Epigenetic Regulation Of Interferon Mediated Anti-tumor Immune Response

Part I Epigenetic Enzyme Inhibitor Enhances Interferon-mediated Antitumor Immune Response and its Underlying MechanismIn the progression of tumor development,comparing to gene mutation,it is more common that CpG methylation of gene promoter region increases to suppress gene expression.DNA modifieation in genome is to add methyl group to the nucleotides.DNA methylation always happens in tumor to inhibit tumor suppressive genes expression(TSGs)which is very important.In hepatocellular carcinoma(HCC),there is correlation between the methylation of CpG region and survival ratio of patients.Therefore,inhibitors of DNA methyltransferase was used to treat tumor for its reduction of tumor growth and induction of tumor cell apoptosis.In recent years,with the development of tumor immunotherapy,in addition,DNA methyltransferase inhibitors can also induce tumor immunogens expression,which help immunotherapy through increasing immunogenicity of tumor.One of DNMT inhibitors,Azacitidine(5-Aza),can activate IFN-α signaling pathway through reducing CpG methylation of endogenous retroviruses(ERVs),which activate inflammatory status of tumor cells through dsRNA sensoring.Thus,the signal released by tumor can attract more immune cells to the tumor sites,and signaling type of activation or inhibition is very important.HCC cell after treated with DNMTi and HDACi inhibitor for a period,IFN-α was added to incubate with cells for 12 hours and expression elevation of ERVs,CTAs and ISGs were detected comparing to IFN-α treatment alone.Then,activation of downstream of IFN-α signaling pathway such as STAT was confirmed.After microarray chip analysis,we speculated some epigenetic modification enzymes,DNMTI and HDAC3,which were with higher expression in tumor tissues from microarray chip data,had similar trends of effects on tumor immunogenicity induction as inhibitors.The effects of inhibitors may change the H3K27me3 modification on TSS regions of immunogenic genes and stemness genes,which lead to the different expression pattern.We further confirmed that the induction of immunogenicity of tumor tissue in nude mice in vivo model can attract and activate more types of immune cells to the tumor sites,which may help tumor immunotherapy.In mechanism,it can change the methylation of CpG region and increase the effects with IFN-α.These results suggested that epigenetic enzymes have potentials in increasing the effects of tumor immunotherapy with IFN-α treatment as well.Our study combined tumor treatment by IFN-α and inflammatory status of tumor itself to induce endogenous elements expression and activate innate immune signaling pathway.The combination of IFN-α and epigenetic inhibitors can foirm positive loop to induce inflammation in tumor cells that are with less immune responses.We confirmed the inhibitors with higher IFN-a effects were involved in inducing immunogenicity and reducing sternness in mice in vivo model.We have further revealed the functions of adaptive immune cells infiltration in mice in vivo model.The difference of tumor formation in WT and RIG-I mice showed RIG-I signaling pathway is important in tumor immunogenicity and stermness.We will try to further illustrate how DNMTi affect inflammatory gene transcription dependent on interferon related pathway,which may offer a new way in tumor treatments by interferon.Part Ⅱ Regulation of Interferon-induced Immune Response by BAF180 of Chromatin Remodeling Complex SWi/SNFEpigenetic modification such as chromatin remodeling is involved in carcinogenesis.Our study Is focus on the function of BAF180,which is a subunit of SWI/SNF complex,in the regulation of inflammation and tumor.The mutation of BAF180 may affect carcinogenesis through DNA repair and genome stability procedure in clear cell renal cell carcinoma(ccRCC).However,it was reported that mutation of BAF180 may induce inflammatory signaling pathway,which attracts immune cells including CD8+T cells to the tumor sites.We have first confirmed the condition that BAF180 can affect the induction of inflammation in mouse peritoneal cell and MEF cell.When cells meet second stimulus,such as VSV infection,BAF180 can negatively regulate IL-6 and ISG15 production in cells which was first treated with IFN-a for a period.In tumor microenvironment,chronic inflammation in treatment such as IFN-a may induce immune tolerance in tumor,which obviously reduce the effects of immunotherapy.Then,we found that BAF180 silencing can induce the activation of STAT and Caspase signaling pathways,which was confirmed by western blot.In mechanism,when the expression of BAF180 was reduced,H3K27me3 modification was increasing at promoter regions of IL-6 and ISG15 genes.Usually,H3K27me3 helps to inhibit gene expression,but,in our experiments,there were two stimuli,so the upregulation of H3K27me3 may induce cells similar to their naive chromatin status,in which,cells are easy to respond to stimulus like dsRNA sensor signaling.To further identify our hypothesis,we performed Co-IP with anti-BAF180 antibody,and got H3K27me3 band in cell lysates by immunoblot.It was proved that H3K27me3 could bind to BAF180,which further raise two possible mechanisms.One is that BAF180 may bind to methyltransferase and block modification to histone.The other is that BAF180 can bind to H3K27me3 on chromatin and block the function of methyltransferase on chromatin sites within limited space after the first stimulation and maintain cells at immune tolerance status.We have illustrated BAF180 also can negatively regulate immunogenic genes expression through H3K27me3 modification.We will further illustrate the specificity of the interferon regulation on inflammatory genesand try to use construction methods to reveal more details in the future study,which may offer the possibility in combinated treatments in tumor.