Co-expression of Bmi1 and EZH2 as an independent poor prognostic factor in esophageal squamous cell carcinoma

Bmi1 polycomb ring finger oncogene (Bmi1) and the enhancer of zeste homolog 2 (EZH2) are members of polycomb repressive complex (PRC) 1 and PRC2, respectively. PRC1 represses tumor suppressor genes such as p16INK4a and p14ARF in a PRC2-dependent manner. There have been few studies on Bmi1 or EZH2 expression in esophageal squamous cell carcinoma (ESCC). We investigated Bmi1 and EZH2 expression in 164 cases of ESCCs using immunohistochemistry, and evaluated the correlation with clinicopathologic features and their prognostic significance. Bmi1 and EZH2 were more highly expressed in tumor than in adjacent normal tissue (p<0.001). High expression of Bmi1 or EZH2 alone was not correlated with any clinicopathologic parameter and did not influence the prognosis. However, the group with high expression of both Bmi1 and EZH2 showed the poorest prognosis in overall survival (p=0.027) and disease-free survival (p=0.007). Also, it was an independent prognostic factor in overall survival (p=0.047). High expression of both Bmi1 and EZH2, not each alone, is an independent poor prognostic factor in ESCCs, supporting the repression of tumor suppressor gene by Bmi1 in an EZH2-dependent manner. This result suggests that both Bmi1 and EZH2, not each alone, could be potent candidates of new target therapy in ESCCs.     

A complex Polycomb issue: the two faces of EZH2 in cancer

In the April 1, 2012, issue of Genes & Development, Simon and colleagues (pp. 651-656) demonstrated that the disruption of Ezh2 in mice is sufficient to cause T-acute lymphoblastic leukemia (T-ALL). Moreover, in concert with concurrent studies, the authors revealed that similar mechanisms are involved in human T-ALL. These data contrast with previous findings showing that increased EZH2 activity promotes cancer.     

EZH2基因单核苷酸多态性对中国汉族人群散发性胃癌易感性的影响

目的 研究EZH2基因单核苷酸多态性与汉族人群散发性胃癌易感性关系以及胃癌临床病理特征与EZH2蛋白表达的关系.方法 采用基因测序仪对安徽地区311例胃癌患者及425例正常对照组血样EZH2 rs734004、rs734005、rs2072407、rs6464926和rs12670401多态性测序,用组织芯片技术和免疫组化检测238例同一来源胃癌组织和30例正常胃黏膜EZH2蛋白表达.结果 EZH2 rs12670401基因型CC以及rs6464926基因型TT在胃癌患者中比例较高,可增加胃癌发生风险(P=0.007,aOR=1.786,95% CI=1.172～2.723;P=0.013,aOR=1.721,95% CI=1.123～2.638).胃癌患者中EZH2杂合基因型rs2072407TC、rs734005TC、rs734004CG比例低于对照组,可降低胃癌发生风险.(P=0.034,aOR=0.712,95% CI=0.520～0.975;P=0.033,aOR=0.709,95% CI=0.518～0.972;P=0.035,aOR=0.711,95% CI=0.518～0.976).连锁不平衡分析LD进一步证实上述观察.胃癌不同组织学类型间以及不同临床参数间EZH2免疫组化染色强度差异无显著性.结论 EZH2基因单核苷酸多态性与中国汉民族人群散发性胃癌易感性相关.     

Distinct expression of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma

Polycomb gene products play a crucial role in the development of highly malignant phenotypes and aggressive cancer progression in a variety of cancers; however, their role in hepatocellular carcinoma remains unclear. First, we analyzed the impact of EZH2 and BMI1 modulation on cell growth of HepG2 cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays revealed marked growth inhibition after EZH2 or BMI1 knockdown. In addition, simultaneous knockdown of these 2 genes further augmented cell growth inhibitory effects. Next, we conducted immnuohistochemical assessment of 86 hepatocellular carcinoma surgical specimens, evaluating the correlation between EZH2 and BMI1 protein expression and clinicopathologic features. High-level EZH2 and BMI1 expression was detected in 57 (66.3%) and 52 tumor tissues (60.5%), respectively. Among these, 48 tumor tissues (55.8%) showed colocalization of EZH2 and BMI1 in almost all tumor cells. The cumulative recurrence rate, but not survival rate, was significantly higher in patients positive for EZH2 (P = .029) and BMI1 (P = .039) than in their negative counterparts, as determined by Kaplan-Meier analysis. These data indicate that EZH2 and BMI1 may cooperate in initiation and progression of hepatocellular carcinoma.     

JARID1B deletion induced apoptosis in Jeko-1 and HL-60 cell lines

Aims: To investigate the involvement of JARID1B histone methyltransferase in the epigenetic change of euchromatic promoter in mantle cell lymphoma (MCL) and acute leukemia. Methods: We retrospectively analyzed the protein of JARID1B and tri-methylated histone H3 lysine 4 (H3K4), histone H3 lysine 9 (H3K9), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. JARID1B was depleted by small interfering RNA (siRNA), and cell apoptosis and cell proliferation were detected by flow cytometry and MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], histone tri-methylated H3K4 and histone acetylated H3, H4, cyclin D1, Bcl-2, procaspase-3, C-myc were studied by Western blot. Results: We demonstrated that JARID1B was upregulated and histone tri-methylated H3K4 was downregulated in MCL compared to proliferative lymphadenitis, P < 0.05. The expression of histone methylated H3K9 was similar in both. Histone methylation of H3K4 was positively correlated with Ki67 in MCL (Kappa = 0.757, P < 0.05). This study showed that depletion of JARID1B cleavage apoptotic proteins of Bcl-2, procaspase-3, C-myc and resulted in loss cell viability and inducing apoptosis in Jeko-1 and HL-60 cell lines. JARID1B siRNA improved tri-methyl H3K4 and histone acetylated H3 and inhibited cyclin D1, but did not affect histone acetylated H4. Conclusions: This study revealed hyper JARID1B expression and hypo histone H3K4 tri-methylation in MCL. We identify depletion JARID1B as a demethylase which is capable of removing three methyl groups from H3K4 and up-regulating histone acetylation of H3 in both cell lines. Interestingly, depletion of JARID1B inhibits Cyclin D1, which is one of the genes contributes to MCL pathogenesis. JARID1B might be one of therapeutic targets in acute leukemia and MCL.     

大鼠EZH2基因过表达质粒以及催化亚基缺失质粒构建及鉴定

目的 构建特异性的大鼠EZH2过表达质粒以及EZH2催化亚基SET domain缺失质粒,并在293T细胞中评估其表达水平.方法 提取大鼠心脏组织RNA,逆转录为cDNA,重叠PCR扩增催化亚基SET domain缺失的EZH2的cDNA,以其为模板进行PCR扩增,将PCR产物和载体双酶切,切胶回收目的片段,经T4连接酶连接,连接产物转入感受态细胞,涂板挑取单克隆摇菌测序,提取质粒,采用脂质体转染法转染293T细胞.结果 Western Blot法和RT-PCR法检测结果表明,质粒在293T细胞中实现EZH2基因的过表达.结论 构建的2种质粒为进一步研究EZH2基因与心肌肥厚的关系及EZH2催化亚基的作用奠定了基础.     

Jarid2 and PRC2, partners in regulating gene expression

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.     

Expanding the molecular spectrum of gene fusions in endometrial stromal sarcoma: Novel subunits of the chromatin remodeling complexes PRC2 and NuA4/TIP60 as alternative fusion partners

Endometrial stromal sarcomas (ESS) are morphologically and molecularly heterogeneous. We report novel gene fusions (EPC1::EED, EPC1::EZH2, ING3::PHF1) identified by targeted RNA sequencing in five cases. The ING3::PHF1-fusion positive ESS presented in a 58-year-old female as extrauterine mesocolonic, ovarian masses, and displayed large, monomorphic ovoid-to-epithelioid cells arranged in solid sheets. The patient remained alive with disease 13 months after surgery. The three ESS with EPC1::EED occurred in the uterine corpus in patients with a median age of 58 years (range 27–62 years). One tumor showed a uniform epithelioid nested morphology, while the other two were composed of monomorphic spindle cells in fascicles with elevated mitotic figures, focal tumor cell necrosis, and lymphovascular invasion. At a median follow-up of 20 months, two patients developed local recurrence, including one with concomitant distant metastasis, while one patient remained free of disease. All three patients were alive at the last follow-up. The EPC1::EZH2-fusion positive ESS presented in a 52-year-old female in the uterus, and displayed uniform spindled cells arranged in short fascicles, with focally elevated mitotic activity but without necrosis. The patient remained free of disease 3 months after surgery. All cases were diffusely positive for CD10; four diffusely express estrogen and progesterone receptors. Our study expands the molecular spectrum of EPC1 and PHF1-related gene fusions in ESS to include additional novel subunits of the PRC2 and/or NuA4/TIP60 complexes. These cases displayed a monomorphic epithelioid or spindled phenotype, spanning low-grade and high-grade cytomorphology, all expressing CD10 and commonly ER and PR, and are prone to local and/or distant spread.     

胃癌组织中EZH2和p53蛋白的表达及临床意义

目的探讨EZH2和p53蛋白在胃癌组织中的表达,及其在胃癌发生、发展过程中的作用和临床病理学意义。方法采用免疫组化EnVision法检测EZH2和p53蛋白在199例胃癌、25例高级别上皮内瘤变、24例低级别上皮内瘤变、46例肠上皮化生、17例慢性萎缩性胃炎和23例正常胃黏膜组织中的表达情况。结果在胃癌和上皮内瘤变组织中EZH2蛋白表达明显高于肠上皮化生、慢性萎缩性胃炎和正常胃黏膜组织。胃癌组织中EZH2蛋白的表达在进展期胃癌高于早期胃癌,差异有统计学意义。EZH2蛋白在胃癌组织中的表达与肿瘤的大小、淋巴结转移、组织学分期和临床分期均有关;但与患者的性别、年龄、肿瘤的部位、肿瘤的类型和分化以及患者的病死率均无关。p53蛋白在胃癌组织中表达最高,几乎不表达于其他病变和正常胃黏膜组织。p53表达水平与患者的年龄、性别、肿瘤大小、淋巴结转移、肿瘤分化、胃癌的类型和患者的生存情况相关。EZH2蛋白表达与p53蛋白表达之间呈正相关。结论 EZH2在胃癌中的表达增加与p53相关,提示胃癌中EZH2与p53可能相互协调,促进胃癌的发生、发展。