FH535,a β-catenin Pathway Inhibitor,represses Growth And Angiogenesis Of Pancreatic Cancer Xenografts

Objective:Abnormal Wnt/β-catenin pathway activation is closely related to the development of many cancers,including pancreatic cancer.Studies have proved that β-catenin correlated with abnormal Wnt/β-catenin pathway activation.FH535 is β-catenin pathway inhibitor.In the present study,we investigated the mechanism of FH535 represses pancreatic cancer xenograft growth and angiogenesis.Meta-analysis and clinical samples were used to confirm the relationship between abnormal Wnt/β-catenin pathway activation and pancreatic cancer outcome.Providing a basis for further development of FH535 as a promising therapeutic agent for the treatment of pancreatic cancer.Methods:1.Microarray analyses were conducted to profile global gene expression patterns.The differences in genes expression were analyzed.2.Chromatin immunoprecipitation sequencing(ChIP-Seq)data analysis and identification of β-catenin–targeted genes.3.Gene Ontology(GO)analyses were performed for the differentially expressed genes and β-catenin–targeted genes.4.Real-time PCR and flow cytometry were used to test CD24 and CD44 expression at mRNA level and protein level.5.Protein arrays was used to test the expression of angiogenesis–related genes.6.Milliplex assay was used to measure VEGF,IL-6,IL-8,and TNF-α levels in culture.7.HUVEC tube formation potency was sued to test angiogenesis in vitro.8.Pancreatic cancer xenograft model was used to analysis xenograft growth.9.β-catenin,Ki-67,and CD34 expression in pancreatic cancer was tested by immunohistochemistry.MVD calculated by CD34.10.Meta-analysis was performed a confirm the relationship between abnormal β-catenin pathway activation and the outcome of pancreatic cancer on a larger scale.Kaplan–Meier curves were constructed to analyze suivival date.Results:1.Gene Ontology(GO)analyzed Microarray analyses data and Chromatin immunoprecipitation sequencing(ChIP-Seq)data.We confirmed the differentially expressed genes encode a wide range of functions,including cell division,DNA damage and repair,and stem cell maintenance;The β-catenin–targeted genes not only regulate cellular biological processes themselves,but also participate in cancer microenviroment remodeling,such as angiogenesis and extracellular martrix organization.2.FH535 repressed the mRNA and protein expression of both CD24 and CD44 and downregulated CD24+/CD44+ populations,the presumed pancreatic CSCs.3.Evaluated the mRNA expression of the proangiogenic-related genes after FH535 treatment;the VEGF,VEGFB,FLT4,ANG,ANGPT2,AKT1,PLAU,CXCL8,IL10,IL17 F,TGFA,TGFB1,FGF1,FGF13,FGFR3,PGF,HGF,IGF1,PDGFA,THPO,TNF-α,IFN-γ,TIMP1,MMP2 were downregulated at mRNA level.And evaluated the protein expression of the proangiogenic-related genes after FH535 treatment;the Bfgf,IFN-γ,TIMP1,TIMP2,THPO,VEGFA,VEGFD,ANGPT1,ANGPT2,IL-1β,MCP-4,Upar,VEGFR2,VEGFR3 were downregulated at protein level.FH535 downregulated the proangiogenic genes ANGPT2,FLT4,IFN-γ,PLAUR,THPO,TIMP1,and VEGF expression at both mRNA and protein level.FH535 downregulated the VEGF,IL-6,IL-8,and TNF-α levels in culture medium.4.FH535 repressed HUVEC tube formation potency,therefore,FH535 repressed pancreatic cancer angiogenesis in vitor.5.The tumor volume and body weight of FH535-treated group significant low to the control,FH535 had an anti-tumor effect against pancreatic cancer xenograft.6.FH535 repressed MVD in pancreatic cancer xenograft,therefore,FH535 repressed pancreatic cancer angiogenesis in vitor.7.Unclear β-catenin–positive group had higher MVD and Ki-67 than unclear β-catenin–negative group.Patients with higher MVD had shorter OS.MVD and Ki-67 had positive correlation.Unclear β-catenin–positive group had shorter OS than unclear β-catenin–negative group.8.Abnormal β-catenin expression was associated with significantly increased pancreatic cancer mortality risk,suggesting abnormal β-catenin pathway activation was associated with unfavorable outcome,rendering the abnormal β-catenin pathway activation was associated with unfarvorable outcome.Conclusion:FH535 plays a pivotal role in pancreatic CSC stemness maintenance and angiogenesis.Our present results indicated that β-catenin pathway inhibitor FH535,through impaired pancreatic CSC stemness,depressed the proangiogenic genes expression and downregulated the secreted proangiogenic factors repressed pancreatic cancer xenograft growth and angiogenesis,shedding new lights on the therapy of pancreatic cancer.