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Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities.

Enhancer of zeste homolog 2 (EZH2), the critical component of polycomb group protein family, has been demonstrated to be overexpressed in various types of human cancer, including hepatocellular carcinoma, breast, bladder and lung cancer. The mechanism of how EZH2 promotes oncogenesis has also been well studied. However, little is known about the role of EZH2 in colorectal cancer (CRC). The main purpose of the present study was to analyze the association between EZH2 expression and the clinicopathological features of CRC. Therefore, the mRNA and protein expression levels were analyzed in tumor tissues and adjacent non-cancerous tissues by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The expression of EZH2 was demonstrated to be significantly increased in tumor tissues compared with adjacent noncancerous tissues, according to the results of western blot analysis and RT-qPCR in the majority of cases. Patients with low EZH2 expression had a longer overall survival rate compared with those with high EZH2 expression. An analysis of the association between clinicopathological features and EZH2 expression indicated that high EZH2 expression was significantly associated with tumor stage, tumor size, histological differentiation and lymph node metastasis. Multivariate analysis demonstrated that high EZH2 expression was an independent predictor of overall survival. In conclusion, to the best of our knowledge, the data presented in the present study is the first to indicate that EZH2 is upregulated in CRC and may serve as a predictor of poor outcome for patients with CRC.

Enhancer of zeste homologue 2 (EZH2), a member of the polycomb group protein family, plays a crucial role in the regulation of embryonic development and has been associated with the regulation of the cell cycle. Recently, several studies have shown that EZH2 is highly expressed in aggressive tumours, including human breast cancer, prostate cancer, and lymphomas. We thus analysed EZH2 expression using real-time reverse transcription-polymerase chain reaction, and correlated its expression status with various clinicopathological parameters in 66 patients with hepatocellular carcinoma (HCC). We found high expression of EZH2 in human liver cancer cell lines. Furthermore, EZH2 gene-expression levels in tumour tissue specimens (0.34+/-0.52) were significantly higher (P0.0001) than those in the corresponding nontumour tissue specimens (0.07+/-0.09). The incidence of cancer cell invasion into the portal vein was significantly higher (P0.001) in the high EZH2 expression group (26 of the 33, 79%) than in the low expression group (13 of the 33, 39%). However, there was no significant difference in the disease-free survival rate between the two groups. The findings of this study indicate that EZH2 mRNA expression was upregulated in human HCC and may play an important role in tumour progression, especially by facilitating portal vein invasion.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27. 18 1592 1605 Cited By :364

Polycomb group proteins are epigenetic gene silencers that have been implicated in cancer development and stem cell maintenance.1 Biological and genetic studies indicate that PcG proteins exist in at least two separate protein complexes, polycomb-repressive complex 2 (PRC2) and polycomb-repressive complex 1 (PRC1), which act in concert to promote and maintain gene repression.1 EZH2, the catalytically active component of PRC2, plays an important role in many biological processes through its ability to trimethylate lysine 27 in histone H3. The core enzymatic activity of PRC1 is an E3 ubiquitin ligase activity contributed by RING1B (and enhanced by BMI1), which ubiquitinates histone H2A at lysine 119.1 PcG proteins are displaced from the promoters of one set of target genes while being recruited to the promoters of another set of target genes during lineage specification.1 How extracellular signaling regulates this process is not well-understood. In 2005, Cha and colleagues found Akt mediated phosphorylation of EZH2 results in a decrease of lysine 27 trimethylation and derepression of silenced genes.2 Recently, we reported that phosphorylation of BMI1 at serine 316 by Akt impairs its function by triggering its dissociation from the Ink4a-Arf locus, which results in decreased histone H2A ubiquitination and the inability of BMI1 to promote cell growth and tumorigenesis.3 It is very likely that other PcG proteins, including EZH1 and MEL18, are downstream targets of Akt. Thus, Akt appears to regulate the activities of both the PRC2 and PRC1 complexes in the cell (Fig. 1). Figure 1. PI3K-Akt signaling regulates polycomb group protein and hematopoietic stem cell self-renewal. PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs).1 HSCs have both the capacity to self-renew and to differentiate into many different mature blood cell types. Understanding the molecular mechanisms governing stem cell self-renewal remains the holy grail of stem cell biology and holds great promise for the development of stem cell-based therapies aimed at treating debilitating and life-threatening diseases such as cancer. Self-renewal requires the integration of survival signals and proliferation controls with the maintenance of an undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of HSCs to maintain an undifferentiated state.4 However, how the crosstalk is coordinated has not been well-defined at the molecular level. Activation of the PI3K-Akt signaling impairs HSC self-renewal,5 and we demonstrated that BMI1 is a critical downstream target of PI3K-Akt signaling in HSCs and that Akt-mediated phophorylation of BMI1 inhibits its ability to promote hematopoietic stem/progenitor cell (HSPC) self-renewal.3 At virtually the same time, the van Lohuizen lab reported that Akt-mediated BMI1 phosphorylation enhances its oncogenic potential in an Ink4a and Arf-independent mouse model of human prostate cancer.6 They identified three sites of serine phosphorylation [Ser251, Ser253 and Ser255 (SDSGS)], none of which were consensus Akt phosphorylation sites (RXRXXS or T).6 Nonetheless, it appears that the inhibition of cell proliferation and oncogenesis by Akt-mediated phosphorylation of BMI1, occurs in a site-specific and possibly context-dependent manner. Similar to BMI1, a component of Polycomb-repressive complex 1 (PRC1), PRC2 and its components play important roles in hematopoiesis. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis.7 On the contrary, Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response, suggesting that Ezh1 is an important histone methyltransferase for adult HSC maintenance.8 Therefore, it is possible that Akt-mediated phosphorylation of EZH2 or EZH1 may limit HSC function as well. Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of diseases. The ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Most hematopoietic cell growth factors activate Akt in ex vivo cultures, promoting HSC differentiation. Therefore, treating HSCs with Akt inhibitors may expand HSCs ex vivo. Our understanding of the crosstalk between PI3K-Akt signaling and polycomb group proteins may facilitate the development of innovative clinical strategies that can enhance ex vivo HSC expansion.