| BACKGROUNDColorectal cancer is the 3rd commonly diagnosed types of cancer all over the world,Und the 4th most common causes of cancer dUath[1];thus great attention has been focused on the mechanism of tumorigenesis of gastrointestinal cancer.Signaling pathway especially Wnt/(3-catenin plays an important role in human cancers,particularly colorectal cancer.Greater than 90%of all Colorectal cancers will have a mutation of the Wnt/β-catenin signaling pathway[4].Our previous work demonstrated that these mutations prevent β-catenin degradation and lead to abnormal activation of Wnt signaling.However,how does β-catenin activate Wnt target genes in the cancer cells are not well understood.Recently,we screened a panel of DNA demethylases and surprisingly found that TDG is required for Wnt activation.We found that TDG interacted with TCF4 and CBP,coactivators of p-catenin.TDG is best known for its ability to remove T from G:T lesions.TDG has also been showed associated with several transcription factors,transcription coactivators.It is reported recently that TDG interacted with and functioned as a coactivator of p53 family proteins,TDG suppressed tumor cell growth[13,14].It supposed that TDG might be a tumor suppressor candidate[15].AIMWe hypothesized that TDG plays a role in colorectal cancer.To get a deeper insight into the function and regulation of TDG in the colorectal cancer,we will study mechanism of TDG on Wnt signaling,we will also study the expression of TDG in human colorectal cancer in this study.METHODS1.TOPFlash luciferase reporter assay was used to test the effect of TDG on Wnt promoter activity.Testing the effect of TDG on Wnt promoter activity by knocking down of TDG RT-PCR was carried to test the level of Wnt target genes after knocking down of TDG.To test whether TDG binds the promoters of Wnt signaling pathway factors,Chromatin Immunoprecipitation(ChIP)assay was perfomed.We made different TDG deletions constructions to analyse the functions of deletions on Wnt siganling through TOPFlash luciferase reporter assay.2.We carried Immunoprecipation(IP)to analyze the interaction of TDG/deletions of TDG and TCF4/CBP.We also analyze TDG and TCF4/CBP localization through Immunostainning.3.We made N140A and SUMO binding sites mutations,and analyzed the effects of TDG on Wnt promoter activity and the interactions between TDG mutations and TCF4/CBP through Immunoprecipation.Immunostainning was carried to test the localization of TDG mutations.4.We applied the ICG-001 which is the inhibitor of Wnt signaling to HCT116 cells and analyzed the expression level of TDG.5.Knocking down of TDG by shRNA to test the effects of TDG on proliferation of different colorectal cancer cell lines.We knocked down TDG in HT29 colorectal cancer cells and inject these cells into the flanks of athymic nude mice to test the effects of TDG on xenotraft tumor.6.We collected a lot of hunman colorectal cancer tumor tissues.Immuno-hitochemical stainning was carried to analysed the expression of TDG in colorectal cancer.TDG expression in tumor tissue was compared to that in adjacent and normal tissues based on paired t-tests.7.Microarray and patient clinical data from four colorectal cancer studies(Refs)were downloaded from the Oncomine database.Two-sample t-test was used to compare TDG expression in colorectal adenocarcinoma patients versus normal controls.One-way analysis of variance model was used to compare TDG expression in different tumor stages and in different tumor grades.Proportional hazards model was used to assess the association between TDG expression and patients’ survival time.Samples were also classified into TDG high expression and TDG low expression groups based on the median expression level.Kaplan-Meier curves and log-rank test were used to compare survival time in these two groups.RESULTS1.TDG upregulated the Wnt signaling.Knocking down of TDG by shRNAs inhibited the Wnt signaling.Besides,RT-PCR demonstrated that Survivin,Axin2 and c-myc lower express when knocking down of TDG TDG expression is positively correlated with survivin expression.We found that TDG indeed binded promoter of c-myc,which is the Wnt signaling target gene through Chromatin Immunoprecipitation(ChIP)assay.We also found that knockdown of TDG decreased localized histone acetylation on c-myc promoter as well.In addition,Topflash reporter assay demonstrated that both N terminal and C terminal of TDG are required for activating Wnt signaling.2.TDG interacted with TCF4 and CBP,coactivators of β-catenin,instead ofβ-catenin.TCF4 and CBP HAT domain are binded N terminal of TDG Moreover,TDG was detected in the nucleus and colocalized with TCF4 and CBP.3.N140A and SUMO binding site mutations of TDG inhibited Wnt signaling but had no effects of the interactions of TDG and TCF4/CBP.Moreover,N140A and D133A SUMO binding site mutations were detected both in nucleus and cytoplasm.4.ICG-001 inhibited the expression of TDG as well as Wnt signaling.KLF4 binded TDG promoter and inhibited the expression of TDG,as well as DnTCF4.5.Knocking down of TDG by shRNA inhibited the proliferation of defferent colorectal cancer cell lines.Besides,knocking down of TDG significantly inhibited the growth of xenograft tumors.6.Expression levels of TDG were significantly increased in human colorectal cancer tissues compared with the adjacent tissue(P=0.001)and in normal tissues(P=0.007)through the IHC of clinical colorectal cancer tissues.7.TDG expression is significantly higher in colorectal adenocarcinoma patients than in normal controls(P<0.001).There is no significant difference in TDG expression across different tumor stages.TDG expression appears to be higher in grade 3 patients.Lower TDG expression appears to be associated with longer survival time,although the association is not statistically significant.Based on the proportional hazards model,the hazard ratio is 1.53(95%CI 0.84 to 2.77,P =0.161)based on the data in Smith et al,and is 6.04(95%CI 0.96 to 37.84,P =0.055)based on the TCGA data.We also classified samples into TDG low expression and TDG high expression groups based on the median expression level.As shown in the figure,the survival curve of the low TDG expression group is higher compared to that of the high TDG expression group for both the data in Smith et al and the TCGA data.CONCLUSIONSIn our study,we provide evidence that TDG is required for Wnt activation.TDG interacted with TCF4 and CBP which are the Wnt transcriptianal complex.Wnt signaling also regulated expression of TDG.Knocking down of TDG by shRNAs inhibited Wnt signaling and inhibited the proliferation of colorectal cancer cells and the growth of xenograft tumors.In addition,the expression levels of TDG were significantly increased in human colorectal cancer tissues compared with the adjacent normal tissues.We also found that the expression of TDG was negatively colerated with the survival time of the colorectal cancer patient.These results suggest that TDG is a potential oncogene which enhances Wnt signaling in colorectal cancer cells,and that TDG is a potential marker for human cancers... |
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