HMGA1-TRIP13 Axis Promotes Stemness And Epithelial Mesenchymal Transition Of Perihilar Cholangiocarcinoma In A Positive Feedback Loop Dependent On C-myc

BackgroundCholangiocarcinoma(CCA)is a highly malignant cancer originating from the bile ducts.According to the anatomical location and treatment options for CCA,CCA can be further divided into three subtypes:intrahepatic cholangiocarcinoma(iCCA),perihilar cholangiocarcinoma(pCCA),and distal cholangiocarcinoma(dCCA).These three subtypes of CCA have their own unique risk factors,molecular pathogenesis,treatment options and prognosis.pCCA,accounting for more than 50%of all CCA cases,arises from the second-order intrahepatic bile ducts to the converging point of the cystic duct and the common bile duct.The preferred treatment for pCCA is surgery resection;however,radical resection of pCCA is extremely difficult because of the rapid progression and anatomical complexity of the hilus hepatis.Moreover,most patients present with jaundice,indicating that the stage is too late for radical resection.In general,for atients with advanced or unresectable CCA,the median overall survival is less than 1 year.Even after radical resection,the 5-year overall survival rate is less than 30%.Indeed,the effects of radiotherapy and chemotherapy of pCCA are also very limited.Moreover,studies on pCCA are far behind those of other more common tumors such as lung cancer and colon cancer in this era of precision treatment.Several reasons can be attributed to,including(?)the prevalence of pCCA is relatively low,making a large cohort difficult to establish;(?)pCCA specimens are hard to obtain because of its special anatomical location,resulting in rare reports on pCCA high-throughput experiments;(?)most patients lose surgical opportunity and the survival times are so short to perform any experimental treatment.Thus,further studies of the pathogenesis and therapeutic options of pCCA are necessary.Targeted therapy and precision treatments are emerging treatment methods based on effective biomarkers and an in-depth understanding of tumor progression.However,this is extremely hard as to pCCA because of the difficulty of specimen obtainment and establishment of large cohort.Even in the low proportion of patients who underwent resection,rate of radical resection is very low.This increases the heterogeneity of surgical treatment and results in difficulties in collecting a homogeneous cohort to verify biomarkers or treatments.From our experience,we obtained a validation cohort of patients with pCCA who underwent radical resection(n=106 patients),and confirmed several potential biomarkers of pCCA.However,more prognostic biomarkers of pCCA are needed to predict the post-operational risk and guide the individual reatment for patients with pCCA.High mobility group A1(HMGA1)protein is a small nuclear protein that acts as a structural transcription factor.Under normal conditions,high expression of HMGA1 can occur during embryogenesis and in normal embryonic stem cells and adult stem cells.In mature differentiated tissues,HMGA1 is barely detected;however,some ectopic events in cancer,such as oncogenic transcription factors,epigenetic changes,and chromosomal translocation events,can induce abnormal up-regulation of HMGA1.Ectopic expression of HMGA1 has been widely reported in different tumors.And the overexpression of HMGA1 is associated with progression or poor prognosis in several types of cancers,including pancreatic adenocarcinoma and hepatocellular carcinoma.Moreover,HMGA1 has been shown to activate a variety of genes involved in tumorigenesis,tumor proliferation,migration,invasion,and epithelialmesenchymal transition(EMT).In iCCA,HMGA1 was expressed and can enhance the tumorigenicity.However,the clinical significance of HMGA1 in pCCA has not been elucidated.Changes in the number of chromosomes or aneuploidy are also characteristic of the occurrence of high-grade malignant tumors,which are caused by defects in the control of chromosome segregation by mitotic checkpoints during mitosis.Accurate chromosome segregation is essential to avoid chromosomal aneuploidy,which is a common feature of human malignancies,accounting for approximately 90%of human solid tumors and more than 50%of hematopoietic tumors.As a key modulator of chromosome segregation,thyroid hormone receptor interactor 13(TRIP 13)is expressed in various adult normal tissues and plays key roles in inactivation of the mitotic checkpoint complex(MCC).TRIP 13 is an oncoprotein in several types of cancers and is abnormally expressed in various human tumors,including head and neck cancer,hepatocellular carcinoma,colorectal cancer,breast cancer,etc..As a central protein in MCC inactivation,TRIP 13 promotes tumor progression mainly by altering the conformation of the terminal macromolecule.Overexpression of TRIP 13 in nonmalignant cells can increase the tumorigenicity of the cells;however,the role of TRIP 13 in pCCA are still unclear.Accordingly,in this study,we evaluated the expression and clinical significance of HMGA1 in a large cohort of patients with pCCA and examined the oncogenic functions of HMGA1.With in vitro and in vivo experiments and bioinformatics,we identified TRIP 13 as the key protein in HMGA1-induced stemness,EMT and metastasis of pCCA.In addition,we investigated the underlying mechanism of how HMGA1-TRIP13 axis promoted the progression of pCCA,and demonstrated that HMGA1-TRIP13 axis had a positive feedback loop dependent on c-Myc involvement.Part ?HMGA1 was correlated with unfavorable prognosis of pCCA,and promoted its progressionObjectives:Cholangiocarcinoma(CCA)is a highly malignant digestive system tumor originated from bile duct epithelium.Perihilar cholangiocarcinoma(pCCA)arises from the second-order intrahepatic bile ducts to the converging point of the cystic duct and the common bile duct,which is the most common type of the three subtypes of cholangiocarcinoma.In intrahepatic cholangiocarcinoma(iCCA),HMGA1 was over-expressed and can enhance the tumorigenicity.However,the clinical significance of HMGA1 in pCCA has not been elucidated.In this part of the study,we will analyze the expression of HMGA1 in pCCA to clarify the relationship between HMGA1 and clinicopathological features and prognosis of pCCA.By studying the effect of HMGA1 on the function of pCCA cell lines,we explore the carcinogenic effect of HMGA1 on pCCA.Methods:We first collected information on 325 patients with pCCA diagnosed in Qilu Hospital of Shandong University and underwent surgical resection from 2013 to 2018.According to certain inclusion criteria,106 patients with pCCA who underwent radical resection were futher selected from the main cohort as the validation cohort of this study.The transcriptome sequencing profiles of 8 pairs of pCCA tissues and adjacent normal tissues were used to identify the differentially expressed genes in pCCA.The expression of HMG family members in transcriptome sequencing profiles was analyzed.The expression of HMGA1 in pCCA and adjacent normal tissues was detected by qRT-PCR.Western blot was used to detect the expression of HMGA1 in fresh pCCA tissues and adjacent normal tissues.Immunohistochemistry was used to detect the expression of HMGA1 in 106 cases of pCCA.Survival curves were plotted using the Kaplan Meier method and survival rates were compared using the log-rank test.Univariate analysis and multivariate analysis were used to study the relationship between HMGA1 and clinicopathological conditions of patients with pCCA,and to explore the clinical significance of HMGA1.The expression of HMGA1 in different bile duct epithelial cell lines HIBEpiC,QBC-939,FRH-0201,RBE and HCCC-9810,and gallbladder carcinoma cell lines GBC-SD,NOZ and SGC-996 was detected.HMGA1 was silenced by shRNAs and overexpressed by lentivirus carrying LV-GV492-HMGA1.QBC-939(sh-HMGA1)and FRH-0201(GV492-HMGA1)cell lines were used to evaluate the effects of HMGA1 on cell proliferation,invasion and migration in vitro.The stable cell lines QBC-939(sh-HMGA1)and FRH-0201(GV492-HMGA1)were established and injected subcutaneously into nude mice for tumor formation.We further studied the 3D sphere formation experiment of QBC-939(sh-HMGA1,GV492-HMGA1)cells and the expression of epithelial mesenchymal transition(EMT)markers in QBC-939(sh-HMGA1)and FRH-0201(GV492-HMGA1).Findings:In HMG family,only HMGA1 was significantly up-regulated in pCCA compared with adjacent bile duct tissues.Immunohistochemistry was used to detect the expression of HMGA1 in tumor tissues of patients with pCCA,suggesting that HMGA1 may be a prognostic biomarker of pCCA.In multivariate analysis,HMGA1 was confirmed to be an independent prognostic biomarker of pCCA.In vitro,we verified that HMGA1 can promote pCCA cell proliferation,invasion,metastasis,3D sphere formation and EMT.Subcutaneous tumor formation experiments in nude mice also further verified the role of HMGA1 in promoting tumor proliferation in vivo.Conclusion:High mobility protein HMGA1 was highly expressed in pCCA,and HMGA1 was an independent prognostic biomarker of pCCA.HMGA1 can promoted pCCA cell proliferation,invasion,metastasis,3D sphere formation and EMT.This provided new research ideas for the molecular targeted therapy of pCCA.Part ? HMGA1 induces the progression of pCCA by promoting the transcription and expression of TRIP13Objectives:In the previous part of the study,we found that HMGA1 is a poor biomarker affecting the prognosis of pCCA.However,the downstream target of HMGA1 in pCCA is not clear.In previous studies,3 factors,including the thyroid hormone receptor interactor 13(TRIP 13),were identified in HMGA1 regulation,which were proved to be effective in promoting the progression of breast cancer.We speculate that HMGA1 may also affect the cell function of pCCA by regulating some cytokines,which in turn affects the prognosis of patients with pCCA.In this part,we aim to explore the downstream target factors of HMGA1 and their effects on pCCA.Method:The up-regulated genes in the previous transcriptome sequencing profiles were analyzed by thermography and hierarchical cluster analysis and compared with the protein factors regulated by HMGA1 in previous studies.The correlation between LRRC59/KIFC1/TRIP13 and HMGA1 mRNA levels was analyzed by using TCGA database.By regulating the expression of HMGA1,the mRNA expression of LRRC59,KIFC1 and TRIP13 was detected.The correlation between HMGA1 and TRIP 13 mRNA exp ression in fresh pCCA tissues was studied.The expression of TRIP 13 in 106 patients with pCCA was detected by immunohistochemical method,which was divided into low expression and high expression.We further examined the correlation between HMGA1 and TRIP13.We detected the expression of TRIP 13 in different biliary system cell lines.The expression of TRIP 13 in pCCA and adjacent normal tissues was detected by qRTPCR.Western blot was used to detect the expression of TRIP 13 in fresh pCCA tissues and adjacent normal tissues.Survival curves were plotted using the Kaplan Meier method and survival rates were compared using the log-rank test.Univariate and multivariate analysis were used to study the relationship between TRIP 13 and clinicopathological features of pCCA,and to explore the clinical significance of TRIP13.In vitro,we studied the effects of TRIP 13 on the pCCA cell proliferation,invasion,migration,3D sphere formation and EMT marker expression.Through in vitro and in vivo experiments,we further explored the role of TRIP 13 in the promotion of pCCA by HMGA1.Findings:TRIP 13 was a downstream cytokine regulated by HMGA1 in pCCA.It was highly expressed in pCCA and was positively correlated with the expression of HMGA1.The immunohistochemical method was used to detect the expression of TRIP 13 in the tumor tissues of patients with pCCA,suggesting that TRIP 13 may be a prognostic biomarker for pCCA.In multivariate analysis,it was confirmed that TRIP 13 was an independent prognostic biomarker for pCCA.The high expression of both TRIP13 and HMGA1 was a more sensitive prognostic factor than TRIP 13 or HMGA1 alone.In in vitro experiments,we verified that TRIP 13 can promote the pCCA cell proliferation,invasion,metastasis,3D sphere formation and EMT.In vitro and in vivo experiments,we confirmed that TRIP 13 was required in the progression of pCCA induced by HMGA1.Conclusion:TRIP 13 was a downstream cytokine of HMGA1 in pCCA,and it was positively correlated with the expression of HMGA1.The high expression of both TRIP 13 and HMGA1 was a more sensitive prognostic factor than TRIP 13 or HMGA1 alone.TRIP 13 could promote the pCCA cell proliferation,invasion,metastasis,3D sphere formation and EMT.TRIP 13 was required in the progression of pCCA induced by HMGA1.Part ? HMGA1-TRIP13 axis promotes stemness and epithelial mesenchymal transition of perihilar cholangiocarcinoma in a positive feedback loop dependent on c-MycObjectives:In our previous study,F-box/WD repeat domain protein 7(FBXW7)can inhibit CCA stemness and EMT.A recent study showed that the FBXW7 expression was inhibited by TRIP 13 in glioblastoma.However,the correlation between FBXW7 and TRIP 13 in the progression of pCCA has not yet been clarified.c-Myc is a well-known oncoprotein associated with stemnesss and a target for ubiquitination of FBXW7.We previously reported that c-Myc can promote the progress of pCCA.We speculate that TRIP 13 may affect the expression of c-Myc through FBXW7,and participate in the stemness and EMT of pCCA,thereby affecting the prognosis of pCCA patients.In this part,our purpose is to explore the role of FBXW7 in the progress of pCCA promoted by TRIP13.It is planned to explore the relationship between HMGA1TRIP13-c-Myc in pCCA and the mechanism that affects the progress of pCCA.Methods:We first detected the correlation between TRIP 13 and FBXW7 expression in pCCA cell lines QBC-939 and FRH-0201 by western blot and dual luciferase.The TRIP13 and FBXW7 mRNA levels in HMGA1 silenced/overexpressed xenografts were detected by qRT-PCR.To study the effect of silencing TRIP 13 and/or FBXW7 on the pCCA cells proliferation,invasion,migration,dryness and EMT.By silencing TRIP 13 and/or FBXW7,the changes in protein and mRNA levels of c-Myc,and the changes in c-Myc gene expression by ubiquitination inhibitor MG132 were detected.Through c-Myc inhibitor and lentivirus silencing c-Myc,the gene transcription and expression level changes of HMGA1 and TRIP 13 were detected.Western blot was used to detect the expression of HMGA1 and TRIP 13 in different time periods after silencing HMGA1 or TRIP13.We further studied the effects of c-Myc activator,cMyc inhibitor,TRIP 13 inhibitor and HMGA1 inhibitor on the expression of c-Myc,HMGA1 and TRIP13,and on the stemness,EMT,migration and invasion of pCCA cells.Findings:TRIP 13 could inhibit the expression and transcription of FBXW7 in QBC-939 and FRH-0201 cells,respectively.Silencing TRIP 13 suppressed pCCA proliferation and knocking down FBXW7 had contrary effects.Silencing FBXW7 promoted pCCA migration and invasion,and silencing TRIP 13 reduces the above effects.Silencing TRIP 13 gene attenuated the effects of silencing FBXW7 on the pCCA cells proliferation,migration,invasiveness and EMT.Silencing FBXW7 or TRIP 13 genes had a regulatory effect on the expression of c-Myc,but had almost no effect on the expression of c-Myc mRNA.Silencing the FBXW7 gene reduced the ubiquitination level of c-Myc.Both c-Myc inhibitor and silencing c-Myc could reduce the expression of HMGA1 and TRIP13 in pCCA cells.Moreover,c-Myc promoted the transcription of TRIP13 and HMGA1 in QBC-939 cells.Inhibitors of c-Myc,TRIP13 and HMGA1 all inhibited the expression of c-Myc,TRIP13 and HMGA1,reduced the number of stem cells of pCCA cells,and weakened the EMT of pCCA cells and the migration and invasion of pCCA cells under Wnt3a stimulation.Conclusion:FBXW7 inhibited the stemness and EMT of pCCA cells induced by TRIP 13.The HMGA1-TRIP13 axis promoted pCCA cell invasion,stemness and EMT in a positive feedback loop dependent on c-Myc.Inhibitors of HMGA1,TRIP13 or c-Myc blocked their feedback and inhibit the progression of pCCA.The link between HMGA1-TRIP13-c-Myc may be a promising method for the treatment of pCCA.