Expression And Function Of Thymine-DNA Glycosylase In Pancreatic Cancer

BackgroundPancreatic cancer(PC)is one of the most common malignant tumors with high mortality and poor prognosis.The symptoms of PC typically occur late,that makes patients are diagnosed at advanced stages.Current therapeutic options for PC are limited to surgical resection,systemic chemotherapy,and radiotherapy,but none of them are effective enough and the 5-year survival rate of patients with PC is less than 5%.In China,incidence and mortality of PC increase rapidly,and PC has become the ninth and sixth leading cause of cancer-related deaths for men and woman,respectively.Therefore,there is an urgent need to find out novel strategies for achieving better treatment outcome in PC patients.As the first oncogene isolated from human cancers,Ras is aberrantly activated in many cancers including PC.Oncogenic Ras signaling can promote DNA hypermethylation of tumor suppressor genes to facilitate cancer development.Recently,people learned that 5-methylcytosine,the main epigenetic modification of DNA,can be demethylated in a positive manner.Similar to DNA methylation,DNA demethylation also closely relates to carcinogenesis and cancer development.Studying the role of active DNA demethylation in cancer and its mechanism becomes significantly meaningful.and tested its activity using the luciferase reporter system.Then we will determine the transcription factor role in TDG expression.TDG expression will be recorded after overexprssion of the transcription factor by western blot and RT-PCR.We will also verify the interaction between TDG promoter and the transcription factors by chromatin immunoprecipitation method and then further compare the changes of interaction before and after Ras expression.Third,we compared the expression of TDG in Ras-transformed cells by over expression or knockdown the transcription factor.In order to verify the function and mechanism of TDG in PC development,we did the following studies.First,we will construct the TDG overexpression vector and establish cell lines stably expressing TDG,then compare cell growth ability in vitro and tumorigenesis ability in mice to the control cells.Second,western blot,flow cytometry and immunochemistry will be applied to determine to changes of cell proliferation and apoptosis by detecting the markers like Ki-67 and cleaved caspase-3 before and after TDG expression.Death acceptor Fas had found to be a target of TDG.Third,the expression of Fas will be determined by RT-PCR,western blot and flow cytometry in TDG overexpressed cells and Fas ligand(FasL)will be used to verify the expression of Fas by determining apoptosis through flow cytometry.Then the interaction between TDG and Fas promoter will be analyzed by chromatin immunoprecipitation assay.The status of promoter methylation,hydroxymethylation,histone modification and related transcription factor of Fas gene will also be analyzed after TDG overexpression.Finally,based on the above studies,we will test the anticancer potential of compounds that can affect the activity of TDG or its regulator.ResultsIn this study,we found that TDG was a downstream responder of Ras signaling pathway.Tumor suppressor gene ING4 can activate TDG transcription by specifically binding to its promoter.Active Ras promoted ING4 degradation in a calpain dependent manner.Furthermore,TDG bound to the promoter of death receptor Fas then activated Fas transcription by recruiting JMJD3,a histon demethylase of H3K27Me3.Finally,calpain inhibitor retarded in vivo tumor growth by stabilizing ING4 to activate TDG expression,ObjectiveCytosine methylation catalyzed by DNA methyltransferases(DNMTs)has been well studied.In contrast,the investigation of active DNA demethylation in mammalian somatic cells was still in its infancy.Even less is known about how Ras signaling regulates active DNA demethylation in human cancer cells.In this study,we plan to answer the question that if thymine DNA glycosylase(TDG)is regulated by Ras signaling pathway,if so,how it is regulated.We will also compare the expression of TDG in various pancreatic tissues and analyze the relationship between TDG expression and PC development.Then we will clarify the role of TDG in PC cells and the mechanism.Finally,we will treat PC cells with inhibitor or agonist of TDG or its regulators to verify our hypothesis and estimate the translational potential of them.MethodsIn order to study RAS signal pathway on the regulation of TDG expression and the mechanism,we did the following studies.First,comparing the TDG protein and mRNA expression level before and after RAS transformation by using western blot and RT-PCR method.Second,comparing the TDG protein and mRNA expression level in pancreatic cancer cells with active k-Ras mutation before and after Ras knock-down via RNAi by western blot and RT-PCR.Third,determining the EDG expression level in human pancreatic cancer tissue though immunohistochemistry method and analysing clinical significance of TDG expression.In order to study the mechanism of TDG expression regulated by Ras signal pathway,we did the following studies.First,cloning serious different length promoter fragment of TDG and verifying the shortest TDG active promoter with Luciferase reporter gene system.Then we will analyze the effect of Ras signal pathway on this promoter.Based on this,we then screen the potential transcription factors which can bind to the promoter through bioinformatics analysis.By using the siRNA of these potential transcription factors,we will identify the exact transcription factors involved in TDG expression regulation.Second,we will do mutation on the transcription factor specific binding site restore Fas expression and apoptotic response.ConclusionTDG is a tumor suppressor gene and its expression is suppressed by activated Ras signaling pathway in PC,activating TDG expression by using calpain inhibitors could be a potential option for the target therapy of PC and other Ras-driven cancers.