【作者】 杨晓静；

【导师】 邵荣光； Yu Qiang；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 癌症对于人类健康有巨大威胁并且是导致人类死亡的最常见的疾病之一。经过前一段时间里表观遗传学研究的快速发展,癌症现在已经被认为是无数遗传学和表观遗传学的变异在细胞内累计的最终结果。表观遗传修饰对于肿瘤形成和发展有主要的推动作用。所以靶向表观遗传修饰药物的研究受到人们高度重视。尽管我们已经掌握了这个理论,但是仍有一个问题需要被解答,那就是怎样将研究结果转换到临床的应用。最古老但也是最有效的方法就是使用化学化合物来实现这个目标。本研究中我确认了一个崭新的作用于表观遗传调控的化合物3-Deazaneplanocin A（DZNep）,并且将其应用于多种肿瘤细胞后发现了DZNep诱导凋亡的能力和它的作用机制。DZNep可以靶向PRC2蛋白,我们使用DZNep作为工具发现一些重要的PRC2靶基因,并且确认他们在肿瘤生物学中的功能。我也选择一个已知的肿瘤抑制基因CDKN1C来加以研究,详细分析其转录起始位点的表观遗传修饰模式。并且这个EZH2靶基因在乳腺癌患者中有强的预后价值。1.筛选化合物库后发现3-Deazaneplanocin A（DZNep）并确认其诱导肿瘤细胞E2F1依赖凋亡的活性肿瘤的发生取决于细胞生长和程序死亡是否能保持精确平衡。Rb/E2F1通路在调控细胞周期中起到重要作用。已经有报道发现表观遗传抗肿瘤药物SAHA能通过激活E2F1下游靶基因Bim诱导肿瘤特异的凋亡。这里我展开大规模药物筛选以发现更多的类似SAHA可以通过E2F1通路诱导凋亡的药物。我用基于HCT 116 p53 null ER-E2F1细胞株的筛选平台筛选了一个包括了4000个化合物National Cancer Institute（NCI）化合物库。经过不同方法和细胞系的筛选和确认,我发现一个活性化合物3-Deazaneplanocin A（DZNep）。DZNep诱导E2F1依赖凋亡的能力通过检测不同凋亡标志被确认。DZNep可以促进E2F1促凋亡的靶基因表达,这些高度表达的基因最终导致细胞凋亡。2通过药理学方法抑制Polycomb-repressive complex 2介导的基因抑制可选择性诱导肿瘤细胞凋亡Polycomb-repressive complex 2（PRC2）介导的组蛋白甲基化在肿瘤基因异常沉默中起到重点作用,并且成为癌症治疗的潜在靶点。这里我发现S-adenosylhomocysteine水解酶抑制剂3-Deazaneplanocin A（DZNep）有效诱导肿瘤细胞凋亡但对正常细胞没有作用。我发现DZNep有效消除细胞内PRC2组分包括EZH2,SUZ12和EED蛋白并且抑制相关的组蛋白H3赖氨酸27位三甲基化（但不是组蛋白H3赖氨酸9位三甲基化）。通过RNA干扰分析,全基因组表达分析和染色质免疫沉淀研究,我确认一组突出的基因在乳腺肿瘤中被PRC2抑制,并且它们的表达可被DZNep重新激活。我进一步展示DZNep优先的激活包括新发现的凋亡作用子FBXO32的一系列基因,都对DZNep诱导的乳腺肿瘤细胞凋亡起作用。我的结果显示DZNep作为崭新的改变染色质的化合物具有独一无二的特点,并且提出了通过药理学DZNep处理来逆转PRC2介导的基因抑制将可能是一种崭新的治疗肿瘤的方法。3.肿瘤抑制基因CDKN1C（p57KIP2）是EZH2靶标并且在乳腺肿瘤中通过不同组蛋白修饰协同作用而被抑制CDKN1C(编码p57^KIP2)是周期素依赖性激酶（CDK）抑制剂肿瘤抑制家族中的一员,它在人类肿瘤通常通过启动子区域的DNA甲基化而被抑制。在这一部分工作中我发现CDKN1C在乳腺肿瘤细胞中的抑制与DNA甲基化状态无关。我证明CDKN1C转录通过Polycomb蛋白EZH2及其相关组蛋白赖氨酸27位甲基化而抑制。用S-adenosylhomocysteine水解酶抑制剂3-Deazaneplanosin A（DZNep）处理导致EZH2蛋白表达和H3K27me3的抑制,如果联合使用组蛋白去乙酰酶抑制剂Trichostatin A（TSA）来有效逆转组蛋白修饰将导致一个CDKN1C表达的惊人升高。单独消除EZH2并不足以激活CDKN1C但是加入TSA就可以达到这个目的。这就显示了组蛋白甲基化和去乙酰化协同而抑制CDKN1C表达。进一步研究显示CDKN1C表达抑制在包括乳腺肿瘤的多种人类肿瘤中存在。并且联合高EZH2和低CDKN1C表达水平可以预测一个乳腺肿瘤的特别不良预后。总的来说这些发现揭示在乳腺肿瘤中CDKN1C表达抑制的一个崭新的机制,并且发现其作为EZH2靶点的预后价值。我在这里要特别指出了一个在乳腺肿瘤中通过药理学途径重新激活CDKN1C肿瘤抑制基因的有效方法。更多还原

【Abstract】 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.更多还原