Journal of Biochemistry Advance Access published August 31, 2014 Kondo Y
Targeting histone methyltransferase EZH2 as cancer treatment Running title: EZH2 in cancer
Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Corresponding to: Yutaka Kondo. M.D., Ph. D. Department of Epigenomics Nagoya City University Graduate School of Medical Sciences 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Abbreviations: EZH2, enhancer of zeste; PRC2, polycomb repressive complex 2; T-ALL, T-acute lymphoblastic leukemia; iPSC, induced pluripotent stem cell; CSC, cancer stem cell; GSC, glioma stem cell ; ER, estrogen receptor; PAF, PCNA-associated factor; AR, androgen receptor; STAT3, signal transducer and activator of transcription 3; GATA4, GATA binding protein 4; HDAC, histone deacetylase; DZNep, 3-Deazaneplanocin A; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; EED, embryonic ectoderm development; SUZ12, suppressor of zeste 12 homolog; SAH-EZH2, stabilized α-helix of EZH2; lncRNA, long non-coding RNA
Disclosure statement The author declares no conflict of interest.
© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
Yutaka Kondo
Kondo Y
Summary
It is widely accepted that epigenetic alterations are associated with different stages of tumor formation and progression in many cancers. Therefore, epigenetic abnormalities in cancers are
complex 2 (PRC2) is a key epigenetic regulator that catalyzes tri-methylation of lysine 27 on histone H3 (H3K27me3) via the histone methyltransferase, EZH2, which confers stemness and regulates differentiation during embryonic development. Given these roles of EZH2 and H3K27me3, plastic and dynamic features of cancer cells, especially cancer stem cells, may be closely associated with this epigenetic mechanism. In addition, recent sequencing technology revealed that there are many recurrent mutations in polycomb-related genes, including EZH2, in different types of cancers. Therefore, researchers focused on targeting EZH2 as a novel cancer treatment and identified small compounds that inhibit EZH2 activity. Some of them are now under clinical trial in B-cell lymphoma. However, the underlying mechanisms by which PRC2 precisely regulate epigenetic alterations at certain genomic loci under different cellular conditions remain unclear. In this review, I focus on the recent advancements in EZH2 research, especially its dynamic regulation of epigenetic alterations in tumor cells, including the cancer stem cell population, and discuss perspectives and challenges for cancer treatment in the near future.
Key words: EZH2, Polycomb, Epigenetics, Cancer, Treatment
2
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emerging as important biomarkers and may have therapeutic potential. The polycomb repressive
Kondo Y
1. Introduction Since appropriate regulation of gene expression is important in biological processes including differentiation, dysregulation of gene silencing results in the development of diseases. In general,
chromatin components. Among them, histone modifications have long been thought to have a functional influence on the regulation of transcription. DNA methylation generally occurs in the cytosine-guanine sequence (CpG) in adult cells and is tightly associated with gene silencing. Aberrant epigenetic regulation is known to be associated with different stages of tumor formation and progression. In particular, two important epigenetic silencing mechanisms have been studied for a while, namely polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation-mediated gene silencing, the latter of which is sometimes associated with H3K9 di- or tri-methylation (H3K9me2/3) (1-3). Dysregulation of these two epigenetic mechanisms is frequently observed in many types of cancers. One of the significant differences between the two mechanisms may be the stability of transcriptional repression. H3K27me3-mediated gene silencing can change dynamically in response to extracellular signals as opposed to DNA methylation within dense CpG islands, which is generally highly stable without artificially altering key factors in the cells (4,5). Thus, epigenetic regulation by PRC2, via its catalytic subunit EZH2, is likely to be an important mediator of tumor cell plasticity, which is required for the adaptation of cancer cells to their microenvironment. In consideration that tumor progression to metastasis is thought to occur through epigenetic evolution of tumor cells, better understanding of the roles of PRC2/EZH2 in cancer is required in order to reduce tumor cell plasticity. In this review, recent studies on PRC2/EZH2 in cancer are introduced, and their implications for treatment of human cancers are 3
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
gene silencing is associated with modifications of histones, DNA methylation, and other
Kondo Y
discussed.
2. Dysregulation of PRC2-H3K27me3 in Human Cancers In vertebrates, the PRC2-mediated H3K27me3 process has been identified as a key epigenetic
expression of developmental genes (6-8).
Dysregulation of H3K27me3 is frequently observed
in many types of cancers as well (9). EZH2 has histone methyltransferase activity, and catalyzes di-methylation (H3K27me2) and tri-methylation (H3K27me3) of H3K27 (Fig. 1). EZH2 is frequently overexpressed in many types of tumors, and a higher level of EZH2 expression is sometimes associated with aggressiveness of tumors (10,11). In addition, recent sequencing projects have uncovered mutations in EZH2. Recurrent mutations of the tyrosine 641 (Y641) and alanine 677 (A677) residues of EZH2 have been reported in lymphomas (12,13). Although the Y641 mutant was initially reported to be a loss-of-functional mutation (14), subsequent precise biochemical analysis demonstrated that this mutant led to increased activity of EZH2 (12,15). Similar to the Y641 mutant, the A677 mutation led to aberrantly elevated H3K27me3, and decreased H3K27me1 and H3K27me2 (13). These discoveries imply that increased EZH2 activity induces genome-wide H3K27me3 and may act as an oncogene via repression of tumor suppressor genes. Further, loss of EZH2/PRC2 impaired tumor formation in mouse models, indicating that EZH2 is required for tumorigenesis (16). However, inconsistent with those reports, homozygous and heterozygous deletions and inactive mutations were found in myelodysplastic syndromes, suggesting that EZH2 may also have a tumor suppressor function (17-19). Moreover, studies using mouse models showed that loss of EZH2 in hematopoietic stem cells is sufficient to cause aggressive T-acute lymphoblastic leukemia (T-ALL) (20,21). These divergent roles of EZH2 in human malignancies suggest context and tumor cell-type 4
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
modification during development, including neural stem cell differentiation, by regulating the
Kondo Y
specificities. The contribution of hyperactive or hypoactive EZH2 during tumor formation may reflect the complex and important roles that EZH2/PRC2-associated genes play in cell fate decisions (22). Since faulty differentiation is an important aspect of cancer, this may be reflected by the involvement of EZH2 in different stages of carcinogenesis.
epigenetic reprogramming may drive development of neoplasia that resembles Wilms tumor, a childhood blastoma (23). These tumors showed global changes in DNA methylation patterns and aberrant expression of imprinted genes, which correlated with altered DNA methylation. In addition, failed repression of PRC targets in iPSC was closely associated with the development of this type of tumor. Taken together, these recent findings indicate that dysregulation of PRC2-H3K27me3 plays a pivotal role in tumor development.
3. Plastic Epigenetic Regulation of Glioblastomas Since the PRC2-H3K27me3 regulatory mechanism dynamically changes gene expression in response to extracellular signals, it may confer plastic behavior on a certain population of cancer cells within tumors (Fig. 2). This dynamic plasticity of tumor cells is conceivably required for adaptation to their microenvironment, which in turn might contribute to the establishment of intratumor heterogeneity (24-26). A subpopulation of cells within tumors may be derived from a limited source of cancer cells, called cancer stem cells (CSCs), which may have plasticity and respond to signals from their microenvironment (25,27,28). Such stem-like cancer cells have been well characterized in glioblastoma multiforme (a tumor with extremely variable histopathology, thus “multiforme”), in which they are referred to as glioma stem cells (GSCs) (29-32). GSCs and normal neural stem cells appear to share common features that include self-renewal 5
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
A recent study using an induced pluripotent stem cell (iPSC) system revealed that failed
Kondo Y
and the capability to differentiate into multiple lineages (33). GSCs are considered able to differentiate aberrantly into diverse cell types via dynamic epigenetic regulation in response to signals within the tumor microenvironment. Consequently, the existence of GSCs and plastic epigenetic regulation might be linked to the morphological and lineage heterogeneities that are
and controls the development of organisms by regulating the expression of developmental genes (7,8), tumor cells might usurp this epigenetic process to mediate adaptation to tumor environments. Recently, we revealed that biological interconversion between GSCs and differentiated non-GSCs is reversible and functionally plastic, and accompanied by the gain or loss of PRC2-mediated H3K27me3 on pluripotent or development-associated genes (e.g. Nanog, Wnt1, BMP5), together with alterations in the subcellular localization of EZH2 (5). Inhibition of EZH2 disrupted the morphological conversion and impaired tumorigenicity of GSCs in NOD-SCID mice. Thus, epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Consistently, differential cellular distribution of EZH2 has also been reported in hematological malignancy: nuclear localization in leukemia stem cells and cytoplasmic localization in leukemic cells with low self-renewing capacity (34). Thus, dynamic regulation of PRC2 is closely associated with malignant progression of cancers. These data give evidence that PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, and provide a new paradigm in cancer treatment.
4. Non-canonical Roles of EZH2 in Tumorigenesis The oncogenic function of EZH2 is known to depend on transcriptional silencing of 6
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
frequently observed in glioblastomas. Given that PRC2-mediated H3K27me3 confers stemness
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tumor-associated genes. However, several recent studies showed some additional functions of EZH2, such as transcriptional activation of target genes, which are sometimes associated with malignant progression (Fig. 3). A study of estrogen receptor (ER)–positive breast cancer cells showed that EZH2 interacts
pathways, and phenotypically promotes cell cycle progression (35). Intriguingly, in ER-negative basal-like breast cancer cells, EZH2 activates NF-κB target genes by formation of a ternary complex with the NF-κB components, RelA and RelB (36). Another research group studied the interaction between Wnt signaling and EZH2, and identified a novel function of PCNA-associated factor (PAF), a component of translesion DNA synthesis, in modulating Wnt signaling (37). When Wnt signaling is activated, PAF dissociates from PCNA and binds directly to β-catenin. Then, PAF recruits EZH2 to the β-catenin transcriptional complex and specifically enhances Wnt target gene transactivation. These three studies showed that EZH2 functions as a transcriptional activator, which is independent of its histone methyltransferase activity (35-37). EZH2 can also methylate non-histone proteins, and gene activation mechanisms through EZH2 methyltransferase activity for non-histone proteins have also been reported. In prostate cancer, phosphorylation of EZH2 at Ser21 (pS21) by AKT induces gene activation via interaction with the androgen receptor (AR) (38). Further, pS21suppresses the methyltransferase activity of EZH2 and impedes the binding of unphosphorylated EZH2 to histone H3 (39), whereas the binding of pS21 EZH2 stimulates methylation of the androgen receptor and transcriptional activation of a certain set of genes. In this case, EZH2 functions without the other PRC2 components. pS21 EZH2 by AKT signaling also facilitates signal transducer and activator of transcription 7
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
directly with ER and β-catenin, functionally enhances gene transactivation by estrogen and Wnt
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3 (STAT3) methylation, resulting in enhancement of STAT3 activity through increased tyrosine phosphorylation of STAT3 in glioma (40). Although the mechanism by which pS21 EZH2 recognizes its partner protein to form a complex is still unclear, pS21 may be a molecular switch from a canonical repressor to that of transcriptional coactivator.
proteins have also been reported. In fetal hearts, EZH2 interacts with the GATA binding protein 4 (GATA4) transcription factor and directly methylates it at K299. Methylation of GATA4 attenuates its transcriptional activity by reducing its interaction with and acetylation by P300, a histone acetyltransferase (41). This study demonstrated that EZH2 mediates transcriptional repression through methylation of a non-histone protein. These studies show that EZH2 works as a multifunctional regulatory protein in human malignancies and that the roles of EZH2 in tumorigenesis may differ depending on tumor cell type. These variations in EZH2 function highlight the complexity and uniqueness of the epigenome in the context of different cancers.
5. Targeting EZH2 as a Cancer Treatment After the success of DNA demethylating agents and histone deacetylase (HDAC) inhibitor in treatment of hematological malignancies, scientific and industrial interests in epigenetic drugs as a cancer therapy has greatly increased. As mentioned above, H3K27me3-PRC2 is causally associated with a number of human cancers. Therefore, small molecule inhibitors that target PRC2 or EZH2 in cancer cells, which depend on PRC2 enzymatic activity for proliferation and survival, may be good cancer treatment candidates. Pharmacological inhibition of EZH2/PRC2 by 3-Deazaneplanocin A (DZNep), an S-adenosylhomocysteine (SAH) hydrolase inhibitor, has been shown to reduce H3K27me3 and 8
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Gene suppressing mechanisms through EZH2 methyltransferase activity for non-histone
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inhibit cancer cell growth (42). DZNep induces the accumulation of SAH in the cells, which inhibits cellular methyltransferase activity, and results in inhibition of PRC2 catalytic activity (43,44). Therefore, the suppression of methyltransferase activity by this chemical is not selective.
UNC1999; Table 1) that directly and selectively inhibit PRC2 enzymatic activity (44-47). Among these chemicals, the EPZ6438 compound is currently undergoing a phase I trial in patients with advanced solid tumors or with relapsed or refractory B-cell lymphoma (48). These chemicals bind to the S-adenosylmethionine (SAM) pocket of the EZH2 SET domain, which possesses methyltransferase activity, and selectively inhibit EZH2. Intriguingly, the basic chemical core structures of all these compounds are similar to each other, although they were identified by different types of screening, suggesting that structural variations in inhibiting EZH2 activity via binding to the SAM pocket might be limited. These selective inhibitors are only effective in cell lines with gain-of-functional EZH2 mutation, although decreased levels of H3K27me3 were equally observed in both EZH2-mutated and -wild type cancer cells. These data suggest that EZH2-mutated cells display oncogene addiction and that EZH2 inhibition may be more beneficial to patients with EZH2-mutated cancers compared with wild type cases. These findings further imply that the anti-tumor effect of EZH2 inhibition is not correlated with decreased level of H3K27me3 in certain types of cancers. Recently, another approach to inhibit the H3K27 methyltransferase activity of EZH2 has been reported. The enzymatic activity of EZH2 requires association with other partners, including the embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) proteins, in PRC2. The newly discovered stabilized α-helix of EZH2 (SAH-EZH2) peptides disrupt the EZH2-EED interaction and reduce EZH2 protein levels, which result in selective inhibition of 9
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Recently, several groups reported compounds (GSK126, EPZ005687, EPZ6438, El1,
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H3K27 methyltransferase activity of PRC2 (49). This mechanism is distinct from that of the above-mentioned small molecule EZH2 inhibitors which target the enzyme catalytic domain. Intriguingly, SAH-EZH2 peptides are also effective in cells with gain-of-functional EZH2 mutation, but have minimal effects in cells without any EZH2 mutation. These data suggest that
cancers. Given the evidence that specific inhibition of EZH2 by short hairpin RNA (shRNA) efficiently inhibits the growth of many types of cancer cells including EZH2 wild type cells by inducing cellular senescence (3,50,51), the other functions of EZH2 rather than methylation of histone H3 may also be attractive therapeutic targets for certain types of cancers. Thus, it is desirable to develop new strategies to target oncogenic pathways associated with the diverse EZH2 function.
6. Perspectives It is recognized that dysregulation of EZH2/PRC2 plays an important role in the molecular pathology of many types of cancers. However, knowledge about the precise mechanisms of dysregulation of EZH2/PRC2 in cancer is still limited, especially how the epigenome is specifically altered at certain loci and how this affects the phenotypes of each cancer. Recent genome-wide studies have shown that the large number of non-coding RNAs that are transcribed from the human genome include a group termed long non-coding RNAs (lncRNAs)(52). LncRNAs are known to regulate gene expression through providing a scaffold for chromatin modifying proteins, such as methylases, demethylases, and deacetylases, and recruiting these proteins to target loci that can be situated close together in the genome (cis-regulation) or genomically distant (trans-regulation) (53-55). Recent studies demonstrated 10
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inhibitors of PRC2 function may be applicable predominantly for patients with EZH2-mutated
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that many lncRNAs are bound to PRC2 in a variety of cellular contexts (56-59). Although it is plausible that PRC2 is an RNA-binding protein with high affinity but low specificity, proper assembly of a functional complex scaffolded by lncRNAs may regulate localization and modulate functions for PRC2 in cis and in trans (60). Further investigations are required to
that are important in cancer. In summary, epigenetic regulation affects multiple transcription factors including some master regulators and is not limited to a single cell lineage. In order to eradicate cancer cells, control of such epigenetic regulators might be the future direction of cancer treatment. Elucidation of the roles of non-coding RNA in epigenetic regulation might be a key to the development of selective cancer therapies which are based on reversing epigenetic abnormalities or freezing the plasticity of cancer cells.
Acknowledgments This work was supported by the P-Direct from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
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clarify the functional roles of lncRNAs in order to elucidate the gene regulatory mechanisms
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Table 1. Selectively EZH2 inhibitors Chemicals
Ki (nM)†
Sensitive cancer
EZH2
EZH1
EZH2
EZH1
to chemical
9.9
680
0.57
89
DLBCL with EZH2
Effect
Clinical trials
H3K27me3 ↓, me2 ↓
－
H3K27me3 ↓, me2 ↓
－
DLBCL with EZH2
H3K27me3 ↓, me2 ↓,
Phase I/II
mut*
me1 ↓
DLBCL with EZH2
H3K27me3 ↓, me2 ↓
－
H3K27me3 ↓
－
H3K27me3 ↓
－
mut* EPZ005687
ND
ND
24
1200
DLBCL with EZH2 mut*
EPZ-6438
El1
11
9.4
392
1340
2.5
ND
ND
ND
mut* UNC-1999
Targeting histone methyltransferase EZH2 as cancer treatment Running title: EZH2 in cancer
Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Corresponding to: Yutaka Kondo. M.D., Ph. D. Department of Epigenomics Nagoya City University Graduate School of Medical Sciences 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Abbreviations: EZH2, enhancer of zeste; PRC2, polycomb repressive complex 2; T-ALL, T-acute lymphoblastic leukemia; iPSC, induced pluripotent stem cell; CSC, cancer stem cell; GSC, glioma stem cell ; ER, estrogen receptor; PAF, PCNA-associated factor; AR, androgen receptor; STAT3, signal transducer and activator of transcription 3; GATA4, GATA binding protein 4; HDAC, histone deacetylase; DZNep, 3-Deazaneplanocin A; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; EED, embryonic ectoderm development; SUZ12, suppressor of zeste 12 homolog; SAH-EZH2, stabilized α-helix of EZH2; lncRNA, long non-coding RNA
Disclosure statement The author declares no conflict of interest.
© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 1
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
Yutaka Kondo
Kondo Y
Summary
It is widely accepted that epigenetic alterations are associated with different stages of tumor formation and progression in many cancers. Therefore, epigenetic abnormalities in cancers are
complex 2 (PRC2) is a key epigenetic regulator that catalyzes tri-methylation of lysine 27 on histone H3 (H3K27me3) via the histone methyltransferase, EZH2, which confers stemness and regulates differentiation during embryonic development. Given these roles of EZH2 and H3K27me3, plastic and dynamic features of cancer cells, especially cancer stem cells, may be closely associated with this epigenetic mechanism. In addition, recent sequencing technology revealed that there are many recurrent mutations in polycomb-related genes, including EZH2, in different types of cancers. Therefore, researchers focused on targeting EZH2 as a novel cancer treatment and identified small compounds that inhibit EZH2 activity. Some of them are now under clinical trial in B-cell lymphoma. However, the underlying mechanisms by which PRC2 precisely regulate epigenetic alterations at certain genomic loci under different cellular conditions remain unclear. In this review, I focus on the recent advancements in EZH2 research, especially its dynamic regulation of epigenetic alterations in tumor cells, including the cancer stem cell population, and discuss perspectives and challenges for cancer treatment in the near future.
Key words: EZH2, Polycomb, Epigenetics, Cancer, Treatment
2
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
emerging as important biomarkers and may have therapeutic potential. The polycomb repressive
Kondo Y
1. Introduction Since appropriate regulation of gene expression is important in biological processes including differentiation, dysregulation of gene silencing results in the development of diseases. In general,
chromatin components. Among them, histone modifications have long been thought to have a functional influence on the regulation of transcription. DNA methylation generally occurs in the cytosine-guanine sequence (CpG) in adult cells and is tightly associated with gene silencing. Aberrant epigenetic regulation is known to be associated with different stages of tumor formation and progression. In particular, two important epigenetic silencing mechanisms have been studied for a while, namely polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation-mediated gene silencing, the latter of which is sometimes associated with H3K9 di- or tri-methylation (H3K9me2/3) (1-3). Dysregulation of these two epigenetic mechanisms is frequently observed in many types of cancers. One of the significant differences between the two mechanisms may be the stability of transcriptional repression. H3K27me3-mediated gene silencing can change dynamically in response to extracellular signals as opposed to DNA methylation within dense CpG islands, which is generally highly stable without artificially altering key factors in the cells (4,5). Thus, epigenetic regulation by PRC2, via its catalytic subunit EZH2, is likely to be an important mediator of tumor cell plasticity, which is required for the adaptation of cancer cells to their microenvironment. In consideration that tumor progression to metastasis is thought to occur through epigenetic evolution of tumor cells, better understanding of the roles of PRC2/EZH2 in cancer is required in order to reduce tumor cell plasticity. In this review, recent studies on PRC2/EZH2 in cancer are introduced, and their implications for treatment of human cancers are 3
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
gene silencing is associated with modifications of histones, DNA methylation, and other
Kondo Y
discussed.
2. Dysregulation of PRC2-H3K27me3 in Human Cancers In vertebrates, the PRC2-mediated H3K27me3 process has been identified as a key epigenetic
expression of developmental genes (6-8).
Dysregulation of H3K27me3 is frequently observed
in many types of cancers as well (9). EZH2 has histone methyltransferase activity, and catalyzes di-methylation (H3K27me2) and tri-methylation (H3K27me3) of H3K27 (Fig. 1). EZH2 is frequently overexpressed in many types of tumors, and a higher level of EZH2 expression is sometimes associated with aggressiveness of tumors (10,11). In addition, recent sequencing projects have uncovered mutations in EZH2. Recurrent mutations of the tyrosine 641 (Y641) and alanine 677 (A677) residues of EZH2 have been reported in lymphomas (12,13). Although the Y641 mutant was initially reported to be a loss-of-functional mutation (14), subsequent precise biochemical analysis demonstrated that this mutant led to increased activity of EZH2 (12,15). Similar to the Y641 mutant, the A677 mutation led to aberrantly elevated H3K27me3, and decreased H3K27me1 and H3K27me2 (13). These discoveries imply that increased EZH2 activity induces genome-wide H3K27me3 and may act as an oncogene via repression of tumor suppressor genes. Further, loss of EZH2/PRC2 impaired tumor formation in mouse models, indicating that EZH2 is required for tumorigenesis (16). However, inconsistent with those reports, homozygous and heterozygous deletions and inactive mutations were found in myelodysplastic syndromes, suggesting that EZH2 may also have a tumor suppressor function (17-19). Moreover, studies using mouse models showed that loss of EZH2 in hematopoietic stem cells is sufficient to cause aggressive T-acute lymphoblastic leukemia (T-ALL) (20,21). These divergent roles of EZH2 in human malignancies suggest context and tumor cell-type 4
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
modification during development, including neural stem cell differentiation, by regulating the
Kondo Y
specificities. The contribution of hyperactive or hypoactive EZH2 during tumor formation may reflect the complex and important roles that EZH2/PRC2-associated genes play in cell fate decisions (22). Since faulty differentiation is an important aspect of cancer, this may be reflected by the involvement of EZH2 in different stages of carcinogenesis.
epigenetic reprogramming may drive development of neoplasia that resembles Wilms tumor, a childhood blastoma (23). These tumors showed global changes in DNA methylation patterns and aberrant expression of imprinted genes, which correlated with altered DNA methylation. In addition, failed repression of PRC targets in iPSC was closely associated with the development of this type of tumor. Taken together, these recent findings indicate that dysregulation of PRC2-H3K27me3 plays a pivotal role in tumor development.
3. Plastic Epigenetic Regulation of Glioblastomas Since the PRC2-H3K27me3 regulatory mechanism dynamically changes gene expression in response to extracellular signals, it may confer plastic behavior on a certain population of cancer cells within tumors (Fig. 2). This dynamic plasticity of tumor cells is conceivably required for adaptation to their microenvironment, which in turn might contribute to the establishment of intratumor heterogeneity (24-26). A subpopulation of cells within tumors may be derived from a limited source of cancer cells, called cancer stem cells (CSCs), which may have plasticity and respond to signals from their microenvironment (25,27,28). Such stem-like cancer cells have been well characterized in glioblastoma multiforme (a tumor with extremely variable histopathology, thus “multiforme”), in which they are referred to as glioma stem cells (GSCs) (29-32). GSCs and normal neural stem cells appear to share common features that include self-renewal 5
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
A recent study using an induced pluripotent stem cell (iPSC) system revealed that failed
Kondo Y
and the capability to differentiate into multiple lineages (33). GSCs are considered able to differentiate aberrantly into diverse cell types via dynamic epigenetic regulation in response to signals within the tumor microenvironment. Consequently, the existence of GSCs and plastic epigenetic regulation might be linked to the morphological and lineage heterogeneities that are
and controls the development of organisms by regulating the expression of developmental genes (7,8), tumor cells might usurp this epigenetic process to mediate adaptation to tumor environments. Recently, we revealed that biological interconversion between GSCs and differentiated non-GSCs is reversible and functionally plastic, and accompanied by the gain or loss of PRC2-mediated H3K27me3 on pluripotent or development-associated genes (e.g. Nanog, Wnt1, BMP5), together with alterations in the subcellular localization of EZH2 (5). Inhibition of EZH2 disrupted the morphological conversion and impaired tumorigenicity of GSCs in NOD-SCID mice. Thus, epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Consistently, differential cellular distribution of EZH2 has also been reported in hematological malignancy: nuclear localization in leukemia stem cells and cytoplasmic localization in leukemic cells with low self-renewing capacity (34). Thus, dynamic regulation of PRC2 is closely associated with malignant progression of cancers. These data give evidence that PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, and provide a new paradigm in cancer treatment.
4. Non-canonical Roles of EZH2 in Tumorigenesis The oncogenic function of EZH2 is known to depend on transcriptional silencing of 6
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
frequently observed in glioblastomas. Given that PRC2-mediated H3K27me3 confers stemness
Kondo Y
tumor-associated genes. However, several recent studies showed some additional functions of EZH2, such as transcriptional activation of target genes, which are sometimes associated with malignant progression (Fig. 3). A study of estrogen receptor (ER)–positive breast cancer cells showed that EZH2 interacts
pathways, and phenotypically promotes cell cycle progression (35). Intriguingly, in ER-negative basal-like breast cancer cells, EZH2 activates NF-κB target genes by formation of a ternary complex with the NF-κB components, RelA and RelB (36). Another research group studied the interaction between Wnt signaling and EZH2, and identified a novel function of PCNA-associated factor (PAF), a component of translesion DNA synthesis, in modulating Wnt signaling (37). When Wnt signaling is activated, PAF dissociates from PCNA and binds directly to β-catenin. Then, PAF recruits EZH2 to the β-catenin transcriptional complex and specifically enhances Wnt target gene transactivation. These three studies showed that EZH2 functions as a transcriptional activator, which is independent of its histone methyltransferase activity (35-37). EZH2 can also methylate non-histone proteins, and gene activation mechanisms through EZH2 methyltransferase activity for non-histone proteins have also been reported. In prostate cancer, phosphorylation of EZH2 at Ser21 (pS21) by AKT induces gene activation via interaction with the androgen receptor (AR) (38). Further, pS21suppresses the methyltransferase activity of EZH2 and impedes the binding of unphosphorylated EZH2 to histone H3 (39), whereas the binding of pS21 EZH2 stimulates methylation of the androgen receptor and transcriptional activation of a certain set of genes. In this case, EZH2 functions without the other PRC2 components. pS21 EZH2 by AKT signaling also facilitates signal transducer and activator of transcription 7
Downloaded from http://jb.oxfordjournals.org/ at Galter Health Sciences Library, Northwestern Univ. on September 8, 2014
directly with ER and β-catenin, functionally enhances gene transactivation by estrogen and Wnt
Kondo Y
3 (STAT3) methylation, resulting in enhancement of STAT3 activity through increased tyrosine phosphorylation of STAT3 in glioma (40). Although the mechanism by which pS21 EZH2 recognizes its partner protein to form a complex is still unclear, pS21 may be a molecular switch from a canonical repressor to that of transcriptional coactivator.
proteins have also been reported. In fetal hearts, EZH2 interacts with the GATA binding protein 4 (GATA4) transcription factor and directly methylates it at K299. Methylation of GATA4 attenuates its transcriptional activity by reducing its interaction with and acetylation by P300, a histone acetyltransferase (41). This study demonstrated that EZH2 mediates transcriptional repression through methylation of a non-histone protein. These studies show that EZH2 works as a multifunctional regulatory protein in human malignancies and that the roles of EZH2 in tumorigenesis may differ depending on tumor cell type. These variations in EZH2 function highlight the complexity and uniqueness of the epigenome in the context of different cancers.
5. Targeting EZH2 as a Cancer Treatment After the success of DNA demethylating agents and histone deacetylase (HDAC) inhibitor in treatment of hematological malignancies, scientific and industrial interests in epigenetic drugs as a cancer therapy has greatly increased. As mentioned above, H3K27me3-PRC2 is causally associated with a number of human cancers. Therefore, small molecule inhibitors that target PRC2 or EZH2 in cancer cells, which depend on PRC2 enzymatic activity for proliferation and survival, may be good cancer treatment candidates. Pharmacological inhibition of EZH2/PRC2 by 3-Deazaneplanocin A (DZNep), an S-adenosylhomocysteine (SAH) hydrolase inhibitor, has been shown to reduce H3K27me3 and 8
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Gene suppressing mechanisms through EZH2 methyltransferase activity for non-histone
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inhibit cancer cell growth (42). DZNep induces the accumulation of SAH in the cells, which inhibits cellular methyltransferase activity, and results in inhibition of PRC2 catalytic activity (43,44). Therefore, the suppression of methyltransferase activity by this chemical is not selective.
UNC1999; Table 1) that directly and selectively inhibit PRC2 enzymatic activity (44-47). Among these chemicals, the EPZ6438 compound is currently undergoing a phase I trial in patients with advanced solid tumors or with relapsed or refractory B-cell lymphoma (48). These chemicals bind to the S-adenosylmethionine (SAM) pocket of the EZH2 SET domain, which possesses methyltransferase activity, and selectively inhibit EZH2. Intriguingly, the basic chemical core structures of all these compounds are similar to each other, although they were identified by different types of screening, suggesting that structural variations in inhibiting EZH2 activity via binding to the SAM pocket might be limited. These selective inhibitors are only effective in cell lines with gain-of-functional EZH2 mutation, although decreased levels of H3K27me3 were equally observed in both EZH2-mutated and -wild type cancer cells. These data suggest that EZH2-mutated cells display oncogene addiction and that EZH2 inhibition may be more beneficial to patients with EZH2-mutated cancers compared with wild type cases. These findings further imply that the anti-tumor effect of EZH2 inhibition is not correlated with decreased level of H3K27me3 in certain types of cancers. Recently, another approach to inhibit the H3K27 methyltransferase activity of EZH2 has been reported. The enzymatic activity of EZH2 requires association with other partners, including the embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) proteins, in PRC2. The newly discovered stabilized α-helix of EZH2 (SAH-EZH2) peptides disrupt the EZH2-EED interaction and reduce EZH2 protein levels, which result in selective inhibition of 9
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Recently, several groups reported compounds (GSK126, EPZ005687, EPZ6438, El1,
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H3K27 methyltransferase activity of PRC2 (49). This mechanism is distinct from that of the above-mentioned small molecule EZH2 inhibitors which target the enzyme catalytic domain. Intriguingly, SAH-EZH2 peptides are also effective in cells with gain-of-functional EZH2 mutation, but have minimal effects in cells without any EZH2 mutation. These data suggest that
cancers. Given the evidence that specific inhibition of EZH2 by short hairpin RNA (shRNA) efficiently inhibits the growth of many types of cancer cells including EZH2 wild type cells by inducing cellular senescence (3,50,51), the other functions of EZH2 rather than methylation of histone H3 may also be attractive therapeutic targets for certain types of cancers. Thus, it is desirable to develop new strategies to target oncogenic pathways associated with the diverse EZH2 function.
6. Perspectives It is recognized that dysregulation of EZH2/PRC2 plays an important role in the molecular pathology of many types of cancers. However, knowledge about the precise mechanisms of dysregulation of EZH2/PRC2 in cancer is still limited, especially how the epigenome is specifically altered at certain loci and how this affects the phenotypes of each cancer. Recent genome-wide studies have shown that the large number of non-coding RNAs that are transcribed from the human genome include a group termed long non-coding RNAs (lncRNAs)(52). LncRNAs are known to regulate gene expression through providing a scaffold for chromatin modifying proteins, such as methylases, demethylases, and deacetylases, and recruiting these proteins to target loci that can be situated close together in the genome (cis-regulation) or genomically distant (trans-regulation) (53-55). Recent studies demonstrated 10
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inhibitors of PRC2 function may be applicable predominantly for patients with EZH2-mutated
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that many lncRNAs are bound to PRC2 in a variety of cellular contexts (56-59). Although it is plausible that PRC2 is an RNA-binding protein with high affinity but low specificity, proper assembly of a functional complex scaffolded by lncRNAs may regulate localization and modulate functions for PRC2 in cis and in trans (60). Further investigations are required to
that are important in cancer. In summary, epigenetic regulation affects multiple transcription factors including some master regulators and is not limited to a single cell lineage. In order to eradicate cancer cells, control of such epigenetic regulators might be the future direction of cancer treatment. Elucidation of the roles of non-coding RNA in epigenetic regulation might be a key to the development of selective cancer therapies which are based on reversing epigenetic abnormalities or freezing the plasticity of cancer cells.
Acknowledgments This work was supported by the P-Direct from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
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clarify the functional roles of lncRNAs in order to elucidate the gene regulatory mechanisms
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Table 1. Selectively EZH2 inhibitors Chemicals
Ki (nM)†
Sensitive cancer
EZH2
EZH1
EZH2
EZH1
to chemical
9.9
680
0.57
89
DLBCL with EZH2
Effect
Clinical trials
H3K27me3 ↓, me2 ↓
－
H3K27me3 ↓, me2 ↓
－
DLBCL with EZH2
H3K27me3 ↓, me2 ↓,
Phase I/II
mut*
me1 ↓
DLBCL with EZH2
H3K27me3 ↓, me2 ↓
－
H3K27me3 ↓
－
H3K27me3 ↓
－
mut* EPZ005687
ND
ND
24
1200
DLBCL with EZH2 mut*
EPZ-6438
El1
11
9.4
392
1340
2.5
ND
ND
ND
mut* UNC-1999
