Effects Of Cell Migration Inducing Protein On Metastasis And Metabolism In Anoikis-resistant Prostate Cancer Cells

This dissertation is divided into three parts:Part l Establishment of Anoikis-resistant Model in Prostate Cancer Cells and Exploration of The Biological Behaviors Objective:To establish anoikis-resistant model in prostate cancer cells and identify the biological behaviors on anoikis-tolerance,migration,invasion and metabolism of the model.Methods:Human prostate cancer(PCa)PC-3 and DU 145 cells were continuously suspension cultured in ultra-low attachment plates for 7 days,then transferred into normal plates 'with attachment culture for 2 days and the re-adherent cells were regarded as anoikis-resistant.Apoptosis rates of parental and anoikis-resistant PCa cells with suspension culture for 48 hours were detected by flow cytometry with the FITC/P1 apoptosis detection kit.Transwell migration or invasion assays were employed to analyze cell migration or invasion of parental and anoikis-resistant PCa cells.Pyruvate,lactate and ATP levels of parental or anoikis-resistant PCa cells were detected by pyruvate assay kit,lactic acid assay kit and ATP assay kit,respectively.Western blot was used to detect differential protein expressions of pro-metastatic MMP2,VEGF and metabolism-related PDK4,LDHA in parental and anoikis-resistant PCa cells.Results:Flow cytometry indicated that anoikis-resistant PCa cells displayed markedly less detachment-induced apoptosis compared with that of parental cells.Transwell assays demonstrated that anoikis-resistant PCa cells presented remarkably more migration and invasion capacities than that of parental cells.Biochemical detections of cellular pyruvate,lactate and ATP showed that anoikis-resistant PCa cells exhibited obviously increased pyruvate production,lactate generation and ATP level when compared with parental cells.Accordingly,significantly elevated expressions of MMP2,VEGF,PDK4 and LDHA were observed in anoikis-resistant PCa cells compared with that of parental cells as illustrated by Western blot.Conclusions:Anoikis-resistant PCa cells exhibit significantly enhanced tolerance to detachment-induced apoptosis,migration,invasion capacities and elevated metabolic reprogramming than parental cells,which may result from increased expressions of MMP2,VEGF,PDK4 and LDHA in anoikis-resistant cells.Part 2 Filtration and Identification of Cell Migration Inducing Protein in Anoikis-resistant Prostate Cancer CellsObjective:To filtrate and identify the potential molecular target related to metastasis and metabolism in anoikis-resistant prostate cancer cells.Methods:Differentially expressed genes(DEGs)between anoikis-resistant PC-3 cells and corresponding parental cells were filtrated through whole human genome microarray analysis.On the basis of GO term and KEGG pathway enrichment analysis,10 candidate genes were verified by qRT-PCR and the object of the present study was further determined according to previous reports.Western blot was afterwards applied to affirm the differential expression of the target gene between anoikis-resistant and parental PCa cells.Immunohistochemistry(IHC)analysis to a tissue microarray(TMA)containing 90 pairs of PCa tissues and corresponding pericarcinous specimens was adopted to investigate the expression of the target gene and its correlations with clinical progression.Results:Totally 2370 DEGs were presented through whole human genome microarray analysis:1183 genes were up-regulated while 1187 genes were down-regulated significantly in anoikis-resistant PC-3 cells.The qRT-PCR affirmed that CYB5R2,SERPINE2,CEMIP,CCL2,ETV5 were up-regulated while LGSN,ENPP1,SORBS2,DCN,CSPG4 were down-regulated in the process of anoikis-resistance.Western blot also demonstrated that expressions of both CEMIP and its upstream ?-catenin markedly increased in anoikis-resistant PCa cells.The IHC analysis of TMA illustrated that expression of CEMIP was exceedingly higher in PCa tissues compared with that of pericarcinous counterparts,besides,expression of CEMIP in pericarcinous tissues was positively correlated to that in carcinoma tissues.Conclusions:Elevated expression of CEMIP is closely associated with anoikis-resistance of PCa cells and may be positively correlated to clinical progression.Part 3 Biological Effects and Expression Regulation Mechanisms of CEMIP in Anoikis-resistance of Prostate Cancer CellsObjective:To explore the biological effects and expression regulation mechanisms of CEMIP in anoikis-resistance of prostate cancer cells.Methods:The CEMIP CRISPR/Cas9 knockout(CEMIP-KO)plasmid and corresponding negative control(NC)plasmid were transfected into anoikis-resistant PCa cells respectively.Apoptosis rates of CEMIP-KO and NC anoikis-resistant PCa cells with suspension culture for 48 hours were detected by flow cytometry with the PE/7-AAD apoptosis detection kit.Transwell migration or invasion assays were employed to analyze cell migration or invasion of NC and CEMIP-KO cells.Pyruvate,lactate and ATP levels of NC or CEMIP-KO cells were detected by biochemical detection kits.Western blot was used to assess protein expressions of MMP2,VEGF,PDK4 and LDHA in NC and CEMIP-KO cells.After suspension culturing PCa cells for 1,3,5,7 days,expressions of total AMPKa and p-AMPKa were assessed by Western blot.When treating anoikis-resistant PCa cells with AMPK inhibitor Dorsomorphin,expressions of p-AMPK?,p-GSK3?,?-catenin and CEMLP were detected by Western blot.Expressions of ?-catenin were inhibited by siRNA for anoikis-resistant PC-3 cells while Wnt?-catenin pathway inhibitor XAV939 was applied in anoikis-resistant DU145 cells,subsequently,expressions of ?-catenin and CEMIP were detected by Western blot.Results:After knocking-out of CEMIP in anoikis-resistant PCa cells,flow cytometry indicated that CEMIP-KO cells presented dramatically increased detachment-induced apoptosis while Transwell assays illustrated impaired migration and invasion capacities in CEMIP-KO cells.In addition,cellular pyruvate,lactate and ATP levels significantly decreased in CEMIP-KO cells.Remarkably decreased expressions of MMP2,VEGF,PDK4 and LDHA were also observed in CEMIP-KO cells.For Western blot,expressions of p-AMPKa of PCa cells increased in a time-dependent manner upon suspension culture while p-AMPKa levels decreased in re-adherent PCa cells but were still higher than that of parental cells.However,there was not notable altering in the expressions of total AMPKa among all the samples.When treated with the AMPK inhibitor Dorsomorphin,expressions of p-AMPKa in anoikis-resistant PCa cells significantly decreased and in consequence,expressions of p-GSK3?,P-catenin and CEMIP markedly declined.When disrupting expressions of ?-catenin by siRNA or XAV939 in anoikis-resistant PCa cells,Western blot showed significantly decreased CEMIP expressions.Conclusions:CEMIP may not only influence anoikis-resistance,migration,invasion of anoikis-resistant PCa cells by mediating MMP2 and VEGF expressions,but also affect metabolism reprogramming by regulating PDK4 and LDHA expressions.Meanwhile,activation of AMPK/GSK3?/?-catenin cascade promotes the up-regulation of CEMIP in anoikis-resistance of PCa cells.