interference and overexpression vectors to knock down or overexpress SOX9 in cholangiocarcinoma cells,and examined the effect of SOX9 on the proliferation of CCA cells by CCK8,colony formation assay.8.To establish a subcutaneous tumorigenic model in nude mice to investigate the effect of SOX9 on the proliferation ability of CCA cells.Findings:1.8 pairs of pCCA and paraneoplastic tissues with high-throughput sequencing showed that SOX family members were significantly more expressed in CCA tissues than in paraneoplastic tissues and SOX9 was the most significant difference.2.In TCGA database,SOX9 expression was significantly higher in CCA tissues than in normal tissues.3.qRT-PCR results confirmed that the mRNA expression levels of SOX9 in iCCA,pCCA and dCCA tissues were significantly higher than those in paraneoplastic tissues.4.Western blot results confirmed that the protein expression levels of SOX9 were significantly higher in the three cholangiocarcinoma subtypes than in the paraneoplastic tissues.5.IHC scores showed that SOX9 was expressed in iCCA,pCCA and dCCA tissues,and the IHC scores of SOX9 in all three subtypes of CCAs were significantly higher than those in paracancerous tissues.6.The results of univariate analysis showed that:high expression of SOX9 was significantly associated with poor prognosis in patients with iCCA,pCCA and dCCA.7.SOX9 overexpression promoted the proliferative capacity of CCA cells,while knockdown of SOX9 obtained the opposite result.8.Overexpression of SOX9 promoted the proliferation of CCA cells in a nude mouse subcutaneous tumorigenesis model,and knockdown of SOX9 inhibited the proliferation of CCA cells in a nude mouse subcutaneous tumorigenesis model.Conclusion:1.SOX9 was differentially expressed in cancerous and paraneoplastic tissues of iCCA,pCCA and dCCA patients,i.e.,the expression in cancerous tissues was significantly higher than that in paraneoplastic tissues.The expression of SOX9 was significantly higher in cancer tissues than in paracancerous tissues.2.High SOX9 expression was significantly associated with poor prognosis in patients with iCCA,pCCA and dCCA.3.SOX9 promotes the proliferative capacity of CCA cells.