EZH2: an emerging role in melanoma biology and strategies for targeted therapy

Histone modifications are increasingly being recognized as important epigenetic mechanisms that govern chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2), responsible for tri‐methylation of lysine 27 on histone 3 (H3K27me3) that leads to gene silencing. This highly conserved histone methyltransferase is found to be overexpressed in many different types of cancers including melanoma, where it is postulated to abnormally repress tumor suppressor genes. Somatic mutations have been identified in approximately 3% of melanomas, and activating mutations described within the catalytic SET domain of EZH2 confer its oncogenic activity. In the following review, we discuss the evidence that EZH2 is an important driver of melanoma progression and we summarize the progress of EZH2 inhibitors against this promising therapeutic target.     

Roles of the EZH2 histone methyltransferase in cancer epigenetics

EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. This methylated H3-K27 chromatin mark is commonly associated with silencing of differentiation genes in organisms ranging from plants to flies to humans. Studies on human tumors show that EZH2 is frequently over-expressed in a wide variety of cancerous tissue types, including prostate and breast. Although the mechanistic contributions of EZH2 to cancer progression are not yet determined, functional links between EZH2-mediated histone methylation and DNA methylation suggest partnership with the gene silencing machinery implicated in tumor suppressor loss. Here we review the basic molecular biology of EZH2 and the findings that implicate EZH2 in different cancers. We also discuss EZH2 connections to other silencing enzymes, such as DNA methyltransferases and histone deacetylases, and we consider progress on deciphering mechanistic consequences of EZH2 overabundance and its potential roles in tumorigenesis. Finally, we review recent findings that link EZH2 roles in stem cells and cancer, and we consider prospects for integrating EZH2 blockade into strategies for developing epigenetic therapies.     

Substrate preferences of the EZH2 histone methyltransferase complex

Histone methylation plays an important role in chromatin dynamics and gene expression. Methylation of histone H3-lysine 27 by the EZH2 complex has been linked to the silencing of homeotic genes and the inactivation of the X chromosome. Here we report a characterization of the substrate preferences of the enzyme complex using a reconstituted chromatin and enzyme system. We found that the linker histone H1, when incorporated into nucleosomes, stimulates the enzymatic activity toward histone H3. This stimulatory activity may be explained by protein-protein interactions between H1 and components of the EZH2 complex. In addition, we found that the EZH2 complex exhibits a dramatic preference for dinucleosomes when compared with mononucleosomes and that the stimulation of H3 methylation by H1 requires dinucleosomes or oligonucleosome substrates. Furthermore, in contrast with a recent study suggesting that Embryonic Ectoderm Development EED isoforms may affect substrate specificity, we found that EZH2 complexes reconstituted with different EED isoforms exhibit similar substrate preference and specificity. Our work supports the hypothesis that linker histone H1 and chromatin structure are important factors in determining the substrate preference of the EZH2 histone methyltransferase complex.     

EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 catalyzing histone H3 lysine 27 tri-methylation (H3K27me3), is frequently up-regulated in human cancers. In this study, we identified the tumor suppressor Deleted in liver cancer 1 (DLC1) as a target of repression by EZH2-mediated H3K27me3. DLC1 is a GTPase-activating protein for Rho family proteins. Inactivation of DLC1 results in hyper-activated Rho/ROCK signaling and is implicated in actin cytoskeleton reorganization to promote cancer metastasis. By chromatin immunoprecipitation assay, we demonstrated that H3K27me3 was significantly enriched at the DLC1 promoter region of a DLC1-nonexpressing HCC cell line, MHCC97L. Depletion of EZH2 in MHCC97L by shRNA reduced H3K27me3 level at DLC1 promoter and induced DLC1 gene re-expression. Conversely, transient overexpression of GFP-EZH2 in DLC1-expressing Huh7 cells reduced DLC1 mRNA level with a concomitant enrichment of EZH2 on DLC1 promoter. An inverse relation between EZH2 and DLC1 expression was observed in the liver, lung, breast, prostate, and ovarian cancer tissues. Treating cancer cells with the EZH2 small molecular inhibitor, 3-Deazaneplanocin A (DZNep), restored DLC1 expression in different cancer cell lines, indicating that EZH2-mediated H3K27me3 epigenetic regulation of DLC1 was a common mechanism in human cancers. Importantly, we found that DZNep treatment inhibited HCC cell migration through disrupting actin cytoskeleton network, suggesting the therapeutic potential of DZNep in targeting cancer metastasis. Taken together, our study has shed mechanistic insight into EZH2-H3K27me3 epigenetic repression of DLC1 and advocated the significant pro-metastatic role of EZH2 via repressing tumor and metastasis suppressors.     

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.