| Background:Pancreatic cancer is one of the most common malignant tumors,and pancreatic ductal adenocarcinoma?PDAC?accounts for 80-90%.Metastasis is an important cause of death.About 90%of PDAC patients died from early metastasis,with a 5-year survival rate of only7%,although significant advance has been made in the fields of radiotherapy,chemotherapy and immunotherapy.Epithelial-mesenchymal transition?EMT?plays an important role in the metastasis of PDAC.Studies have shown that EMT is an independent risk factor for the prognosis of pancreatic cancer,for that the higher degree of interstitialization,the worse the prognosis.Remarkably,high expression of EMT-associated markers can be detected in the very early stages of pancreatic cancer development,such as intraepithelial neoplasia and acinar-catheter metaplasia.Therefore,in-depth exploration of the regulatory factors and mechanisms of the EMT process is of great significance for a comprehensive understanding of the metastasis of pancreatic cancer,and help to find new targets for the treatment of pancreatic cancer metastasis.EMT is an adjustment of tumor cells accompany with metabolism reprogramming to adapt to microenvironment.Recent efforts have shown that tumor cell metabolism reprogramming may play an important regulatory role in EMT phenotype.The metabolic status of tumor cells is significantly correlated with EMT,but the underlying mechanism need more exploration.Aldehyde dehydrogenase?ALDHs?is a family of key enzymes that regulate endogenous aldehyde metabolism.It contains 19 subtypes and is widely involved in various metabolic processes.The expression status,especially high in a variety of cancer stem cells,is closely related to invasion and migration,therapeutic resistance.However,the relationship between ALDH family members and the EMT phenotype of pancreatic cancer cells is still unclear.Furthermore,the molecular basis and therapeutic significance remain further investigation.Purposes:1.Analysis of metabolic regulatory genes associated with EMT gene sets in pancreatic cancer.2.To investigate the role of ALDH1A3 in invasion and migration of pancreatic cancer.And reveal the underlying mechanism.3.To explore the value of ALDH1A3 in diagnosis and prognosis of patients with pancreatic cancer.4.Screening for ALDH1A3 specific inhibitors and assessing its role in pancreatic cancer metastasis.Methods:1.The genes differentially expressed in pancreatic cancer and adjacent tissue and closely related to EMT status were screened out through the analysis of pancreatic cancer data in the GEO database?GSE71729,GSE62165,GSE15471 and GSE11838?.Then the relationship between DEGs and patient survival was further analyzed using TCGA database.2.Immunohistochemical staining of tissue microarray?HPan-Ade170Sur-01,includes99 cancer patients and 71 adjacent mucosa tissues?was used to analyze the correlation between ALDH1A3 and pathological information and survival of pancreatic cancer patients.3.Knock-down the expression of ALDH1A3 in pancreatic cancer cells?CAPAN1,HS766T and PANC1?by lentivirus,reveal the characteristics of those cells by MTS,colony formation assay and transwell experiment.4.To reveal the mechanism involved in the tumorigenesis of pancreatic cancer by detecting the RNA?CDH1,CDH2,SNAI1,SNAI2,VIM,ZEB1,ZEB2,CTNNB1,TWIST1and SIP1?and protein expression levels?E-cadherin,N-cadherin and Snail1?.5.Screening for small molecule inhibitors specific for ALDH1A3 by molecular docking.Using Thermal surge assay and Isothermal Titration Calorimetry?ITC?to verify the specific binding of small molecules to ALDH1A3 recombinant protein.to Assessing the specific binding in Pancreatic Cancer Cells by Thermal Drift Experiment.6.Verifing the influence of ALDH1A3 inhibitor-YD1701 in tumorigenesis of pancreatic cancer by in vitro and in vivo experiments.7.The experimental data was processed using SPSS 22.0 and GraphPad Prism 5,and was shown as?±s.The comparison between groups was performed by independent sample t test.The Kaplan-Meier survival analysis was performed by Log-Rank test.The contingency table analysis was performed using chi-square test.And univariate/multivariate analysis of variance for prognostic predictors was based on Cox proportional hazard?CPH?regression model.The maximum likelihood method and Wald test parameter were used for parameter estimation and test.Test level:?=0.05.Results:1.Bioinformatic analysis identified that 19 genes were significantly different expressed between tumor and adjacent tissue in multiple pancreatic cancer datasets?GSE71729,GSE62165,GSE15471,and GSE11838?and positively correlated with EMT activation.Analysing TCGA database revealed that the EMT pathway is significantly enriched in pancreatic cancer patients with high ALDH1A3 expression?NES?=2.03,FDR=0.004),and patients with high expression?OS=660 d,n=86 vs.OS=840 d,n=84?of ALDH1A3 owned shorter survival time?P=0.029?.2.ALDH1A3 was involved in the regulation of invasion and migration in pancreatic cancer.Through in vitro experiments,we found that the invasion and migration abilities of pancreatic cancer cells are significantly reduced after knocking down ALDH1A3.3.The protein level of ALDH1A3 in pancreatic cancer cell lines was higher than that in normal pancreatic epithelial cells?HpDE6-C7?.After knocking down ALDH1A3,EMT pathway related genes changed significantly.E-cadherin was increased.N-cadherin,Snail1and Slug were reduced.4.ALDH1A3 was positively correlated with the malignancy of pancreatic cancer.Immunohistochemical staining analysis of pancreatic cancer tissue microarray showed that the protein expression level of ALDH1A3 in pancreatic cancer tissues was significantly higher than that in adjacent tissues?5.537±0.1363,n=98 vs.4.752±0.1688,n=71.P=0.0004?,and positively correlated with the clinical stage?P=0.0136?Multivariate survival analysis based on Cox proportional hazards?CPH?models defines high ALDH1A3expression as an unfavorable prognostic marker and risk factor in pancreatic cancer patients.5.We confirmed that the ALDH1A3 inhibitor?YD1701?is specifically binding with ALDH1A3 through thermal surge(Kd=9.75×10-6),ITC(N:2.02±0.0797 Sites,K:7.36 E5±1.61 E5 M-1,?H:2.441 E4±1478 cal/mol,?S:109 cal/mol/deg)and thermal drift experiments.In vitro experiments confirmed that ALDH1A3 inhibitor can significantly inhibit the invasion and migration of pancreatic cancer.We constructed orthotopic pancreatic cancer mouse model and found that YD1701 could prolong orthotopic transplanted mice survival time,as well as reducing the efficiency of lung metastasis rate.Conclusions:1.The expression level of ALDH1A3 is significantly correlated with clinical stage,survival time and prognosis.The higher the expression,the later the stage and the worse prognosis.2.ALDH1A3 can regulate EMT by Snail1/E-cadherin pathway thus inhibit the invasion and migration of pancreatic cancer cells.3.YD1701 can directly bind to ALDH1A3 and inhibit its enzymatic activity.4.ALDH1A3 might be a promising therapeutic target,for that YD1701 can inhibit pancreatic cancer invasion and migration. |
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