EZH2作为抗肿瘤免疫治疗靶点研究进展

多梳蛋白复合体(PcG)的核心亚基zeste基因增强子同源物2(Enhancer of zeste homolog2,EZH2)是一种组蛋白甲基转移酶,参与维持细胞密度、干细胞多能性、细胞周期调节等重要的生理作用。研究发现,EZH2在多种肿瘤组织中高表达,是促进肿瘤发生和发展的致癌因子。由于EZH2在正常组织中低表达或者不表达,使其新近被鉴定为一种肿瘤相关抗原。已经在EZH2蛋白分子中鉴定出多条特异性抗原肽,这些抗原肽能激发机体免疫细胞对EZH2表达异常增高肿瘤细胞的杀伤活性。上述研究提示,EZH2可能是一种新的抗肿瘤治疗分子靶点,并在肿瘤免疫治疗中具有潜在的应用价值。就该领域的最新研究进展作一简要综述。     

EZH2通过抑制miR-608基因表达促进结肠癌细胞增殖

探讨EZH2对结肠癌细胞增殖调控作用以及具体作用机制。通过对结肠癌细胞系SW480以及HCT116进行EZH2基因沉默,以检测EZH2对结肠癌细胞的增殖调控作用。MTT检法测细胞的增殖,流式细胞仪检测细胞周期及荧光定量PCR检测周期相关基因Cyclin D1、P15、P21以及mi R-608的表达变化。si RNA转染后,结肠癌细胞中EZH2的表达明显下降(p0.001),细胞增殖受到明显抑制(p0.05,p0.01或p0.001);si RNA组与阴性对照相比,G_1期细胞比例增高,G_2/M、S期细胞相对减少,cyclin D基因表达下调(p0.01,p0.05),P15、P21表达上调(p0.01)。通过荧光定量PCR发现,结肠癌细胞增殖抑制基因mi R-608在EZH2基因沉默组表达量显著上调(p0.001)。萤光素酶活性测试结果表明,EZH2在SW480和HCT116细胞中直接调控miR-608的基因转录(p0.001)。此外,miR-608基因沉默阻断siEZH2对结肠癌细胞增殖的调控作用。本研究发现了EZH2对结肠癌细胞增殖的显著促进作用,其具体作用机制在于抑制miR-608基因表达。因此,EZH2可望成为结肠癌潜在的治疗靶标。     

EZH2与肿瘤的研究进展

EZH2作为甲基化转移酶,通过催化组蛋白H3K27三甲基化,介导基因表达沉默。EZH2作为PRC2复合物的核心成分,广泛参与细胞分化、维持干细胞多能性、在肿瘤发生过程中发挥重要作用。EZH2在多种肿瘤细胞中高表达,其表达水平与肿瘤病人的预后具有相关性。作为潜在的抗肿瘤治疗靶点,EZH2的表达调控及其EZH2抑制剂的研发已经得到深入研究。本文综述了近年来有关EZH2与肿瘤防治研究的新进展。     

Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义

目的探讨Bmi-1和EZH2基因在小细胞肺癌中的表达及其意义。方法收集武汉大学人民医院病理科2006-2009年非小细胞肺癌存档蜡块40例,采用免疫组织化学S-P法检测40例非小细胞肺癌及癌旁组织中Bmi-1和EZH2基因的表达水平,并采用HPIAS-1000高清晰度彩色病理图文报告管理系统对Bmi-1和EZH2基因的表达进行定量分析,用SPSS11.5软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 1.Bmi-1在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织Bm i-1的表达明显高于癌旁组织,差异有显著性(P<0.05)。2.EZH2基因的表达EZH2基因在非小细胞肺癌中呈高表达,癌旁组织中呈低表达。非小细胞肺癌组织中EZH2基因的表达明显高于癌旁组织(P<0.05)。结论 EZH2和Bmi-1基因对肺癌的生长有着不同的调节作用,在肺癌的发生、发展中发挥重要的作用。     

EZH2在卵巢癌中的研究进展

果蝇zeste基因增强子人类同源2(enhancer of zeste homolog 2,EZH2)是近年来肿瘤领域研究的热门靶点基因,其在卵巢恶性肿瘤中表达增高,与卵巢癌的发生发展及预后密切相关。EZH2作为一种重要的负性表观遗传调控蛋白,可通过催化组蛋白3赖氨酸27三甲基化(H3K27me3)和调控新型钙黏蛋白分子CD13等影响卵巢细胞的增殖、迁移和侵袭,对EZH2的深入研究将为治疗卵巢恶性肿瘤提供新的分子靶点。本文就该领域的最新研究进展作一简要综述。     

微RNA-101在肿瘤中的作用

微RNA(microRNA)是内源性发挥转录后调控功能的单链小RNA,种类繁多,在细胞周期调控、凋亡调控、肿瘤的进展等方面起重要作用。近期研究发现,微RNA在功能上表现为癌基因与抑癌基因与前列腺癌、膀胱癌、肝癌、乳腺癌等肿瘤的发生、发展关系密切。EZH2基因的生物学行为与癌基因类似,微RNA-101主要通过调控EZH2的表达量和表观遗传学特征调控其功能。微RNA-101的缺失或表达下调促使组织细胞分裂、增殖,最终可能导致肿瘤的发生与浸润。因此,探寻微RNA-101的生物学作用具有重要意义。     

EZH2在心脏与血管发育中的作用

Zeste基因增强子人类同源物2(enhancer of zeste homolog 2, EZH2)是多梳蛋白抑制复合物2(polycomb repressive complex 2, PRC2)的主要元件之一，利用组蛋白甲基化酶活性发挥经典作用，抑制靶基因的表达。此外，EZH2通过甲基化其他蛋白，作为蛋白支架分子募集转录相关分子介导转录激活，与lncRNA及miRNA相互作用等非经典途径调控各项生命活动，与干细胞分化和组织器官发育关系密切。EZH2及其功能相关分子在心脏发育、血管发生等过程中发挥着至关重要的作用。靶向敲除小鼠心脏Ezh2基因会影响心肌组织及内皮源性组织的正常发育，造成广泛性的心脏发育缺陷。EZH2参与调控正常组织和肿瘤组织的血管生成，维持新生血管完整性，并参与调控内皮间质化和内皮造血转化。本文探讨了EZH2在心脏和血管发生领域的影响效应、调控机制，及其与相关疾病的关系。     

EZH2抑制剂与肿瘤治疗

果蝇Zeste基因增强子人类同源物2(Drosophila zeste gene enhancer of human homolog 2,EZH2)是一种参与基因负性表观遗传调控的甲基转移酶。研究发现,EZH2在多种肿瘤组织中高表达,并与肿瘤的发生发展和侵袭转移密切相关,由此成为抗肿瘤治疗新的分子靶点。目前已研发出多种EZH2抑制剂用于肿瘤的基础和临床治疗研究,并取得令人鼓舞的结果。本文将对EZH2小分子抑制剂的研究进展做简要综述。     

EZH2蛋白在食管上皮永生化细胞系和恶性转化细胞系中的表达

目的研究EZH2蛋白在食管上皮永生化细胞系(SHEE)和恶性转化细胞系(SHEEC)中的表达。方法采用免疫细胞化学染色、免疫印迹分析和流式细胞术检测两种细胞系EZH2蛋白的表达。结果两种细胞EZH2蛋白染色均呈阳性,阳性反应定位于细胞核,部分细胞胞浆也有阳性着色。免疫印迹分析表明SHEEC细胞和SHEE细胞的总蛋白、核蛋白在分子质量约90ku的位置均出现特异性印迹条带。SHEE细胞中EZH2蛋白的表达强于SHEEC细胞(P<0.05)。流式细胞术显示EZH2蛋白在两种细胞中均有表达,两者的平均荧光强度无明显差别;阳性细胞率均较高,其中SHEE细胞阳性率高于SHEEC细胞。结论EZH2蛋白在SHEE细胞和SHEEC细胞中高表达可能参与了它们的恶性改变;而EZH2蛋白在两种细胞系中的差异表达可能与细胞的分化程度及来源于胚胎食管上皮细胞相关。     

siRNA下调EZH2 基因对肾癌细胞系769-P增殖的影响

目的：运用小干扰RNA下调果蝇zeste 基因增强子人类同源物（enhancer of zeste homolog 2,EZH2）在肾癌细胞系769-P 中 的表达,明确其对肾癌细胞增殖的影响。方法：将处于对数生长期769-P 细胞分为实验组（experiment group）、阴性对照组(negative group)、空白对照组（blank group）,合成靶向EZH2 基因的小干扰RNA片段（EZH2-siRNA）和无效序列片段后,通过脂质体介导分 别转染至实理组和阴性对照组,空白对照组未做任何处理。以qRealtime-PCR 检测EZH2 基因mRNA 水平的变化情况,以MTT 法检测各组细胞增殖变化;流式细胞术（FCM）检测转染后细胞周期变化情况。结果：实理组中EZH2 在mRNA 表达水平明显受 抑制;MTT实验中第4 天始,实验组中769-P 细胞的增殖能力开始受抑制,第5 天时实验组细胞抑制更明显,与阴性对照组和空 白组比较差异有统计学意义（P < 0.05）。siRNA 转染后实验组中G0/G1 期细胞比例明显增多(81.32± 3.14)%,与阴性对照组 (44.13± 1.52)%和空白对照组(45.71± 2.32)%差异有统计学意义。结论：EZH2-siRNA 可有效下调并抑制肾癌细胞769-P的增殖, EZH2在肾癌的发生、发展中发挥了重要作用,为下一步研究肾癌基因治疗提供了理论支持。     

Decreased expression of EZH2 reactivates RASSF2A by reversal of promoter methylation in breast cancer cells

EZH2, the catalytic subunit of polycomb repressor complex 2, has oncogenic properties, whereas RASSF2A, a Ras association domain family protein, has a tumor suppressor role in many types of human cancer. However, the interrelationship between these two genes remains unclear. Here, we showed that the downregulation of EZH2 reduces CpG island methylation of the RASSF2A promoter, thereby leading to increased RASSF2A expression. Our findings also showed that knockdown of EZH2 increased RASSF2A expression in the human breast cancer cell line MCF‐7 in cooperation with DNMT1. This was similar to the effect of 5‐Aza‐CdR, a DNA methylation inhibitor that reactivates tumor suppressor genes and activated RASSF2A expression in our study. The EZH2 inhibitor DZNep markedly suppressed the proliferation, migration, and invasion of MCF‐7 cells treated with ADR and TAM. EZH2 inhibits the expression of tumor suppressor gene RASSF2A via promoter hypermethylation. Thus, it plays an important role in tumorigenesis and is a potential therapeutic target for the treatment of breast cancer.     

Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

Enhancer of Zeste homolog 2 (EZH2) is overexpressed in recurrent nasopharyngeal carcinoma and is regulated by miR-26a, miR-101, and miR-98

There is increasing evidence supporting the role of members of the polycomb group (PcG) gene family in tumor development and progression. However, their precise role in tumorigenesis and mechanisms of their regulation remain to be elucidated. Using nasopharyngeal carcinoma (NPC) as a disease model, a comprehensive analysis was undertaken on the clinical significance of EZH2 expression, identification of the cellular processes regulated by EZH2, and the mechanisms of its deregulated expression. Herein, we report EZH2 as being associated with a higher risk of relapse in NPC patients (P=0.002). Genome-wide microarray and bioinformatics identified several vital cellular processes (such as differentiation, development, and apoptosis) to be regulated by EZH2, corroborated by in vitro lethality, and delayed tumor formation in vivo upon EZH2 depletion. The combination of global microRNA (miR) profiling in primary NPC specimens, and in silico analyses provided several candidate miRs that could regulate EZH2. Using a luciferase-based assay, miR-26a, miR-101, and miR-98 were validated as bona fide regulators of EZH2 expression. In particular, miR-98 was underexpressed in relapsed patient samples, strongly suggesting an important role for the miR-98 and EZH2 axis in NPC biology.     

Phosphorylation of EZH2 by CDK1 and CDK2

Histone H3 lysine 27 trimethylation (H3K27me3) catalyzed by the enzymatic subunit EZH2 in the Polycomb repressive complex 2 (PRC2) is essential for cells to ‘memorize’ gene expression patterns through cell divisions and plays an important role in establishing and maintaining cell identity during development. However, how the epigenetic mark is inherited through cell generations remains poorly understood. Recently, we and others demonstrate that CDK1 and CDK2 phosphorylate EZH2 at threonine 350 (T350) and that T350 phosphorylation is important for the binding of EZH2 to PRC2 recruiters, such as noncoding RNAs (ncRNAs) HOTAIR and XIST, and for the effective recruitment of PRC2 to EZH2 target loci in cells. These findings imply that phosphorylation of EZH2 by CDK1 and CDK2 may provide cells a mechanism that enhances EZH2 function during S and G2 phases of the cell cycle, thereby ensuring K27me3 on de novo synthesized H3 incorporated in nascent nucleosomes before sister chromosomes are divided into two daughter cells. Additionally, a potential role of T350 phosphorylation of EZH2 in differing EZH2 from its homolog EZH1 in catalyzing H3K27me3 as well as the interplay between phosphorylation at T350 and other residues (e.g. phosphorylation by p38 at threonine 372 (T372)) in governing EZH2 activity in proliferating versus non-dividing cells are also discussed. Together, CDK phosphorylation of EZH2 at T350 may represent a key regulatory mechanism of EZH2 function that is essential for the maintenance of H3K27me3 marks through cell divisions.     

A covalently bound inhibitor triggers EZH2 degradation through CHIP-mediated ubiquitination

Enhancer of zeste homolog 2 (EZH2) has been characterized as a critical oncogene and a promising drug target in human malignant tumors. The current EZH2 inhibitors strongly suppress the enhanced enzymatic function of mutant EZH2 in some lymphomas. However, the recent identification of a PRC2- and methyltransferase-independent role of EZH2 indicates that a complete suppression of all oncogenic functions of EZH2 is needed. Here, we report a unique EZH2-targeting strategy by identifying a gambogenic acid (GNA) derivative as a novel agent that specifically and covalently bound to Cys668 within the EZH2-SET domain, triggering EZH2 degradation through COOH terminus of Hsp70-interacting protein (CHIP)-mediated ubiquitination. This class of inhibitors significantly suppressed H3K27Me3 and effectively reactivated polycomb repressor complex 2 (PRC2)-silenced tumor suppressor genes. Moreover, the novel inhibitors significantly suppressed tumor growth in an EZH2-dependent manner, and tumors bearing a non-GNA-interacting C668S-EZH2 mutation exhibited resistance to the inhibitors. Together, our results identify the inhibition of the signaling pathway that governs GNA-mediated destruction of EZH2 as a promising anti-cancer strategy.