Identification And Characterization Of 3-Deazaneplanocin A (DZNep) As A Novel Chromatin Modification Agent Targeting Cancer Epigenetic Process

Cancer is a great threat to human health and is one of the most lethiferous diseases.With the fast growing epigenetic research during last decade,cancer now is known as a phenotypic end point of numerous genetics and/or epigenetic alternation that have accumulated within cells.It is clear that epigenetic modifications are major contributors for formation and progression of tumor,so targeting epigenetic modifications has been put under the spotlight of investigation.Although having this principle in hand,there is one question has to be addressed that is how to translate research paper to clinic benefit. The eldest but still most efficient way is using chemical compound to achieve this aim.In this study I identified a novel compound 3-Deazaneplanocin A(DZNep) affecting epigenetic regulation and applied it in multiple cancer cells to point out its working mechanism.With the exciting finding that DZNep target PcG proteins,we use DZNep as a tool to fish out some important PcG target genes and identify their function in cancer biology.We also pick up one well known tumor suppressor CDKN1C and detailed characterized the epigenetic modification pattern around its transcription start site.And this EZH2 target gene show strong prognosis value in breast cancer patients.1.Screening compound library and identifying 3-Deazaneplanocin A (DZNep) can induce cell apoptosis in a E2F1 depend mannerThe development of cancer depends on delicate balance between cell proliferation and programmed cell death.Rb/E2F1 pathway plays an important role in regulation cell cycle. It has been reported that epigenetic anticancer drug SAHA can induce cancer specific apoptosis through activating E2F1 downstream target Bim.Here I carry out a drug screen to search more SAHA-like compounds which can induce apoptosis through E2F1 pathway.A National Cancer Institute(NCI) compound library including around 4000 compounds was submitted to the established screen platform based on HCT 116 p53 null ER-E2F1 cell line.After screen and validation through different method and cell line system,I have identified one compound named 3-Deazaneplanocin A(DZNep).The ability of DZNep to induce E2F1-dependent apoptosis is confirmed through analyzing different apoptosis markers.DZNep can promote E2F1 pro-apoptosis target genes expression and these highly active genes led to apoptosis.2.Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cellsPolycomb-repressive complex 2(PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy.Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A(DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells.We found that DZNep effectively depleted cellular levels of PRC2 components EZH2,SUZ12 and EED while also inhibited associated histone H3 Lys 27 methylation(but not H3 Lys 9 methylation).By integrating RNA interference(RNAi), genome-wide expression analysis,and chromatin immunoprecipitation(CHIP) studies,we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep.We further demonstrate that the preferential reactivation of a set of these genes by DZNep,including a novel apoptosis affector, FBXO32,contributes to DZNep-induced apoptosis in breast cancer cells.Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy.3.Tumor Suppressor CDKN1C(p57^KIP2) is an EZH2 target and transcriptionally repressed in breast cancer cells through coordinated action of histone modificationsCDKN1C(encoding p57^KIP2) is a member of cyclin-dependent kinase(CDK) inhibitor tumor suppressor family that is often transcriptionally inactivated in human cancer via promoter DNA methylation.In this study,we show that CDKN1C repression in breast cancer cells is not associated with the status of DNA methylation.We demonstrate that CDKN1C transcription is repressed by Polycomb protein EZH2 and associated histone H3 lysine 27 trimethylation(H3K27me3).Treatment of breast cancer cells with Sadenosylhomocysteine hydrolase inhibitor 3-Deazaneplanosin A(DZNep) which inhibits EZH2 protein expression and H3K27me3,together with histone deacetylase inhibitor Trichostatin A(TSA) caused a robust induction of CDKN1C expression through effective reversal of histone modifications.Depletion of EZH2 alone is insufficient to activate CDKN1C but does so in the presence of TSA,suggesting that histone methylation and deacetylation coordinate to repress CDKN1C expression.We further show that CDKN1C expression is underexpressed in multiple human cancers including breast cancer,and the combination of high EZH2 and low CDKN1C expression predicts an extreme poor prognosis of breast cancer.Taken together,these findings reveal a novel mechanism underlying CDKN1C repression in breast cancer and its prognostic value as an EZH2 target.In particular we point out an effective approach for pharmacological reactivation of CDKN1C tumor suppressor in breast cancer.