EZH2和H3K27me3在食管鳞状细胞癌中的表达及意义

目的 分析EZH2和H3K27me3在食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）组织中的表达及与食管鳞状细胞癌临床病理指标之间的关系,并评价其表达对食管鳞状细胞癌患者预后的意义。方法 利用免疫组织化学法检测65例ESCC患者癌组织及相应的癌旁组织中EZH2及H3K27me3的蛋白表达,分析两者表达与ESCC患者临床病理学指标之间的相关性,并分析两者之间表达的相关性。利用Kaplan-Meier方法及Log rank检验分析两者表达与食管癌生存率之间的关系。结果 癌旁组织中EZH2和H3K27me3蛋白阳性表达率分别为44.6%和26.2%,ESCC组织中分别为70.8%和47.7%,EZH2和H3K27me3蛋白表达均与ESCC患者的组织学分级及淋巴结转移状态呈正相关关系,且EZH2和H3K27me3蛋白表达之间存在正相关关系。EZH2和H3K27me3表达阳性的ESCC患者五年总生存率均明显低于EZH2和H3K27me3表达阴性的患者;EZH2和H3K27me3双阳性的ESCC患者五年总生存率明显低于EZH2和H3K27me3单阳性和双阴性的ESCC患者。Cox回归结果显示,淋巴结转移、组织学分级、EZH2和H3K27me3表达是ESCC患者的独立预后因素。结论 EZH2和H3K27me3在ESCC中均高表达,并与ESCC患者的组织学分级和淋巴结转移状态相关,可能是ESCC患者的不良预后因子。     

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.     

新兴抗肿瘤靶点EZH2作用机制及研究进展

zeste基因同源蛋白2(enhancer of zeste homolog 2,EZH2)是由EZH2基因编码的一种赖氨酸特异性组蛋白甲基转移酶（histone-lysine N-methyltransferase,HRX),是多梳家族蛋白(polycomb-group proteins,PcGs)家族的重要组分。该基因正常状态下参与胚胎干细胞发育,维持正常细胞分化,在两性发育中驱动女性一个X染色体的沉默,在造血过程中维持B淋巴细胞和T淋巴细胞的未分化状态。病理状态下,EZH2成为一种基因抑制因子,当它过度表达时,组蛋白的三甲基化导致许多正常开启的肿瘤抑制因子被关闭。在多种肿瘤组织中EZH2含量明显高于癌旁组织,预后差的肿瘤中EZH2表达含量明显高于预后良好的肿瘤。因此,抑制EZH2的表达很可能是未来癌症治疗的一个有希望的策略。本文就目前研究状况做一简要综述。     

H2AK119Ub1 and H3K27Me3 in molecular staging for survival prediction of patients with pancreatic ductal adenocarcinoma

Polycomb group (PcG) proteins Ring1B and EZH2, which have been characterized as catalyzing the two epigenetic modifications H2AK119 monoubiquitination (H2AK119Ub1) and H3K27 trimethylation (H3K27Me3), are well-known epigenetic silencers implicated in embryonic development and tumorigenesis. However, the status of polycomb-associated histone modifications and their clinical implications in pancreatic cancer remain unclear. Here, we performed immunohistochemistry on tissue microarrays (TMAs) containing 80 pairs of human pancreatic cancer specimens to assess the expression levels of Ring1B, H2AK119Ub1, EZH2, and H3K27Me3 in tumors. More than 50% of the tumor cells showed a high expression of H2AK119Ub1, Ring1B, and EZH2, whereas more than 50% of the tumor cells showed a low level of H3K27Me3. Different expression patterns of H2AK119Ub1 and H3K27Me3 in tumors were negatively correlated (r = −0.247, P = 0.027). Both H2AK119Ub1 and H3K27Me3 independently predicted the clinical prognosis. In particular, a combinatorial pattern of elevated H2AK119Ub1 and decreased H3K27Me3 in tumors was significantly correlated with a poorer prognosis. Furthermore, compared to the tumor, lymph node, metastasis (TNM) staging system, histone modifications can discriminate the survival difference more accurately, especially for patients with stage I or stage II tumors. Simultaneous silencing of Ring1B and EZH2 via shRNA depleted H2AK119Ub1 and H3K27Me3 in the pancreatic cancer cells PanC1 and AsPC1, enhanced HOX gene derepression, and inhibited tumor cell growth in vitro and in tumor xenograft models. These results demonstrated that H2AK119Ub1 and H3K27Me3 cooperate in tumors and are associated with the clinical prognosis in combinatorial patterns. We have proposed that epigenetic modifications may serve as discriminatory biomarkers for molecular staging of pancreatic cancer.     

EZH2和H3K27me3的表达对食管癌细胞迁移和侵袭能力的影响

目的:通过转染Zeste同源物增强子2(enhancer of zeste homolog 2,EZH2)过表达或者敲低载体,探讨EZH2和Lys27位点三甲基化组蛋白H3(histone H3 methylated Lys27,H3K27me3)对食管麟状细胞癌(esophageal squamous cell cancer,ESCC)细胞迁移和侵袭能力的影响.方法:应用实时荧光定量PCR、Western blotting法检测ESCC细胞株KYSE30、KYSE170、TE1、Eca109中EZH2 mRNA水平,以及ESCC细胞过表达或者敲低EZH2对H3 K27me3表达水平的影响.用划痕实验及Transwell侵袭实验分析过表达或者敲低EZH2后ESCC细胞的迁移侵袭能力.用实时荧光定量PCR法分析ESCC细胞过表达及敲低EZH2对MMPs mRNA水平的影响.结果:食管癌Eea109及TE1细胞中EZH2和H3K27me3 mRNA和蛋白水平明显高于KYSE30及KYSE170细胞(P＜0.05).过表达EZH2的食管癌KYSE30及KYSE170细胞H3K27me3蛋白的表达水平显著升高(P＜0.05),敲低EZH2后Eca109及TE1细胞H3 K27 me3蛋白的表达水平明显降低(P＜0.05).过表达EZH2后,KYSE30及KYSE170细胞的穿膜数目明显增多[(281.33±4.10)、(241.67 ±4.04) vs(132.00 ±4.00)、(105.33 ±3.51)个,均P＜0.05]、迁移距离明显增大[(63.6±1.2)、(62.5±2.5)vs (23.0±2.3)、(21.2±1.0) μm,P＜0.05].敲低EZH2后Eca109及TE1细胞的穿膜数目显著减少(均P＜0.05),转染shEZH2后Eca109及TE1细胞迁移的距离明显减小(均P＜0.05).结论:EZH2可增加靶基因启动子上组蛋白H3第27位赖氨酸的三甲基化,并增强ESCC细胞的迁移和侵袭能力.     

EZH2 基因在卵巢癌发生发展中的作用及其临床病理意义*

目的:探讨EZH 2 基因对卵巢癌细胞增殖和转移能力的影响,及其在卵巢癌组织中的表达与临床病理学意义。方法:运用EZH 2 小干扰RNA（siRNA ）转染卵巢癌OVCAR- 3 细胞株,Western blot方法分析OVCAR- 3 细胞中EZH 2 的蛋白表达;MTT 实验检测细胞增殖水平,Transwell 小室实验检测细胞侵袭和转移能力。另外,应用RT-PCR 和免疫组织化学法分别检测EZH 2 在卵巢癌组织中的mRNA 和蛋白表达情况。结果:与阴性对照组相比,EZH 2 siRNA 能明显降低OVCAR- 3 细胞的EZH 2 蛋白表达,并显著抑制肿瘤细胞的增殖能力（P=0.032）;转染EZH 2 siRNA 的OVCAR- 3 穿膜细胞数,在侵袭实验中,siEZH 2 组为29.3 ± 5,与对照组（51± 6.8）比较,差异有统计学意义（P=0.027）;在迁移实验中,siEZH 2 组的迁移细胞数为51.6 ± 7.7,显著低于对照组（72.3 ± 11.7,P=0.036）。 RT-PCR 检测发现,卵巢癌组织中的EZH 2 mRNA 表达水平明显高于正常组织。在免疫组化实验中,61.0%的卵巢癌组织呈EZH 2 蛋白高表达,而且与卵巢癌的T 分期、N 分期以及FIGO分期显著正相关（P<0.05）。 另外,单变量生存分析发现EZH 2 高表达与卵巢癌患者短生存期密切相关（P=0.007）;多变量分析显示EZH 2 是卵巢癌的独立预后参数（P=0.047）。 结论:EZH 2 在卵巢癌的发生与进展中发挥着重要的作用,而且EZH 2 高表达是卵巢癌患者预后不良的独立分子指标。      

Prognostic impact of H3K27me3 expression on locoregional progression after chemoradiotherapy in esophageal squamous cell carcinoma

Background  

Trimethylation of lysine 27 on histone H3 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that mediates gene silencing. EZH2 is overexpressed and correlates with poor prognosis in many cancers. However, the clinical implication of H3K27me3 in human malignancies has not been well established. We wished to ascertain whether a correlation exists between the expression of H3K27me3 and clinical outcome in a group of patients with esophageal squamous cell carcinoma (ESCC) treated with definitive chemoradiotherapy (CRT).     

幽门螺杆菌感染与胃癌中PRC2和H3K27me3的表达关系

  目的  研究幽门螺杆菌（Helicobacter pylori,HP）感染对胃癌组织中多梳抑制复合物2（polycomb repressive complex 2,PRC2）和组蛋白H3K27me3表达的影响,探索HP感染在胃癌发生过程中的作用。  方法  利用快速尿素酶检测、Giemsa染色和PCR方法检测复旦大学附属上海市第五人民医院2014年1月至2017年10月行胃癌手术的84例患者HP感染情况,应用免疫组织化学法检测胃癌和癌旁组织PRC2和H3K27me3蛋白的表达,分析两者表达与HP感染之间的相关性。  结果  3种方法检测HP感染率分别为70.24%、61.90%和76.19%。PRC2家族成员果蝇zeste基因增强子同源物2（enhancer of zeste homolog 2,EZH2）、SUZ12、EED和H3K27me3蛋白在胃癌组织中的阳性表达率分别为79.76%（67/84）、77.38%（65/84）、40.48%（34/84）和69.05%（58/84）,均显著高于癌旁组织（22.62%、34.52%、8.33%和14.29%,P < 0.05）。临床资料分析表明,EZH2和H3K27me3蛋白在胃癌中的表达与淋巴结转移呈正相关。在HP阳性胃癌组织中,EZH2、SUZ12、EED和H3K27me3蛋白阳性表达率分别为89.06%（57/64）、84.38%（54/64）、46.88%（30/64）和82.81%（53/64）,均显著高于HP阴性胃癌组织（50.00%、55.00%、20.00%和25.00%,P < 0.05）。  结论  HP感染与胃癌组织中PRC2和H3K27me3蛋白表达上调相关。      

骨肉瘤细胞和组织H3K27me3表达与临床病理特征相关性研究

目的 组蛋白H3K27三甲基化(H3K27me3)在肿瘤发生发展过程中起着重要作用,研究发现H3K27me3在肝癌和前列腺癌等恶性肿瘤中的表达和临床病理特征存在相关性,但其在骨肉瘤中的研究相对较少.本研究分析H3K27me3在骨肉瘤细胞和组织中的表达及其与临床病理的关系,并探讨H3K27me3在骨肉瘤发生和发展中的作用和意义.方法 所有切片均来自于河北医科大学第四医院骨科2005-01-01-2011 01-01手术切除的标本.采用蛋白印迹法检测骨肉瘤MG-63、U2-OS、Sa-OS细胞系及hFOB1.19成骨细胞中H3K27me3表达的水平差异;采用免疫组织化学染色方法检测53例骨肉瘤组织及16例骨软骨瘤组织中H3K27me3的表达水平.KaplawMeier分析比较H3K27me3高表达和低表达患者生存率之间的差异.并应用Cox回归模型分析其表达水平对预后影响.结果 hFOB1.19、MG-63、U2-OS和Sa-OS中H3K27me3蛋白表达水平分别为0.37±0.06、0.86±0.06、0.79±0.07和0.83±0.05,与hFOB1.19细胞中H3K27me3表达相比,3组骨肉瘤细胞系中H3K27me3蛋白表达水平均显著升高,P=0.023;与骨软骨瘤组织相比(56.3％),H3K27me3在骨肉瘤组织中高表达(84.9％),二者差异有统计学意义(P＜0.001),并与肿瘤大小、肺转移、Enneking分期和生存率有关,P值分别为0.037、0.020、0.023和0.046.且其表达水平与患者生存期显著相关,P=0.012.结论 H3K27me3在骨肉瘤细胞及骨肉瘤组织中呈高表达,并与肿瘤的临床病理特征相关,可能会是骨肉瘤患者重要的预后因子及治疗靶点.     

Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity

Introduction

Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.

Methods

Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.

Results

H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.

Conclusions

H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.     

Global histone modification of H3K27 correlates with the outcomes in patients with metachronous liver metastasis of colorectal cancer

Background

We evaluated the methylation patterns of histone H3 lysine 27 (H3K27), H3 lysine 36 (H3K36) and the expression of H3K27 methylase EZH2 in patients with colorectal carcinomas with metachronous liver metastasis to search for biomarkers identifying these patients.

Methods

Double 2-mm core tissue microarrays were made from 54 paraffin-embedded samples of primary colorectal adenocarcinomas and corresponding liver metastases and examined using an immunohistochemical analysis of dimethylation and trimethylation in H3K27, H3K36 and EZH2. Positive tumor cell staining for each histone modification (H-score) was used to classify patients into low- and high-staining groups, which were then examined to identify any correlations between the clinicopathological parameters and the clinical outcomes.

Results

The H-scores of H3K27me2 were lower in the liver metastases than in the corresponding primary tumors, while the H-scores of H3K36me2 were higher in the liver metastases than in the corresponding primary tumors (P < 0.001). H3K27me2 in the primary tumors correlated with tumor size (P = 0.016), H3K36me2 in the primary tumors correlated with histological type (P = 0.038), and H3K36me3 in the primary tumors correlated with lymph node metastasis (P = 0.017). In addition, lower levels of H3K27me2 in the primary tumors correlated with poorer survival rates (P = 0.039). The multivariate survival analysis showed that the H3K27me2 status is an independent prognostic factor for colorectal cancer patients (P = 0.047).

Conclusions

Our findings suggest that the methylation level of H3K27me2 detected with immunohistochemistry may be an independent prognostic factor for metachronous liver metastasis of colorectal carcinomas.     

ALDH1A1 is a novel EZH2 target gene in epithelial ovarian cancer identified by genome-wide approaches

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in the United States. EZH2 silences gene expression through trimethylating lysine 27 on histone H3 (H3K27Me3). EZH2 is often overexpressed in EOC and has been suggested as a target for EOC intervention. However, EZH2 target genes in EOC remain poorly understood. Here, we mapped the genomic loci occupied by EZH2/H3K27Me3 using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and globally profiled gene expression in EZH2-knockdown EOC cells. Cross-examination of gene expression and ChIP-seq revealed a list of 60 EZH2 direct target genes whose expression was upregulated more than 1.5-fold upon EZH2 knockdown. For three selected genes (ALDH1A1, SSTR1, and DACT3), we validated their upregulation upon EZH2 knockdown and confirmed the binding of EZH2/H3K27Me3 to their genomic loci. Furthermore, the presence of H3K27Me3 at the genomic loci of these EZH2 target genes was dependent upon EZH2. Interestingly, expression of ALDH1A1, a putative marker for EOC stem cells, was significantly downregulated in high-grade serous EOC (n = 53) compared with ovarian surface epithelial cells (n = 10, P < 0.001). Notably, expression of ALDH1A1 negatively correlated with expression of EZH2 (n = 63, Spearman r = -0.41, P < 0.001). Thus, we identified a list of 60 EZH2 target genes and established that ALDH1A1 is a novel EZH2 target gene in EOC cells. Our results suggest a role for EZH2 in regulating EOC stem cell equilibrium via regulation of ALDH1A1 expression.     

Development of a novel approach, the epigenome-based outlier approach, to identify tumor-suppressor genes silenced by aberrant DNA methylation

Identification of tumor-suppressor genes (TSGs) silenced by aberrant methylation of promoter CpG islands (CGIs) is important, but hampered by a large number of genes methylated as passengers of carcinogenesis. To overcome this issue, we here took advantage of the fact that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. First, we demonstrated that three of six known TSGs in breast cancer and two of three in colon cancer had Pol II and lacked H3K27me3 in normal cells, being outliers to the general rule. BRCA1, HOXA5, MLH1, and RASSF1A had high Pol II, but were expressed only at low levels in normal cells, and were unlikely to be identified as outliers by their expression statuses in normal cells. Then, using epigenome statuses (Pol II binding and H3K27me3) in normal cells, we made a genome-wide search for outliers in breast cancers, and identified 14 outlier promoter CGIs. Among these, DZIP1, FBN2, HOXA5, and HOXC9 were confirmed to be methylated in primary breast cancer samples. Knockdown of DZIP1 in breast cancer cell lines led to increases of their growth, suggesting it to be a novel TSG. The outliers based on their epigenome statuses contained unique TSGs, including DZIP1, compared with those identified by the expression microarray data. These results showed that the epigenome-based outlier approach is capable of identifying a different set of TSGs, compared to the expression-based outlier approach.     

HER2 overexpression correlates with survival after curative resection of pancreatic cancer

HER2 overexpression has been linked to clinical outcomes in several solid tumors, such as breast cancer. However, the correlation between HER2 overexpression and survival in pancreatic carcinoma remains unclear. The impact of HER2 overexpression on survival in pancreatic ductal cancer was examined. Immunohistochemical staining of 129 pancreatic cancers without hematogenous metastases or peritoneal dissemination treated by macroscopically curative resection were analyzed in association with survival data. To determine HER2 overexpression in this pancreatic cancer series, the polyclonal antibody included in HercepTest, which is used worldwide for clinical examination of HER2 overexpression in breast cancer, was used. Immunoreactivity was classified according to the scale presented in the HercepTest Scoring Guidelines. Twenty-two cases (17.1%) had a score of 0, 28 cases (21.7%) had of a score of 1+, 41 cases (31.8%) had a score of 2+, and 38 cases (29.4%) had a score of 3+. Therefore, HER2 overexpression (score 2+ or 3+) was observed in 79 cases (61.2%). Patients with HER2 overexpression tumors had significantly shorter survival times than those with HER2 normal expression (score 0 or 1+) tumors (median survival time, 14.7 vs 20.7 months, respectively; P  = 0.0078 on the log-rank test). On multivariate survival analysis, HER2 overexpression remained an independent prognostic factor (hazard ratio, 1.806; P  = 0.0258). A significant percentage of pancreatic cancers were demonstrated to have HER2 overexpression, and overexpression of this tyrosine kinase receptor proved to be an independent factor for a worse prognosis. These results should encourage further investigation of treatments using new molecular targeting agents against HER2 protein to improve the survival of pancreatic cancer patients. ( Cancer Sci 2009; 100: 1243–1247)     

Clinical Implications of p57KIP2 Expression in Breast Cancer

Objective: To study the relationship between expression of p57KIP2 and prognosis and other clinicopathologicalparameters in invasive breast cancers. Methods: We assessed the expression of p57KIP2 in 89 cases of invasivebreast cancer and 20 cases of normal breast tissue by immunohistochemical methods and analyzed the resultswith SPSS software (ver. 16.0). Result: The positive expression rates of p57KIP2 protein in the invasive breastcancers and surrounding normal tissue were 30.3% (27/89) and 65% (13/20), respectively. Cases with nop57KIP2 expression exhibited a significantly higher post-operative distant metastasis rate than those with p57KIP2expression (37.9% vs. 14.8%; P = 0.01). DFS analysis showed that p57KIP2-/C-erbB-2+ tumors also exhibited asignificantly higher post-operative distant metastasis rate than the other groups (66.7% vs. 29.2%; P = 0.007),as did p57KIP2-/p53+ tumors (64.3% vs. 22.7%; P = 0.001). Survival analysis revealed that p57KIP2 was associatedwith breast cancer-specific survival overall (P = 0.045, log-rank test). Subgroup analysis demonstrated thatindividuals with p57KIP2-/C-erbB-2+tumors experienced significantly worse post-operative survival than thosewith p57KIP2- /C-erbB-2- or other tumors (P = 0.006, log-rank test). p57KIP2-/p53+ tumors were associated withsignificantly worse post-operative survival than p57KIP2-/p53- or other tumors (P = 0.001, log-rank test). Coxregression analysis showed that p57KIP2 was a non-independent prognostic factor for breast cancer (P = 0.303).Conclusions: p57KIP2 is expressed at low levels in invasive breast cancer and is associated with better overallsurvival rate and disease-free survival in breast cancer patients, but it was a non-independent prognostic factorfor breast cancer. Thus, the connection between p57KIP2/p53 and p57KIP2/C-erbB-2 may provide biomarkers forbreast cancer.