2146

EZH2

enhancer of zeste 2 polycomb repressive complex 2 subunit;EZH2;enhancer of zeste 2 polycomb repressive complex 2 subunit

enhancer of zeste homolog 2|histone-lysine N-methyltransferase EZH2|lysine N-methyltransferase 6

7q35-q36

protein-coding

The identification of women with early-stage breast cancer who will develop distant metastasis may improve clinical management. The transcriptional regulator Enhancer of Zeste-2 (EZH2) is overexpressed in invasive breast carcinoma compared with benign breast tissues, with maximal expression in breast cancer metastasis. In this article, our purpose was to investigate the performance of EZH2 protein detection as a predictor of metastasis in women with early-stage breast cancer, which is unknown. We developed a cohort of 480 women with stage I-IIA breast cancer diagnosed between 1996 and 2002 and recorded detailed sociodemographic, clinical, and pathological information. tumors were histologically characterized and arrayed in tissue microarrays containing 1,443 samples. The nuclear EZH2 expression was investigated by immunohistochemistry and was scored as 1-2 (negative and weak) or 3-4 (moderate and strong) using a validated scoring schema. Scores 1-2 were considered low EZH2; scores 3-4 were considered high EZH2. In this study, we found that after a median follow up of 9 years (range 0.04-14.5 years) 46 of 480 patients (9.6%) developed distant metastasis. High EZH2 was associated with larger size, high histological grade, negative hormone receptors, and first degree family history of breast and/or ovarian carcinoma. While EZH2 could not predict survival in the entire cohort, high EZH2 was a predictor of disease-specific survival in patients with early-stage disease and first degree family history (log rank P value 0.05). Importantly, in this group of patients, high EZH2 was an independent predictor of distant metastasis up to 15 years after primary carcinoma diagnosis (hazard ratio 6.58, 95% CI: 1.40-30.89, P = 0.016) providing survival information above and beyond currently used prognosticators. In conclusion, EZH2 may be a useful biomarker of long-term metastatic risk in women with familial early-stage breast cancer, and warrant further validation studies.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

EZH2 is a Polycomb group protein that exerts oncogenic functions in breast cancer, where its overexpression is associated with metastatic disease. While it reportedly acts a transcriptional repressor through trimethylation of histone H3 at lysine 27, EZH2 may exhibit context-dependent activating functions. Despite associations with worse outcome and metastasis in breast cancer, a functional role of EZH2 in breast cancer metastasis in vivo has not been demonstrated. Furthermore, whether EZH2 regulates cancer cell phenotype and motility are unknown. In this study, we discovered that knockdown of EZH2 induces a phenotypic reprogramming from mesenchymal to epithelial, reduces motility, and blocks invasion in breast cancer cell lines. In vivo, EZH2 downregulation in MDA-MB-231 cells decreases spontaneous metastasis to the lungs. We uncover an unexpected role of EZH2 in inducing the p38 mitogen-activated protein kinase signaling pathway, an important regulator of breast cancer invasion and metastasis. In breast cancer cells, EZH2 binds to phosphorylated p38 (p-p38) in association with other core members of the Polycomb repressive complex 2, EED, and SUZ12, and EZH2 overexpression leads to increased levels of p-p38 and of activated, downstream pathway proteins. The effect on p-p38 was confirmed in vivo, where it correlated with decreased spontaneous metastasis. In clinical specimens of matched primary and invasive breast carcinomas, we found that EZH2 expression was upregulated in 100 % of the metastases, and that EZH2 and p-p38 were coexpressed in 63 % of cases, consistent with the functional results. Together our findings reveal a new mechanism by which EZH2 functions in breast cancer, and provide direct evidence that EZH2 inhibition reduces breast cancer metastasis in vivo.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3, resulting in epigenetic silencing of gene expression and cancer progression. We investigated the expression and function of EZH2 in intrahepatic and extrahepatic cholangiocarcinoma (ICC and ECC). METHODS: The influence of EZH2 on cell growth and apoptosis was assessed by knockdown experiments. Target gene of EZH2 was searched by quantitative RT-PCR. Clinical significance of EZH2 in 86 cholangiocarcinoma patients (45 ICC and 41 ECC) who underwent curative surgery was examined by immunohistochemistry. RESULTS: In vitro analysis, knockdown of EZH2 reduced cell growth, induced G1 arrest, and induced apoptosis, as confirmed by Annexin V staining and increased sub-G1 populations in cholangiocarcinoma cell lines. The expression levels of p16 (INK4a) and p27 (KIP1) were remarkably increased by knockdown of EZH2 in these cell lines. In immunohistochemical study, EZH2 upregulation correlated with tumor diameter (p = 0.0103) in ICC, lymph node metastasis (p = 0.0292) in ECC, and Ki67 index in both ICC (p = 0.0364) and ECC (p = 0.0017). In addition, EZH2 expression was correlated with poor prognosis in both ICC (p = 0.0447) and ECC (p = 0.0227). CONCLUSIONS: The current study demonstrated relationships between EZH2 expression and acceleration of the cell cycle and antiapoptosis, and poor prognosis in cholangiocarcinoma. These results suggest that EZH2 may represent a potential therapeutic target in patients with cholangiocarcinoma.

CONTEXT: Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase mediating chromatin condensation and epigenetic modulation, is overexpressed in various human carcinomas and is associated with adverse clinicopathologic characteristics and biologic behavior. The expression of EZH2 in renal cell carcinomas (RCCs) has not been fully characterized yet. OBJECTIVE: To evaluate the prognostic role of EZH2 in RCC by analyzing the immunohistochemical staining pattern of the marker in relation to pathologic features and clinical outcome. DESIGN: We correlated the immunolabeling of EZH2 with multiple clinicopathologic features, including Fuhrman nuclear grade, pathologic stage, metastatic status, and clinical outcome in 223 clear cell RCCs (CRCCs) and 21 papillary RCCs, by using tissue microarrays of primary and metastatic cases. RESULTS: Most CRCCs (75%) showed positive EZH2 staining, with most primary tumors showing focal staining in comparison to nonfocal staining in metastatic cases. In primary tumors, EZH2 expression was associated with higher nuclear grade and lower pathologic stage. metastatic tumors showed a higher number of positive cases (81% versus 67%) and a more diffuse and more intense pattern of staining than primary CRCCs. For the 22 locally advanced primary tumors (T3/4) and 43 metastatic RCCs, patients who experienced RCC-related deaths significantly overexpressed the marker in comparison to patients who did not experience RCC-related mortality. CONCLUSIONS: By showing that EZH2 expression is associated with increased metastatic potential and a worse clinical outcome, this study suggests that EZH2 can serve as a prognostic biomarker for RCC, thus confirming it as a key molecule driving oncogenesis and metastasis.

BACKGROUND: Despite therapeutic innovations, metastatic colorectal cancer (mCRC) is still characterized by poor prognosis and few molecular markers predict the risk of progression. Polycomb group genes (PcGs) are epigenetic modifiers involved in tumor suppressor gene silencing. PcG member EZH2 mediates gene silencing through histone-H3 lysine-27 methylation. In colorectal cancer (CRC), EZH2 overexpression predicts shorter survival. Recently, four EZH2 single-nucleotide polymorphisms (SNPs) have been described. The present study was aimed at evaluating the correlation between EZH2 SNPs and outcome parameters in mCRC patients. PATIENTS AND METHODS: DNA was extracted from blood samples of 110 mCRC patients treated with first-line 5-fluorouracil, folinic acid, irinotecan (FOLFIRI) and bevacizumab. Genotyping was carried out by real-time PCR. Genotype was used to predict objective response, progression-free survival (PFS) and overall survival (OS). EZH2 messenger RNA levels were evaluated on lymphocytes of a parallel cohort of 50 CRC patients. RESULTS: One allelic variant (rs3757441 C/C versus C/T or T/T) was significantly associated with shorter PFS and OS (P < 0.01 and P < 0.05, respectively). At multivariate analysis, the same variant resulted an independent predictor of PFS and OS (P < 0.05). The C/C variant was associated with significantly higher EZH2 expression (P < 0.05). CONCLUSION: An EZH2 SNP may be useful to predict clinical outcome in mCRC patients.

The enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins, plays an important role in cell proliferation and cell cycle regulation. EZH2 is overexpressed in aggressive forms of prostate, breast, bladder, and endometrial cancers. However, the role of EZH2 expression in gastric cancer has not been fully determined. This study was conducted to investigate the correlation between EZH2 and cell cycle-related molecules, and the clinical value of EZH2 expression in gastric cancer. We analyzed EZH2 expression using Western blotting in AGS, MKN-28, SNU-16, SNU-484, SNU-601, and SNU-638 gastric cancer cell lines. After transfection of EZH2 siRNA into MKN-28 cells, the change in cell cycle-related molecules was assessed by Western blot analysis. expression of EZH2, Ki-67, and p53 was determined by immunohistochemical staining of tissue microarrays from specimens of 137 cases of resected gastric cancer. We found high expressions of EZH2 in all of the tested gastric cancer cell lines. RNA interference of EZH2 induced upregulation of p53 and HDAC1 and downregulation of cyclin D1 and cyclin E. High EZH2 expression was observed in 60.6% of gastric cancers and in 6.7% of non-neoplastic gastric tissues (p < 0.01); 40.1% were positive for p53 in gastric cancers. High EZH2 expression was correlated with Ki-67 and p53 expressions and was significantly associated with distant metastases and non-signet ring cells. Our results suggest that high EZH2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

Epigenetic alterations and microRNA (miRNA) deregulation are common in hepatocellular carcinoma (HCC). The histone H3 lysine 27 (H3K27) tri-methylating enzyme, enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up-regulated in human cancers. In this study we aimed to delineate the implications of EZH2 up-regulation in miRNA deregulation and HCC metastasis. expressions of a total of 90 epigenetic regulators were first determined in 38 pairs of primary HCCs and their corresponding nontumorous livers. We identified EZH2 and its associated polycomb repressive complex 2 (PRC2) as one of the most significantly deregulated epigenetic regulators in primary HCC samples. Up-regulation of EZH2 was next confirmed in 69.5% (41/59) of primary HCCs. Clinicopathologically, EZH2 up-regulation was associated with HCC progression and multiple HCC metastatic features, including venous invasion (P = 0.043), direct liver invasion (P = 0.014), and absence of tumor encapsulation (P = 0.043). We further demonstrated that knockdown of EZH2 in HCC cell lines reduced the global levels of tri-methylated H3K27, and suppressed HCC motility in vitro and pulmonary metastasis in a nude mouse model. By interrogating the miRNA expression profile in EZH2-knockdown cell lines and primary HCC samples, we identified a subset of miRNA that was epigenetically suppressed by EZH2 in human HCC. These included well-characterized tumor-suppressor miRNAs, such as miR-139-5p, miR-125b, miR-101, let-7c, and miR-200b. pathway enrichment analysis revealed a common regulatory role of these EZH2-silenced miRNAs in modulating cell motility and metastasis-related pathways. Our findings suggest that EZH2 exerts its prometastatic function by way of epigenetic silencing of multiple tumor suppressor miRNAs. CONCLUSION: Our study demonstrated that EZH2 epigenetically silenced multiple miRNAs that negatively regulate HCC metastasis.

INTRODUCTION: Elevated Enhancer of Zeste Homologue 2 (EZH2) expression is involved in many human malignancies through epigenetically silencing related genes. However, the study of the EZH2 protein expression in lung cancer remains at the qualitative or semi-quantitative level. The present study is to elucidate the roles of EZH2 in the progression and metastasis of different subtypes of lung cancer at quantitative level. MATERIALS AND METHODS: Lung carcinoma tissue microarray was constructed containing 32 normal adult lung tissues, 113 lung carcinomas and 57 lymph-node metastases. EZH2 protein expression was detected by immunohistochemistry and assessed quantitatively with Leica Q500MC image analysis system. Positive unit (PU) value was used to evaluate the protein expression intensity of positive cells from systematically selected fields under the microscope. RESULTS: Elevated Enhancer of Zeste Homologue 2 PU in lung carcinomas was significantly greater than that in normal lung tissues (p = 0.001). Increased EZH2 expression was correlated with histological subtypes, differentiation, TNM stage, and lymph-node metastases (p < 0.05). EZH2 PU of primary lung carcinomas was smaller than that of lymph-node metastasis (p = 0.002). EZH2 PU was not associated with patients' gender, age, smoking status, tumor location, and tumor size (p > 0.05). CONCLUSIONS: Elevated Enhancer of Zeste Homologue 2 PU is increased with the development of lung cancer. EZH2 may play an important role in the progression and metastasis of lung cancer.

The enhancer of zeste homolog 2 (EZH2) is upregulated and has an oncogenic role in several types of human cancer. However, the abnormalities of EZH2 and its underlying mechanisms in the pathogenesis of nasopharyngeal carcinoma (NPC) remain unknown. In this study, we found that high expression of EZH2 in NPC was associated closely with an aggressive and/or poor prognostic phenotype (P<0.05). In NPC cell lines, knockdown of EZH2 by short hairpin RNA was sufficient to inhibit cell invasiveness/metastasis both in vitro and in vivo, whereas ectopic overexpression of EZH2 supported NPC cell invasive capacity with a decreased expression of E-cadherin. In addition, ablation of endogenous Snail in NPC cells virtually totally prevented the repressive activity of EZH2 to E-cadherin, indicating that Snail might be a predominant mediator of EZH2 to suppress E-cadherin. Furthermore, co-immunoprecipitation (IP), chromatin IP and luciferase reporter assays demonstrated that in NPC cells, (1) EZH2 interacted with HDAC1/HDAC2 and Snail to form a repressive complex; (2) these components interact in a linear fashion, not in a triangular fashion, that is, HDAC1 or HDAC2 bridge the interaction between EZH2 and Snail; and (3) the EZH2/HDAC1/2/Snail complex could closely bind to the E-cadherin promoter by Snail, but not YY1, to repress E-cadherin. The data provided in this report suggest a critical role of EZH2 in the control of cell invasion and/or metastasis by forming a co-repressor complex with HDAC1/HDAC2/Snail to repress E-cadherin, an activity that might be responsible, at least in part, for the development and/or progression of human NPCs.

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.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer deaths in the United States. Single-agent gemcitabine remains the standard treatment of advanced pancreatic adenocarcinoma. A recently discovered histone methyltransferase termed enhancer of zeste homologue 2 (EZH2) was found to be overexpressed in a variety of carcinomas including pancreatic adenocarcinoma. Silencing of E-cadherin was proposed as a mechanism by which enhancer of zeste homologue 2 mediates tumor aggressiveness, and enhancer of zeste homologue 2 depletion has been found to sensitize pancreatic cancer cells to gemcitabine. In this study, we correlated enhancer of zeste homologue 2 with E-cadherin expression in pancreatic adenocarcinoma and evaluated response to gemcitabine in relation to enhancer of zeste homologue 2 expression in tumor cells. Fifty-four pancreatic adenocarcinomas, 13 intraductal papillary mucinous neoplasms, and 6 chronic pancreatitis cases were stained with antibodies against enhancer of zeste homologue 2 and E-cadherin. Enhancer of zeste homologue 2 staining was scored from 1 to 4+ and classified as either low (1-2+ in <25% of tumor nuclei) or high (3-4+ in >25% of tumor nuclei). E-cadherin expression was scored on membrane positivity as follows: 0 (0%-10%), 1 (10%-25%), 2 (25%-75%), and 3 (>75%). High enhancer of zeste homologue 2 expression in pancreatic adenocarcinoma was significantly associated with decreased E-cadherin expression and more aggressive disease. There was significantly longer survival in gemcitabine-treated patients with low versus high enhancer of zeste homologue 2 expression. High enhancer of zeste homologue 2 expression was detected in intraductal papillary mucinous neoplasms with moderate to severe dysplasia, but not in chronic pancreatitis. Our study suggests that E-cadherin down-regulation may lead to enhancer of zeste homologue 2-mediated invasion and metastasis.

The function of EZH2 in tumorigenesis and liver metastasis of pancreatic cancer has never been elucidated in vivo. EZH2 was overexpressed in pancreatic carcinomas and its overexpression was associated with tumor differentiation and pT status. Suppression of EZH2 caused a significant growth inhibition of pancreatic cancer cells in vitro and markedly diminished their tumorigenicity in vivo. Knock-down of EZH2 inhibited liver metastasis of pancreatic cancer in vivo. EZH2 has a crucial role in tumor growth and liver metastasis of pancreatic cancer.

BACKGROUND & AIMS: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is overexpressed by pancreatic ductal adenocarcinoma (PDAC) cells and increases their aggressiveness. We identified microRNAs (miRs) that are regulated by EZH2 and studied their functions in PDAC cells. METHODS: We performed miR profile analysis of PDAC cells incubated with EZH2 inhibitor 3-deazaneplanocin A, and pancreatic ductal epithelial cells that overexpressed EZH2. expression levels of miRs and the targets of miRs were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We expressed different forms of EZH2 to analyze functional domains and used small interfering RNAs to reduce its level in PDAC cells. RESULTS: expression of miR-218 was repressed by EZH2 in PDAC cells. Levels of miR-218 were significantly reduced in primary PDAC tumor samples compared with paired, adjacent nontumor tissue. Overexpression of miR-218 in SW1990 cells reduced their proliferation and tumor formation and metastasis in nude mice. Loss of miR-218 from SW1990 cells increased levels of UDP-glycosyltransferase 8 and miR-218 was found to bind to its 3'-UTR. Levels of UDP-glycosyltransferase protein and messenger RNA were associated with the metastatic potential of PDAC cell lines and progression of tumors in patients. EZH2 was found to silence miR-218 by binding to its promoter, promoting heterochromatin formation, and recruiting the DNAs methyltransferase 1, 3A, and 3B. CONCLUSIONS: EZH2 is up-regulated in PDAC samples from patients and silences miR-218. MicroRNA-218 prevents proliferation of PDAC cells in culture, and tumor growth and metastasis in nude mice. MicroRNA-218 reduces levels of UDP-glycosyltransferase, which is associated with the metastatic potential of PDAC tumors in mice and progression of human PDAC.

AIM: EZH2 expression is a prognostic marker in radically resected pancreatic ductal adenocarcinoma (PDAC) patients. Here we investigated its role in locally advanced/metastatic patients, as well as candidate polymorphisms. MATERIALS & METHODS: EZH2 expression and polymorphisms were evaluated by quantitative reverse transcription PCR in 32 laser microdissected tumors, while polymorphisms were also studied in blood samples from two additional cohorts treated with gemcitabine monotherapy (n = 93) or polychemotherapeutic regimens (n = 247). RESULTS: EZH2 expression correlated with survival and with the rs6958683 polymorphism in the first cohort of patients, but this polymorphism was not associated with survival in our larger cohorts. CONCLUSION: EZH2 is a prognostic factor for locally advanced/metastatic PDACs, while candidate polymorphisms cannot predict clinical outcome. Other factors involved in EZH2 regulation, such as miR-101, should be investigated in accessible samples in order to improve the clinical management of advanced PDAC.

Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Here we show, through gene expression profiling, that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression.

The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.

PURPOSE: Enhancer of zeste homolog 2 (EZH2), a kind of transcriptional repressor, is reportedly over-expressed in metastatic prostate cancer. In this study, we analyzed EZH2 mRNA in circulating tumor cells (CTCs) in peripheral blood as a biomarker in patients with metastatic prostate cancer. PATIENTS AND METHODS: Ber-EP4 coated immunomagnetic beads were used to harvest CTCs, and mRNA was isolated by oligo- dT conjugated immunomagnetic beads. Reverse transcriptase- polymerase chain reaction for EZH2 mRNA was performed and the expression density was measured. The sensitivity of this test for detection of EZH2 mRNA was determined by serial dilutions of a human prostate cancer cell line. Blood samples were collected from 20 patients each with metastatic or localized prostate cancer and 10 healthy volunteers. RESULTS: Sensitivity experiments showed that the test was highly sensitive as it could detect 10 tumor cells per 5 mL. EZH2 mRNA expression was obtained from peripheral blood samples of patients and control subjects. EZH2 mRNA expression density in the metastatic prostate cancer group was significantly higher than in the control (p=0.023) and localized prostate cancer groups (p=0.019). There was no difference between the control and localized prostate cancer groups (p > 0.05). CONCLUSION: EZH2 mRNA expression in circulating epithelial cells represents a promising marker for detecting early metastasis in prostate cancer. However, more specific and sensitive techniques for detection of CTCs are needed to avoid mononuclear cell contamination.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Evidence suggests that overexpression of enhancer of zeste homologue 2 (EZH2) is associated with aggressive behavior in various cancers. However, the clinical, pathological, and prognostic associations of EZH2 expression in the upper urinary tract carcinoma have not been reported. This study aimed to investigate the significance of EZH2 expression in the upper urinary tract carcinoma by immunohistochemical analysis using a tissue microarray. High EZH2 expression was observed in 94 of 171 (55 %) cases and was significantly associated with several adverse prognostic factors, including sessile architecture, high histological grade, presence of lymphovascular invasion, concomitant carcinoma in situ, higher tumor stage, and higher Ki-67 expression (all P < 0.01). EZH2 expression status in primary and metastatic lesions was concordant in all 13 cases examined. Survival analyses were performed using the Kaplan-Meier method and multivariate Cox proportional hazards regression models. High EZH2 expression was significantly associated with a shorter metastasis-free survival (log-rank P = 0.005; multivariate hazard ratio, 1.85; 95 % confidence interval, 0.93-3.71) and cancer-specific survival (log-rank P = 0.006; multivariate hazard ratio, 3.08; 95 % confidence interval, 1.30-7.32) after nephroureterectomy. Our results suggest that EZH2 may serve as a novel prognostic biomarker and a potential therapeutic target in upper urinary tract carcinoma.