Dysregulation of histone methyltransferases in breast cancer - Opportunities for new targeted therapies?

M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 1 4 9 7 e1 5 1 5

available at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/molonc

Review

Dysregulation of histone methyltransferases in breast cancer e Opportunities for new targeted therapies? Ewa M. Michalaka,b,*, Jane E. Visvadera,b,* a

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia b Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia

A R T I C L E

I N F O

A B S T R A C T

Article history:

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine resi-

Received 15 July 2016

dues on histone tails and non-histone targets. These important post-translational modifi-

Received in revised form

cations are exquisitely regulated and affect chromatin compaction and transcriptional

14 September 2016

programs leading to diverse biological outcomes. There is accumulating evidence that ge-

Accepted 14 September 2016

netic alterations of several HMTs impinge on oncogenic or tumor-suppressor functions and

Available online 23 September 2016

influence both cancer initiation and progression. HMTs therefore represent an opportunity for therapeutic targeting in those patients with tumors in which HMTs are dysregulated, to

Keywords:

reverse the histone marks and transcriptional programs associated with aggressive tumor

Histone methyltransferase

behavior. In this review, we describe the known histone methyltransferases and their

Breast cancer

emerging roles in breast cancer tumorigenesis.

Chromatin

ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights

Transcription

reserved.

Inhibitors

1.

Introduction

In eukaryotes, the challenge of condensing 1.8 m of DNA into a cell is solved by packaging it into chromatin. Chromatin is composed of nucleosome subunits: a 147 base-pair segment of DNA wrapped around a histone octamer (two dimers of histones H2A and H2B and a tetramer of H3 and H4). The N- and Cterminal histone tails protruding from the nucleosome core are subject to extensive covalent post-translational modifications, including acetylation, phosphorylation, ubiquitination and

methylation (reviewed in (Audia and Campbell, 2016)). The patterns of histone marks are established and maintained through a dynamic interplay between histone readers, writers, and erasers (Greer and Shi, 2012; Zhang et al., 2015). Distinct modifications, or combinations of modifications can directly impact on chromatin organization and also serve as binding sites for specific modulatory proteins. Different patterns of modifications are associated with distinct transcriptional states, resulting from tightly packaged heterochromatin versus more accessible euchromatin.

* Corresponding authors. ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. E-mail addresses: [email protected] (E.M. Michalak), [email protected] (J.E. Visvader). http://dx.doi.org/10.1016/j.molonc.2016.09.003 1574-7891/ª 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1498


Histone methylation primarily occurs on histone tails of H3 and H4. More than 60 human histone methyltransferases (HMTs) have been identified and catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to amine residues (lysines and arginines) (Table 1). The state of methylation on a given residue (mono-, di- or trimethylation) enables a precise level of biological regulation (Table 1 and Figure 1) (Audia and Campbell, 2016; Barski et al., 2007; Onder et al., 2012; Zhang et al., 2015). Lysines can be mono-methylated (me1), dimethylated (me2) or trimethylated (me3) on their ε-amine group, while arginines can be mono-methylated (me1), symmetrically dimethylated (me2s) or asymmetrically dimethylated (me2a) on their guanidinyl group (reviewed in (Greer and Shi, 2012)). Cross-talk with adjacent modifications on the same histone tail, together with the level of cytosine methylation of the underlying DNA, results in distinct transcriptional states (active, poised and silent domains) and leads to dramatically different functional consequences. Genomewide mapping of histone marks, largely performed in human T cells, has identified distinct patterns of histone methylation in the enhancer and promoter regions as well as in the gene body and correlated these with gene transcription (Figure 2). However, these patterns remain incompletely defined and the literature contains conflicting reports due to genespecific modifications, the particular cell type or organism studied, and whether or not enrichment was correlated with gene transcription at a specific locus or genome-wide. At the level of active genes, however, mono-methylation of H3K4, H3K9, H3K27, H3K36, H4K20 are generally linked to activation (Barski et al., 2007; Heintzman et al., 2007; Vakoc et al., 2006), as is di-methylation of H3K79 (Okada et al., 2005; Onder et al., 2012) and trimethylation of H3K36 (Barski et al., 2007; Vakoc et al., 2006). On the other hand, H3K27 trimethylation is linked to repression and generally co-localizes with H3K27me2 (Barski et al., 2007; Squazzo et al., 2006), and H3K79 trimethylation has been reported at the promoters of both active and repressed genes (Barski et al., 2007; Vakoc et al., 2006). While H3K9 trimethylation is generally associated with heterochromatin, supporting a role in transcriptional silencing, it has also been reported to mark actively transcribed promoters (Squazzo et al., 2006; Vakoc et al., 2006). The effect of arginine marks on chromatin is less clear, however H3R2me2s marks are generally present in euchromatic regions, while H4R3me2s is considered a mark of transcriptional repression and H3R8me2s has been associated with both transcriptional activation and repression (Koh et al., 2015). It is also of interest that H3K9 and H3K27 can be acetylated in a manner that is mutually exclusive from methylation and leads to transcriptional activation (Zhang et al., 2015). In addition, there is mutual exclusivity between the presence of H3K4me3 and H3R2me2a and the marks antagonize one another (Guccione et al., 2007; Kirmizis et al., 2007). Structurally, HMTs can be broadly categorized into three functional enzymatic families, the SET (Suppressor of variegation, Enhancer of zeste, Trithorax)-domaincontaining methyltransferases, the non-SET DOT1-like (DOT1L) lysine methyltransferases, the PRDM family, containing a PR (PRDIBF1-RIZ1 homologous) domain that is structurally and functionally similar to the SET domain, and the PRMT1 family which shares a common methyltransferase domain (Katoh,

2016; Mzoughi et al., 2016; Nguyen and Zhang, 2011; Schotta et al., 2004; Teyssier et al., 2010). In most cases, there is little redundancy between family members, owing to their methyl group- and cell type-specificity. This is highlighted by findings from loss-of-function mouse models and hereditary disorders associated with mental retardation and intellectual disability (Katoh, 2016). Breast cancer is the leading cause of cancer-related death in women world-wide (Kamangar et al., 2006). Comprehensive gene expression profiling has identified five major molecular subtypes of breast cancer: basal-like, luminal A, luminal B, HER2þ/ER and normal-like breast cancer (Perou et al., 2000; Sørlie et al., 2001), however, it is likely that many more subtypes exist (Curtis et al., 2012). There is mounting evidence that dysregulation of HMTs leads to imbalances in histone methylation patterns and contributes to the pathogenesis of a wide array of human cancers, including breast cancer. The hallmarks of sporadic breast cancer are somatic copy number alterations and “driver” mutations i.e., those mutations that confer a proliferative advantage on cells to promote cancer development. Several large-scale sequencing efforts have led to the development of databases such as TCGA and COSMIC, which allow for the cataloging of somatic mutations in cancer. Analyses of these large datasets have revealed that HMTs are frequently mutated in cancer (Ciriello et al., 2015; Kudithipudi and Jeltsch, 2014; Nik-Zainal et al., 2016) and represent 5% of driver genes identified in whole-genome sequences of breast cancers (Nik-Zainal et al., 2016). Notably, basal-like breast cancers bear the highest frequencies of HMT gene amplifications, deletions, and mutations, whereas luminal A tumors have the lowest frequencies in every category of genetic alteration (Liu et al., 2015). These findings are consistent with triple-negative breast cancer (TNBC, a subset of basal-like cancers) exhibiting the highest degree of genomic instability. In addition to somatic mutations, single nucleotide polymorphisms (SNPs) found in genes encoding HMTs are associated with cancer risk susceptibility (Wang et al., 2012; Yoon et al., 2010) and clinical outcome (Crea et al., 2012). Moreover, acquired resistance to treatment is associated with elevated expression of HMTs (Borley and Brown, 2015; Magnani et al., 2012). HMTs therefore represent potential biomarkers or therapeutic targets in those patients in which HMTs are dysregulated. A systematic review of the genetic alterations of HMTs in breast cancer has not been undertaken. In this review, we examine all known human HMT families and discuss their normal roles as well as their clinical relevance in the context of breast cancer. Finally, we outline the status of HMT inhibitors in clinical development and their potential use as a therapeutic strategy.

1.1.

H3K9 methyltransferases

H3K9 methylation is catalyzed by at least six members of the SET-containing SUV39 protein family: SUV39H1, SUV39H2, G9A, G9a-like protein (GLP1), SETDB1 and SETDB2. G9A and GLP1 form a heterodimer and catalyze mono- and dimethylation of H3K9 primarily found associated with silent genes in euchromatin (Shinkai and Tachibana, 2011), while SUV39H1 and SUV39H2 are trimethyltransferases responsible primarily for H3K9me3 at centromeric and pericentromeric

1499


Table 1 e List of all human histone methyltransferases identified to date and their dysregulation in breast cancer. Gene

Synonym

SET domain

Targets

Dysregulation in breast cancer

KMT1A KMT1B KMT1C KMT1D KMT1E KMT1F

SUV39H1 SUV39H2 EHMT2, G9a EHMT1, GLP1 SETDB1 SETDB2

Canonical Canonical Canonical Canonical

H3K9me3 H3K9me3 H3K9me1/me2, H1.2K187, p53me2 H3K9me1/me2, H1.2K187 H3K9me3 H3K9me3

Patani et al. (2011) None reported Si et al. (2015) None reported Chen et al. (2014), Liu et al. (2015) Liu et al. (2015)

KMT2A KMT2B KMT2C

MLL MLL4 MLL3

Canonical Canonical Canonical

H3K4me1/me2/me3 H3K4me1/me2/me3 H3K4me1/me2/me3

KMT2D

MLL2

Canonical

H3K4me1/me2/me3

KMT2E KMT2F KMT2G KMT7 KMT3C KMT3D KMT3E ZMYND21 ZMYND22

MLL5 SETD1A, Set1A SETD1B, Set1B SET7, SET9, SETD7 SMYD2 SMYD1, ZMYND18 SMYD3 SMYD4 SMYD5

Canonical Canonical Canonical Canonical

No methyltransferase activity H3K4me1/me2/me3 H3K4me1/me2/me3 H3K4me1 H3K4, H3K36, ER, p53, RB H3K4me3 MAP3K2, H3K4me3, H4K5, H4K20 Unknown Unknown

None reported None reported Cancer Genome Atlas Network (2012), Ciriello et al. (2015), Liu et al. (2015), Nik-Zainal et al. (2016) Nik-Zainal et al. (2016), Ross et al. (2015), Tan et al. (2015) None reported None reported None reported None reported Liu et al. (2015) None reported Liu et al., (2015), Patani et al. (2011) None reported None reported

KMT2H KMT3A KMT3B WHSC1 WHSC1L1 SETMAR SETD3

ASH1L SETD2 NSD1 NSD2, MMSET NSD3 METNASE

Canonical Canonical Canonical Canonical Canonical Canonical

H3K36me1/me2, H3K36me3 H3K36me1/me2 H3K36me1/me2, H3K36me1/me2, H3K36me1/me2, H3K36me1/me2,

SETD4 SETD5 SETD6

H3K4a

H4K20a H3K4 H3K9 H3K4a

Liu et al. (2015) Nik-Zainal et al. (2016), Tan et al. (2015) None reported Kassambara et al. (2009), Wang et al. (2016) Chen et al. (2014), Liu et al. (2015) None reported None reported

Canonical

Unknown Unknown H2AZK8me1, K310me1 of RELA subunit

None reported None reported None reported

EZH2

Canonical

H3K27me1/me2/me3

KMT6B

EZH1

Canonical

KMT4

DOT1L

KMT5A KMT5B KMT5C

SETD8, PR-SET7 SUV420H1 SUV420H2

PRDM1 PRDM2 PRDM3 PRDM4 PRDM5

KMT6A

H3K27me1/me2/me3

Bachmann et al. (2006), Collett et al. (2006), Kleer et al. (2003), Raaphorst et al. (2003) None reported

H3K79me1/me2/me3

Cho et al. (2015)

Canonical Canonical Canonical

H4K20me1 H4K20me3 H4K20me3

Liu et al. (2016a) None reported None reported

BLIMP-1 KMT8, MTB-ZF, RIZ MECOM, EVI1 PFM1 PFM2

PR-SET PR-SET PR-SET PR-SET PR-SET

No methyltransferase activity H3K9me3/me1 H3K9me1 Unknown Unknown

PRDM6 PRDM7 PRDM8 PRDM9 PRDM10 PRDM11 PRDM12 PRDM13 PRDM14

PFM3 PFM4 PFM5 PFM6 PFM7 PFM8 PFM9 PFM10 PFM11

PR-SET PR-SET PR-SET PR-SET PR-SET PR-SET PR-SET PR-SET PR-SET

Putative H4K20me3 Unknown H3K9me3 H3K4me3 Unknown Unknown Unknown Unknown Unknown

PRDM15 PRDM16 PRDM17

PFM15 PFM13 PFM14

PR-SET PR-SET PR-SET

H3K9me1 Unknown Unknown

Nik-Zainal et al. (2016), Wang et al. (2009) None reported Patel et al. (2011) None reported Deng and Huang (2004), Nishikawa et al. (2007) None reported None reported None reported None reported None reported None reported None reported None reported Moelans et al. (2010a, 2010b), Nishikawa et al. (2007) None reported None reported None reported

H4R3me1, H4R3me2as H3R8 RPS2, p53

Mathioudaki et al. (2011) Oh et al. (2014) None reported

PRMT1 PRMT2 PRMT3

(continued on next page)

1500


Table 1 e (continued ) Gene PRMT4 PRMT5 PRMT6 PRMT7 PRMT8 PRMT9 PRMT10 PRMT11

Synonym

SET domain

Targets

Dysregulation in breast cancer

CARM1

H3R17me2a, H3R26me2aa, H3R42me2aa

FBX011 PRMT9 FBX010

H3R8me2s, H4R3me2 H3R2me2, H2AR29, H3R42me2aa H3R2me2, H4R3me2s, H2AR3 EWS, NIFK Unknown Unknown Unknown

Al-Dhaheri et al. (2011), Habashy et al. (2013), Messaoudi et al. (2006) Hsu et al. (2011), Hu et al. (2015) Dowhan et al. (2012), Phalke et al. (2012) Yao et al. (2014) None reported None reported None reported None reported

a Targets that are disputed or require independent confirmation.

heterochromatin (Martin and Zhang, 2005). SETDB1 and SETDB2 are H3K9 trimethyltransferases, and while SETDB1 is responsible for methylating endogenous retroviral elements (Liu et al., 2014) and the inactive X chromosome (Keniry et al., 2016), SETDB2 contributes to centromere and pericentromere organization in concert with SUV39H1 (Falandry et al., 2010). Additionally, members of the PRDM family methylate H3K9 and are discussed below. In mammals, H3K9 and DNA methylation are strongly associated (Chang et al., 2011; Du et al., 2015), thus the relevance of dysregulation of H3K9 methyltransferases extends to DNA methylation. The strongest evidence that H3K9 methyltransferases contribute to breast tumorigenesis stems from studies on G9a. Deletion of G9a in mice results in embryonic lethality with severe differentiation defects in embryonic stem (ES) cells, demonstrating that G9a is essential for the repression of developmentally regulated genes (Tachibana et al., 2002). In line with this, G9a-dependent H3K9 methylation mediates epigenetic silencing of several tumor suppressor genes and G9a overexpression is observed in a number of cancers (Hua et al., 2014; Li et al., 2014). In addition to H3K9 mono- and dimethylation, G9a has several non-histone targets (Casciello et al., 2015; Huang et al., 2010; Rathert et al., 2008) on which it may exert coactivator and corepressor functions. Interestingly, knockdown of G9a suppressed breast tumor cell growth and lung colonization in a xenograft mouse model in vivo (Dong et al., 2012). G9a is critical for E-cadherin promoter silencing in basal-like breast cancer cell lines (BLBC) and pharmacologic inhibition of G9a using the DNA methyltransferase inhibitor 5Aza-20 -deoxycytidine led to the re-expression of cell adhesion factors such as E-cadherin, implying a potential link between G9a and epithelial-to-mesenchymal transition (EMT) (Wozniak et al., 2007). In contrast, G9a was shown to be downregulated in breast cancer samples and negatively correlated with tumor grade, suggesting that G9a is silenced during breast cancer progression (Si et al., 2015). G9a was reported to physically associate with transcription factors such as GATA3 (Si et al., 2015) and ERa (Zhang et al., 2016), suggesting that dysregulation of G9a expression may have important biological outcomes in breast epithelial cells. There is emerging evidence for dysregulation of other H3K9 methyltransferase family members in a range of human cancers, and these include amplifications and deletions of SETDB1 and SETDB2, respectively (Chen et al., 2014; Liu et al., 2015). However, the functional significance of these alterations

remains to be determined. High expression of KMT1A/ SUV39H1 has been observed in breast cancer but did not correlate with disease progression (Patani et al., 2011), possibly reflecting redundancy between Suv39h1 and Suv39h2 (Peters et al., 2001).

1.2.


1.2.1.

KMT2/MLL family

Members of the histoneelysine N-methyltransferase 2 (KMT2; also known as mixed-lineage leukemia (MLL)) family methylate histone H3 on lysine 4 (H3K4), promoting genome accessibility and transcription initiation. KMT2 proteins reside in large, multi-subunit complexes composed of four core subunits (WDR5, RBBP5, ASH2L and DPY30) as well as unique sets of interacting proteins. Members display distinct substrate specificities as demonstrated by the fact that targeted deletion of each family member in mice results in a severe but distinct phenotype (Rao and Dou, 2015). The exception to this is KMT2E/MLL5, which lacks intrinsic methyltransferase activity (Rao and Dou, 2015). Analysis of large-scale data sets such as TCGA (Kandoth et al., 2013; Liu et al., 2015) and COSMIC (Kudithipudi and Jeltsch, 2014; Rao and Dou, 2015) has identified KMT2 family members as among the most frequently mutated genes in human cancer. MLL2 and MLL3 are regarded as driver genes in breast cancers (Nik-Zainal et al., 2016), while neither MLL1 nor MLL4 play a significant role in this disease. These findings may reflect their different functions in cells: MLL1 (KMT2A) and MLL4 (KMT2B) are responsible for H3K4me3 at gene promoters. In contrast, MLL3 (KMT2C) and MLL2 (KMT2D, also called MLL4 in mice) introduce a single methyl group at H3K4 at the enhancers and promoters of target genes, and can repress genes in some cell types leading to inhibition of cell growth (Rao and Dou, 2015). The function of KMT2D is likely to be contextdependent however, since knockdown of MLL2 reduced proliferation of HER2þ breast cancer cells (Matkar et al., 2015) and migration of MDA-MB-231 breast cancer cells (Kim et al., 2014). MLL2 was shown to associate with PYGO2, which regulates WNT1-target gene expression, leading to expansion of a CD44þCD24 stem cell-like population in breast cancer cell lines (Chen et al., 2010). Moreover, MLL2 is mutated in 30% of metaplastic breast carcinomas (Ross et al., 2015), a rare subset of breast tumors. In contrast to MLL2, MLL3 was found to be in


1501

Figure 1 e Histone methyltransferases in humans and their targets. The lysine (K) (purple boxes) and arginine (R) (orange boxes) methyltransferases are grouped according to the specific residue on the histone tail targeted for modification. Histones are shown are circles, and tails are shown as bold lines. Residues can be mono-, di- or trimethylated in most cases. For some methyltransferases, there is evidence that multiple residues are targeted. Non-histone targets of methylation are not shown.

the top 10 most frequently mutated genes in invasive ductal carcinoma (Ciriello et al., 2015), with mutations found across 5e7% of all breast cancer subtypes (Cancer Genome Atlas Network, 2012). Curiously, mutations in MLL3 do not appear to correlate with patient survival, while deletions in MLL3 or copy number gains are associated with poorer and better overall survival, respectively (Liu et al., 2015). In addition to the MLL proteins, there is also emerging evidence that SetD1A may have tumor suppressive functions (Salz et al., 2015).

1.2.2.

SMYD family

The SMYD family comprises a subset of five proteins defined by a SET domain that is split into two segments by a MYND (Myeloid, Nervy and DEAF-1) domain, followed by a cysteinerich post-SET domain (Kudithipudi and Jeltsch, 2014). The MYND domain encompasses a putative zinc-finger motif that facilitates proteineprotein interactions and is the feature that distinguishes SMYDs from all other SET domaincontaining proteins. SMYD1-3 are the best characterized family members and their SET domains have been confirmed to be catalytically active. SMYD2 and SMYD3 were identified as H3K4me3 methyltransferases but additional roles have been reported with links to cancer. For example, SMYD2 has been reported to methylate ERa, p53 and Rb (Huang et al., 2006; Saddic et al., 2010; Zhang et al., 2013) in addition to histone targets (Table 1), while non-histone targets of SMYD3 include MAP3K2 (MEKK2) (Mazur et al., 2014). SMYD3 also functions as a coactivator of ERa by methylating histone H3K4 at the ERE in the promoter regions of target genes in response to ligand (Kim et al., 2009). Analysis of genome sequencing data of a large number of breast cancers identified amplifications of SMYD2 or SMYD3 in more than 10% of samples (Liu et al., 2015). SMYD2

expression was significantly higher in basal-like tumors, suggesting it may be a marker of poor prognosis. Conversely, SMYD3 expression was significantly lower in basal-like tumors than non-basal subtypes (Liu et al., 2015). Despite amplification, higher SMYD3 expression was associated with improved disease-free survival (DFS) suggesting it has a tumor suppressor role (Patani et al., 2011). SMYD3 amplification may simply be a non-pathogenic passenger alteration, since amplification of chromosome 1q on which SMYD3 is present was observed in 22% of tumors (Liu et al., 2015). Nevertheless, knockdown of SMYD3 in breast cancer cell lines reduced their growth (Hamamoto et al., 2006), suggesting that the differences in these findings may relate to the different targets of SMYD3 methylation in different contexts. In contrast to SMYD2 and SMYD3, there is little evidence implicating SMYD1, SYMD5 or SYMD4 in breast cancer (Hu et al., 2009).

1.3.


Methylation of H3K36 is executed by at least eight SET-domain containing enzymes in humans including ASH1L, SETD2 and NSD1-3 (Wagner and Carpenter, 2012). The biological function of H3K36 methylation in humans is not fully understood, however, it is highly correlated with actively transcribed genomic regions (Vougiouklakis et al., 2015). The nuclear receptor binding SET domain (NSD; also known as multiple myeloma SET domain (MMSET)) family consists of three members that are known to mono- or dimethylate H3K36, although additional substrate specificities have been reported. Dimethylation of H3K36 is found on newly activated genes and is sufficient for gene transcription (Kuo et al., 2011) but does not necessarily correlate with gene transcript levels

1502


Figure 2 e Distribution of histone modifications across a gene. Enrichment of histone modifications at the enhancer and promoter regions and across the gene body are shown for an active gene (blue shading) or inactive gene (red shading). TSS [ transcriptional start site. Only lysine modifications are shown, where there is sufficient information about distribution across genes rather than genome-wide profiles.

(Rao et al., 2005) suggesting that the H3K36 methylation code is complex (Wagner and Carpenter, 2012). Nsd1 knockout mice are embryonically lethal (Rayasam et al., 2003) and Nsd2 knockout mice die shortly after birth with WolfeHirschhorn-like syndrome (Nimura et al., 2009), suggesting that they have non-redundant functions (Vougiouklakis et al., 2015). While there is limited evidence of NSD1 dysregulation in breast cancer (Vougiouklakis et al., 2015), NSD2/MMSET overexpression is associated with tumor aggressiveness (Kassambara et al., 2009). Moreover, NSD2 may play a role in drug resistance, since tamoxifen-resistant breast cancer cell lines have highly elevated NSD2, and only wild type NSD2, but not its methylase-defective mutant conferred resistance to tamoxifen (Wang et al., 2016). Consistent with this, NSD2 overexpression is highly correlated with poor survival in tamoxifentreated patients (Wang et al., 2016). Notably, NSD2/MMSET and the H3K27 methyltransferase EZH2 (discussed below) are coexpressed at high levels in cancers including breast and prostate in which EZH2’s oncogenic activity was reported to require NSD2/MMSET (Asangani et al., 2013). NSD3/WHSC1L1 is found in the 8p11.2 region of the genome. Notably, rearrangements of chromosomal region 8p are frequently found in human cancer, including around 15% of breast cancers, and are associated with poor prognosis (Yang et al., 2010). This may be in part attributed to NSD3, since it was recently reported to be the fourth most frequently amplified methyltransferase in breast cancer (Liu et al., 2015), concordant with another study showing NSD3 amplifications in 15% and 5% of breast and ovarian cancers, respectively (Chen et al., 2014). Overexpression of NSD3/WHSC1L1 in MCF10a cells suggests that it has oncogenic properties (Yang et al., 2010), consistent with the observation that high NSD3

mRNA levels in basal-like breast cancers are associated with worse survival (Liu et al., 2015). NSD3/WHSC1L1 is required for the growth and survival of breast cancer cells in which WHSC1L1 is amplified and overexpressed (Yang et al., 2010). These results are intriguing, since NSD3 was ranked as the most ‘druggable’ driver gene identified from TCGA datasets (Chen et al., 2014). Notably, different isoforms of NSD3 have been reported, but their precise roles in breast cancer are yet to be determined (Yang et al., 2010). SETD2 is solely responsible for all H3K36 trimethylation in humans (Edmunds et al., 2008) and is considered a driver gene of breast cancer (Nik-Zainal et al., 2016). Very little is known about the remaining SET proteins, although amplification of ASH1L was recently reported in over 10% of breast cancers (Liu et al., 2015). Intriguingly, knockdown of both SETD4 (Faria et al., 2013) and SETD6 (O’Neill et al., 2014) significantly suppressed proliferation of breast cancer cell lines, suggesting they may have pro-proliferative functions.

1.4.


Trimethylation of H3K27 is an important repressive chromatin mark and is mediated by the Polycomb repressive complex 2 (PRC2). In mammals, the PRC2 core complex consists of EED, SUZ12, NURF55, Rbap46/48 and the catalytic subunits EZH1 or EZH2, which introduce up to three methyl groups on H3K27. EZH1 and EZH2 proteins are highly conserved, share 67% homology with each other and contain identical SET domains (Laible et al., 1997). EZH1 is the dominant H3K27 methyltransferase in non-proliferative adult tissues, while EZH2 is associated with proliferation (Laible et al., 1997; Margueron et al., 2008; Shen et al., 2008) as evidenced


by the embryonic lethality of Ezh2-deficient mice (O’Carroll et al., 2001), while Ezh1-deficient mice have no overt phenotype (Hidalgo et al., 2012). Targets of Polycomb-mediated silencing include transcription factors and signaling genes that play a significant role in cell fate determination, as well as tumor suppressor genes. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases (DNMTs), thereby linking these two epigenetic repression systems (Kudithipudi and Jeltsch, 2014). EZH2 is by far the most extensively studied histone methyltransferase with over 200 papers published on its role in breast cancer alone. EZH2 is up-regulated in invasive carcinoma and breast cancer metastases and independently predicts survival (Bachmann et al., 2006; Collett et al., 2006; Kleer et al., 2003; Raaphorst et al., 2003). EZH2 overexpression is significantly associated with high cell proliferation, and poorly differentiated, aggressive and triple-negative breast cancer (TNBC) (Bachmann et al., 2006; Collett et al., 2006; Kleer et al., 2003; Raaphorst et al., 2003). High EZH2 also correlates with poor outcome in patients with advanced disease and treated with tamoxifen (Reijm et al., 2011). Accordingly, EZH2 is one of a set of 300 genes whose expression predicts poor outcome in breast cancer (van ’t Veer et al., 2002). In benign breast tissues, elevated levels of EZH2 were detected in patients who later developed breast cancer, indicating that EZH2 upregulation precedes morphological changes and may be a marker for precancerous cells (Ding and Kleer, 2006). Dysregulation of EZH2 and H3K27 trimethylation in cancer arises from gain- and loss-of-function mutations in EZH2, overexpression of EZH2, mutations in other Polycomb complex members or the H3K27 demethylase gene UTX, and mutations in the SWI-SNF chromatin remodeling complex that partially antagonizes Polycomb function. Compared with blood cancers, very few mutations in EZH2 are observed in solid tumors (Kim and Roberts, 2016; Zingg et al., 2015) suggesting that different selection pressures in these tissues lead to dysregulation of EZH2. In stark contrast, there is only one report of lower EZH1 expression in breast cancer and overexpression or mutations of EZH1 have not been reported (Liu et al., 2015). Significant efforts have been made to try to understand the role of EZH2 in cancer and the normal setting. There is evidence to suggest that as tissues differentiate, they switch from using EZH2, which has strong methyltransferase activity, to EZH1, which has low activity (Ezhkova et al., 2011; Stojic et al., 2011). While these findings suggest EZH2 facilitates transformation by blocking differentiation, EZH2 can facilitate differentiation programs of several tissue types including bone and adipocytes (Schwarz et al., 2014; Wang et al., 2010). Ultimately, it seems that EZH2 functions in a cell-specific manner to suppress transcriptional programs that underlie alternate cell fates (Kim and Roberts, 2016) and its dysregulation in cancer results in derepression of inappropriate transcriptional programs. Deletion or knockdown of Ezh2 leads to defects in mammary gland development, consistent with a normal role for EZH2 in stem and progenitor cells (Michalak et al., 2013; Pal et al., 2013). Progesterone leads to increased EZH2 levels and luminal cell expansion, suggesting that hormone-induced chromatin changes likely play a role in the initiation of breast cancer (Pal et al., 2013). Overexpression of EZH2 alone leads to hyperplasia, but is not sufficient to

1503

induce tumors (Li et al., 2009) and requires additional oncogenic events (Gonzalez et al., 2014), consistent with findings that EZH2 upregulation is an early event in breast tumorigenesis (Ding and Kleer, 2006). Unexpectedly, despite earlier work correlating EZH2 overexpression with TNBC and BRCA1-linked breast cancer (Gonzalez et al., 2009; Puppe et al., 2009), deletion of EZH2 accelerated tumors in a mouse model of Brca1deleted breast cancer (Bae et al., 2014) suggesting that EZH2 overexpression and loss are not equivalent. Paradoxically, EZH2 and H3K27 trimethylation are inversely correlated in breast cancers, particularly in the TNBC subtype (Holm et al., 2012; Wei et al., 2008), although high EZH2 and low H3K27me3 predict poor outcome even in ERþ breast cancer (Bae et al., 2014). These results suggest the EZH2 present at high levels in tumor cells may have poorly functional methyltransferase activity. Alternatively, at least in basal-like tumors, the low H3K27me3 abundance may reflect relatively few Polycomb gene targets in the tumor cell of origin compared with the number of genes under Polycomb control in more differentiated breast cancer subtypes (Holm et al., 2012). Since basal-like tumors are thought to arise from a luminal progenitor cell (Lim et al., 2009), this would be consistent with the findings that H3K27me3 is relatively lower in stem/progenitor cells and increases upon luminal lineage specification (Pal et al., 2013). Yet another explanation for the discordance between EZH2 and H3K27me3 levels is that the oncogenic activity of EZH2 may be orchestrated through non-canonical functions or through methylation of non-histone targets by EZH2 (Bae and Hennighausen, 2014; Gonzalez et al., 2014; Katoh, 2016; Xu et al., 2015). Intriguingly, although EZH2 overexpression is often observed in basal-like breast cancer, gene expression profiles of the EZH2-null mammary stem cell-enriched basal cell population displayed similarities with signatures of claudin-low breast cancers (Pal et al., 2013).

1.5.

H3K79 methyltransferase DOT1L

Methylation of H3K79 is associated with active gene transcription (reviewed in (Nguyen and Zhang, 2011)). DOT1L is the sole enzyme that methylates H3K79, and mice deficient in Dot1l are not viable (Jones et al., 2008). Instead of a SET domain, DOT1L harbors an AdoMet binding motif similar to arginine and DNA methyltransferases. High DOT1L protein expression is associated with breast tumors lacking both ER and PR expression and with significantly worse overall survival (Cho et al., 2015). Consistent with a role for DOT1L in tumor progression and metastasis, overexpression of DOT1L in cell lines suggests that it has oncogenic properties and knockdown of DOT1L reduced lung metastasis of MDA-MB-231 tumor cells in vivo (Cho et al., 2015).

1.6.


Methylation of H4K20 is highly cell-cycle regulated and important for several biological processes to ensure genome integrity (Jørgensen et al., 2013). Mono- and dimethylated H4K20 is involved in DNA replication and DNA damage repair, whereas trimethylated H4K20 is a hallmark of silenced heterochromatic regions (Jørgensen et al., 2013). SETD8 mono-

1504


methylates H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate H4K20 di- and trimethylation. This collaboration between SETD8 and the SUV4-20H methyltransferases is crucial, and disruption of the dynamic fluctuations in H4K20 methylation during the cell cycle leads to genomic instability (Jørgensen et al., 2013). In parallel, genome-wide loss of H4K20me3 is observed in multiple types of human cancers (Fraga et al., 2005) and knockdown of SUV420H2 in the nontumorigenic MCF10A cell line increased cell invasion (Yokoyama et al., 2014). Conversely, high expression of SETD8 is significantly associated with poor DFS in breast cancer (Liu et al., 2016b).

1.7.

PRDM family of methyltransferases

The PRDM family of proteins comprises 17 family members in primates. The PR domain is structurally and functionally similar to the SET domain, and exhibits histone methyltransferase activity solely on lysine residues. However, enzymatic activity has been demonstrated for only some members (see Table 1) and functional data is still lacking for many of these proteins. Nevertheless, most PRDM family members have been associated with dysregulation in multiple cancer types (reviewed in (Mzoughi et al., 2016)). In contrast to the well-established tumor suppressor role for PRDM1/Blimp1 in activated B cell-like diffuse large cell lymphoma (Pasqualucci et al., 2006), PRDM1 may have an oncogenic role in breast cancer. PRDM1 does not have intrinsic methyltransferase activity but facilitates gene silencing through association with histone deacetylases (HDACs) (Yu et al., 2000) or other methyltransferases (G9a (Gyory et al., 2004) and PRMT5 (see below) (Ancelin et al., 2006)) to mediate H3K9me2 and H4R3me2s, respectively. PRDM1 has been reported to repress ERa transcription and PRDM1 expression is higher in ER compared with ERþ breast cancers (Wang et al., 2009). Moreover, PRDM1 mutations may be “drivers” in a small fraction of both ER and ERþ breast tumors (NikZainal et al., 2016). PRDM2 encodes two protein products: RIZ1, which contains the 202 amino acid PR domain, and RIZ2, which lacks it. Notably, RIZ1 and not RIZ2 is silenced through methylation of the PRDM2/RIZ promoter in several tumor types, including breast cancer, suggesting a role for RIZ1 in tumor suppression (Du et al., 2001). Accordingly, forced RIZ1 expression in breast cancer cells caused cell cycle arrest and/or apoptosis (He et al., 1998), while silencing of RIZ1 expression enhanced breast cancer cell proliferation (Gazzerro et al., 2006). RIZ2 was recently reported to act as a negative regulator of RIZ1 (Abbondanza et al., 2012). Together these data suggest a positive selection for RIZ2 in cancer progression, but this remains to be formally demonstrated. The PRDM3/MECOM coding gene is a complex locus formed by the fusion of two genes, MDS1 and EVI1, and encodes several transcription factor variants transcribed from two distinct transcription start sites, including MDS1-EVI1 (the PR domain containing PRDM3), EVI1 (PR-less isoform) and EVI1D324. EV11 encodes a nuclear zinc finger DNA-binding protein that is implicated in myeloid leukemia. Chromosome 3q26 on which EVI1 is located, is also frequently amplified in a number of epithelial cancers including breast and ovarian cancers

(Zack et al., 2013). High EVI1 levels are associated with reduced relapse-free, metastasis-free and overall survival in ER but not ERþ breast cancer patients (Patel et al., 2011). Little is known about the molecular function of other PRDM family members (see Table 1). There is evidence that PRDM5 is down-regulated in breast cancer (Deng and Huang, 2004; Nishikawa et al., 2007), although the significance of this observation remains unclear. The 8q13.3 region containing PRDM14 is amplified in 34e75% of human breast cancers and is associated with HER2 expression, high mitotic index and high histological grade (Moelans et al., 2010b; Nishikawa et al., 2007). Moreover, PRDM14 amplification is more frequently seen in high-grade ductal carcinoma in situ (DCIS) than in low/ intermediate-grade DCIS (Moelans et al., 2010a) suggesting PRMD14 amplification may be associated with progression to invasive ductal carcinoma. Introduction of PRDM14 into breast cancer cell lines enhanced their growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 induced apoptosis and increased cell sensitivity to chemotherapeutic drugs (Nishikawa et al., 2007) implicating PRDM14 in chemoresistance. Unfortunately, insights into PRDM14 function in the mouse mammary gland have been unsuccessful (Carofino et al., 2013).

1.8.

PRMT family of arginine methyltransferases

The PRMT family consists of 11 members in humans, with the recent identification of two related family members: PRMT9/ FBX011 and PRMT11/FBX010 (Teyssier et al., 2010). With the exception of PRMT10 and PRMT11/FBX010, family members have been shown to have enzymatic activity and can catalyze arginine methylation on histones and non-histone targets (Krause et al., 2007; Wei et al., 2014). PRMTs are primarily classified as type I and type II enzymes and catalyze the formation of a mono-methylated (MMA) intermediate; subsequently, type I PRMTs (PRMT 1e4, 6, 8 and 9) further catalyze the production of asymmetric dimethylation of arginine residues (aDMA), and type II PRMTs (PRMT5, and 7) catalyze the formation of symmetric dimethylation of arginine residues (sDMA) (Yang and Bedford, 2013). PRMTs regulate a number of cellular processes including DNA repair and RNA splicing by functioning as co-activators for transcription factors, such as steroid/nuclear receptors, p53, E2F1 and methylating members of the DNA damage response (Le Romancer et al., 2008; Teyssier et al., 2010; Yang and Bedford, 2013). In turn, the activity of PRMTs and PRMT splice forms (Goulet et al., 2007; Zhong et al., 2012) is regulated through posttranslation modifications, association with regulatory proteins and subcellular localization (Wei et al., 2014; Yang and Bedford, 2013). PRMT1 is the predominant asymmetric arginine methyltransferase in mammalian cells and not surprisingly, Prmt1 knockout mice are not viable (Yu et al., 2009). Aberrant expression of PRMT1 has been observed in several cancers (reviewed in (Yang and Bedford, 2013)). High PRMT1 expression was shown to be indicative of the disease progression and aggressiveness in breast cancer (Mathioudaki et al., 2011). In agreement with this, PRMT1 was shown to activate expression of the EMT-associated transcription factor ZEB1 through methylation of its promoter (Gao et al., 2016), thus potentially linking

1505


PRMT1 with metastatic capacity. In parallel, knockdown of PRMT1 in MDA-MB-231 breast cancer cells reduced their metastasis in vivo and induced cellular senescence (Gao et al., 2016). In contrast to PRMT1, decreased PRMT2 expression in breast cancer and a lower PRMT2 gene expression signature are strongly associated with metastasis-free survival (Oh et al., 2014). In agreement with a tumor suppressor role for PRMT2, knockdown of PRMT2 appears to increase growth of tumors upon xenotransplantation (Zhong et al., 2014). Although PRMT4/CARM1 has been implicated in breast cancer, its role is controversial. CARM1 expression was somewhat elevated in aggressive breast tumors (Messaoudi et al., 2006) and significantly associated with poor outcome (Habashy et al., 2013). This contrasts with another report, which failed to correlate CARM1 protein expression with tumor grade (Al-Dhaheri et al., 2011). Although CARM1 overexpression increased endogenous CCNE1 mRNA levels (Messaoudi et al., 2006), linking CARM1 with cell proliferation, it reduced estrogen-induced proliferation in MCF7 breast cancer cells (Al-Dhaheri et al., 2011). The discrepancies between these studies may be due to the different cell contexts (cell lines versus tissue samples). PRMT5 is a putative oncogene and its upregulation correlates with tumor progression, as shown for breast, ovarian and prostate cancer (Hsu et al., 2011; Mounir et al., 2016; Yang et al., 2013). PRMT5 is the major type II PRMT and mice deficient in this gene are not viable (Tee et al., 2010). Its targets include key repressive histone methylation marks H3R8me2s and H4R3me2s, and several non-histone targets including TP53, EGFR, PDCD4 and KLF4 (Hsu et al., 2011; Hu et al., 2015; Jansson et al., 2008; Powers et al., 2011). Methylation of H4R3me2s recruits DNMT3A, which in turn binds H4R3me2 and silences genes, coupling histone and DNA methylation

(Zhao et al., 2009). Interestingly, PRMT5 expression was found to be highest in TNBC, suggesting its expression correlates with poor prognosis (Hu et al., 2015). PRMT6 can act as either a transcriptional activator or repressor dependent on context (Dowhan et al., 2012), which may explain contradictory evidence that it can act as a tumor suppressor or oncogene. Loss of PRMT6 results in cell cycle arrest and senescence due to release of p53 and p21 repression (Neault et al., 2012; Phalke et al., 2012; Stein et al., 2012). It therefore seems likely that p53 is silenced in tumors with high PRMT6 levels ( Yang and Bedford, 2013). Accordingly, PRMT6 overexpression correlated with increased tumor aggressiveness in breast tumors (Phalke et al., 2012). In contrast, a low PRMT6 gene expression signature and a correspondingly high PRMT6-dependent gene signature correlated with better overall and distant metastasis-free survival in ERþ breast cancer (Dowhan et al., 2012), suggesting the discrepancy between these different studies may be due to analysis of PRMT6 gene versus protein expression. The human PRMT7 gene is located in a region of the genome that is known to have high copy number aberrations in metastatic breast cancers (Thomassen et al., 2009). PRMT7 expression is higher in basal-like breast cancers compared with other subtypes (Yao et al., 2014) and overexpression of PRMT7 led to increased metastasis in a xenograft mouse model (Baldwin et al., 2015; Yao et al., 2014). PRMT7 interacts directly with both YY1 and HDAC3 to repress E-cadherin expression in vitro, suggesting a possible link with EMT (Yao et al., 2014). There is currently little evidence that the remaining PRMT family members are involved in breast oncogenesis. Although PRMT8 is reported to be the most highly mutated of all the PRMTs (Yang and Bedford, 2013), its expression is highest in the brain so it may not be significantly dysregulated in other tissues.

Table 2 e Selected inhibitors of histone methyltransferases and their status in clinical development. HMT

Small molecule

Status

Clinical trial

Cancer/cell type

Reference

G9a

UNC0638

Preclinical

BC cell line

Vedadi et al. (2011)

MLL

MM-401

Preclinical

Human MLL and cell lines

Cao et al. (2014)

SMYD2

LLY-507

Preclinical

ESCC, HCC and BC cell lines

Nguyen et al. (2015)

SMYD3

EPZ031686 BCI-121 GSK2807

Preclinical Preclinical Tool compounda

Orally bioavailable in mice Human cell lines Not shown

Mitchell et al. (2016) Peserico et al. (2015) Van Aller et al. (2016)

EZH2

EPZ-6438 (E7438)

Phase Phase Phase Phase Phase

BCL, solid tumors MRT MRT BCL GCB-DLBCL, tFL and MM

Knutson et al. (2014)

CPI-1205 GSK2816126

1/2 2 1 1 1

NCT01897571 NCT02601950 NCT02601937 NCT02395601 NCT02082977

Bradley et al. (2014) McCabe et al. (2012)

EZH1/EZH2

DS-3201b

Phase 1

NCT02732275

Non-Hodgkin lymphoma

https://clinicaltrials.gov

DOT1L

EPZ-5676

Phase 1 Phase 1

NCT02141828 NCT01684150

AML, ALL AML, ALL

Daigle et al. (2013)

PRMT3

SCG707

Tool compounda

Human cell lines

Kaniskan et al. (2015)

PRMT5

GSK3326595

Phase 1

Non-Hodgkin lymphoma

Chan-Penebre et al. (2015)

PRMT6

EPZ020411

Tool compounda

Human cell lines

Mitchell et al. (2015)

NCT02783300

ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; BC, breast cancer; BCL, B cell lymphoma; ESCC, esophageal squamous cell carcinoma; GCB-DLBCL, germinal center B-cell like diffuse large B cell lymphoma; HCC, hepatocellular carcinoma; MLL, mixed lineage leukemia; MM, multiple myeloma; MRT, malignant rhabdoid tumor; tFL transformed follicular lymphoma. a Tool compound indicates a promising lead compound with potent inhibitory activity in biochemical assays that has not yet been tested in vitro or in vivo on cells.

1506

1.9.


Targeting HMTs as a therapeutic strategy

Although patient survival continues to improve for breast cancer, this disease continues to be a significant burden and improved treatment options are required. There is mounting evidence that HMTs are dysregulated in cancer due to amplification, deletion or mutation and contribute to cancer initiation and progression. Because epigenetic changes are reversible and HMTs are druggable, inhibitors targeting HMTs represent a unique opportunity for pharmacologic intervention using a novel class of anti-cancer drugs. Although the quest to develop epigenetic drugs poses certain challenges (Audia and Campbell, 2016), this is an area of intense interest and several HMTs including EZH2, MLL3 and NSDs have been put forward as rational drug targets (Chen et al., 2014; Katoh, 2016). Several potent and selective SAM-competitive inhibitors of SMYD3 have been described (Van Aller et al., 2016), and their clinical utility certainly warrants more investigation. Small molecule inhibitors for several PRMTs have been reviewed elsewhere (Song et al., 2016), and they represent an exciting class of potential anticancer drugs. Encouragingly, a potent, selective inhibitor of DOT1L has been described (Daigle et al., 2013) and demonstrated some activity in breast cancer cell lines (Zhang et al., 2014). This inhibitor is currently in clinical trials for leukemia, and it remains to be seen if inhibition of DOT1L will of clinical use in breast cancer. Selective inhibitors of HMTs have already been reported to be effective in vivo (Audia and Campbell, 2016; Brien et al., 2016), but efficacy in breast cancer is currently limited to in vitro experiments in cell lines (Table 2). The HMT for which inhibitors are available and clinically most advanced is EZH2. Several small molecule inhibitors of EZH2 are currently in clinical trials (see Table 2) (Bradley et al., 2014; Kim and Roberts, 2016; Knutson et al., 2014; McCabe et al., 2012). However, these are most efficacious in target cells bearing activating mutations in EZH2, which render them hyper-sensitive to EZH2 inhibition and these types of mutations are rare in solid tumors. Nevertheless, there is emerging evidence that mutations in related pathways predict sensitivity to EZH2 inhibitors (Bitler et al., 2015; Kim et al., 2015; Knutson et al., 2013; LaFave et al., 2015; Souroullas et al., 2016). For example, the proliferation of human ovarian cancer cells with loss-of-function ARID1A mutations was suppressed by treatment with the EZH2 inhibitor GSK126 (Bitler et al., 2015). Notably, sensitizing mutations in related pathways in some cell types do not always predict sensitivity to EZH2 inhibition for other cell types. For example, unlike BAP1 mutations in hematopoietic cells and mesothelioma (LaFave et al., 2015), uveal melanoma cells are insensitive to EZH2 inhibition, regardless of BAP1 mutational status (Schoumacher et al., 2016). Moreover, prolonged EZH2 depletion led to tumor progression (de Vries et al., 2015), suggesting that EZH2 inhibition should be approached with caution. It is likely that the same will apply to inhibitors of other HMTs. Since drug resistance frequently develops regardless of the type of cancer therapy, and upregulation of HMTs has been implicated in drug resistance (Matkar et al., 2015; Wray et al., 2009), understanding adaptive resistance will be key to improving outcomes.

Pharmacological development in this area has been making some progress, but the vast majority of HMTs have not progressed beyond preclinical stage with most work limited to in vitro experiments in cell lines. It may now be possible to circumvent some of the challenges of developing small molecule inhibitors by utilizing newly developed technologies for epigenetic editing. The use of DNA-binding domains (DBD) fused to an enzymatic or scaffolding effector has emerged as one such promising approach. DBDs such as zinc-finger proteins (ZFPs), transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators to genomic sites and manipulation of genomic function. The advantage of using engineered DBDs is that they can target any portion of the genome including regulatory elements and non-coding genes that are not targetable by RNAi. These technologies have already been used to activate and repress gene regulation by depositing specific histone modifications (recently reviewed in (Thakore et al., 2016)). Importantly, engineered ZFP repressors were able to epigenetically silence oncogenes in breast cancer, resulting in reduced growth of breast cancer cells in vitro (Falahi et al., 2013) and in mouse models (Stolzenburg et al., 2015), suggesting this approach may be a feasible alternative strategy in the future. This is a very fast-moving area with new developments enabling novel applications. Nevertheless, it remains to be seen if off-target localization of DBDs (Polstein et al., 2015; Thakore et al., 2015) can be limited and if de novo epigenetic modifications can be stably inherited. It appears that at least for de novo DNA methylation-induced silencing, oncogenic silencing was heritable and stable (Stolzenburg et al., 2015). Different combinations of activating and silencing histone modifications have been proposed to regulate distinct gene regulatory programs, so the net effect of dysregulated HMTs is expected to result in transcriptional dysregulation. Indeed, specific patterns of global histone modifications representing different gene expression programs are associated with distinct clinical outcomes (Seligson et al., 2005) and chemoresistance (Chapman-Rothe et al., 2013). Advances in technology such as ChIP-sequencing have allowed us to map the distribution and co-localization of histone marks; however, our ability to detect coincident marks on nucleosomes is currently limited. Recently, Shema et al. established an assay to simultaneously determine the modification state and genomic position of individual nucleosomes (Shema et al., 2016). Moreover, since CRISPR/Cas9 technology can simultaneously activate or repress multiple genes in the same cell (Zalatan et al., 2015), it should be feasible to delete multiple methyltransferases using this approach to study the combined loss of histone modifications. Indeed, the CRISPR/ Cas9 system has revolutionized cell biology research (Wang and Qi, 2016) and is likely to be useful for the study of HMTs. Such approaches will ultimately help decipher the complexity of combinatorial histone modifications and their contribution to tumorigenesis and guiding therapeutic strategies.


2.

Conclusions

With the advent of large scale genomic and transcriptomic analyses of cancer, our ability to comprehensively interrogate the genome and epigenome of human cancers has greatly improved and is expected to inform future therapeutic strategies. Large scale sequencing efforts are uncovering common somatic mutations in breast cancers (Ciriello et al., 2015; Kudithipudi and Jeltsch, 2014; Nik-Zainal et al., 2016) as well as in other epithelial tumors (Kanchi et al., 2014), helping to stratify tumors into clinically relevant subtypes. Moreover, a recent study of 8000 cancer cases revealed that complex insertions/deletions are often overlooked or misannotated (Ye et al., 2016), indicating that their contribution to pathogenesis is currently underestimated. These studies, coupled with advances in technology, will help to define the “epigenomic landscape” and how it relates to gene expression profiles and phenotype. This review has highlighted what is known about the dysregulation of HMTs in breast cancer. Notably, some HMTs such as EZH2 and MLL function in large complexes with cofactors (Zhang et al., 2015) that may also be mutated in breast cancer. For example, PRC2 components EED and SUZ12 are emerging as tumor suppressors in their own right (Jene-Sanz et al., 2013; Koppens and Van Lohuizen, 2016), leading to further complexity in the interpretation of how HMT dysregulation impacts on phenotypes. Since functional data is still missing for many of the HMTs, the use of knockout or transgenic mice or CRISPR/Cas9 technology will be important for elucidating their function and guiding the development of targeted therapies for clinical use.

Acknowledgments The breast cancer laboratory is supported by the Australian National Health and Medical Research Council (NHMRC) grants no. 1016701, no. 1024852, no. 1086727; NHMRC IRIISS; the Victorian State Government through VCA funding of the Victorian Breast Cancer Research Consortium and Operational Infrastructure Support; and the Australian Cancer Research Foundation. E.M.M. is supported by a National Breast Cancer Foundation Career Development Fellowship and J.E.V. by a NHMRC Australia Fellowship. We thank K. Hogg for critical reading of the manuscript and P. Maltezos for assistance with preparation of figures. R E F E R E N C E S

Abbondanza, C., De Rosa, C., D’Arcangelo, A., Pacifico, M., Spizuoco, C., Piluso, G., Di Zazzo, E., Gazzerro, P., Medici, N., Moncharmont, B., Puca, G.A., 2012. Identification of a functional estrogen-responsive enhancer element in the promoter 2 of PRDM2 gene in breast cancer cell lines. J. Cell. Physiol. 227, 964e975. http://dx.doi.org/10.1002/jcp.22803. Al-Dhaheri, M., Wu, J., Skliris, G.P., Li, J., Higashimato, K., Wang, Y., White, K.P., Lambert, P., Zhu, Y., Murphy, L., Xu, W., 2011. CARM1 is an important determinant of ERa-dependent breast cancer cell differentiation and proliferation in breast

1507

cancer cells. Cancer Res. 71, 2118e2128. http://dx.doi.org/ 10.1158/0008-5472.CAN-10-2426. Ancelin, K., Lange, U.C., Hajkova, P., Schneider, R., Bannister, A.J., Kouzarides, T., Surani, M.A., 2006. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat. Cell Biol. 8, 623e630. http://dx.doi.org/10.1038/ ncb1413. Asangani, I.A., Ateeq, B., Cao, Q., Dodson, L., Pandhi, M., Kunju, L.P., Mehra, R., Lonigro, R.J., Siddiqui, J., Palanisamy, N., Wu, Y.-M., Cao, X., Kim, J.H., Zhao, M., Qin, Z.S., Iyer, M.K., Maher, C.A., Kumar-Sinha, C., Varambally, S., Chinnaiyan, A.M., 2013. Characterization of the EZH2-MMSET histone methyltransferase regulatory axis in cancer. Mol. Cell 49, 80e93. http://dx.doi.org/10.1016/j.molcel.2012.10.008. Audia, J.E., Campbell, R.M., 2016. Histone modifications and cancer. Cold Spring Harb. Perspect. Biol. 8. http://dx.doi.org/ 10.1101/cshperspect.a019521. Bachmann, I.M., Halvorsen, O.J., Collett, K., Stefansson, I.M., Straume, O., Haukaas, S.A., Salvesen, H.B., Otte, A.P., Akslen, L.A., 2006. EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J. Clin. Oncol. 24, 268e273. http:// dx.doi.org/10.1200/JCO.2005.01.5180. Bae, W.K., Hennighausen, L., 2014. Canonical and non-canonical roles of the histone methyltransferase EZH2 in mammary development and cancer. Mol. Cell. Endocrinol. 382, 593e597. http://dx.doi.org/10.1016/j.mce.2013.05.002. Bae, W.K., Yoo, K.H., Lee, J.S., Kim, Y., Chung, I.-J., Park, M.H., Yoon, J.H., Furth, P.A., Hennighausen, L., 2014. The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers. Mol. Carcinog. 54, 1172e1180. http://dx.doi.org/10.1002/mc.22188. Baldwin, R.M., Haghandish, N., Daneshmand, M., Amin, S., ^ te , J., 2015. Protein Paris, G., Falls, T.J., Bell, J.C., Islam, S., Co arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression. Oncotarget 6, 3013e3032. http://dx.doi.org/10.18632/ oncotarget.3072. Barski, A., Cuddapah, S., Cui, K., Roh, T.-Y., Schones, D.E., Wang, Z., Wei, G., Chepelev, I., Zhao, K., 2007. High-resolution profiling of histone methylations in the human genome. Cell 129, 823e837. http://dx.doi.org/10.1016/j.cell.2007.05.009. Bitler, B.G., Aird, K.M., Garipov, A., Li, H., Amatangelo, M., Kossenkov, A.V., Schultz, D.C., Liu, Q., Shih, I.-M., ConejoGarcia, J.R., Speicher, D.W., Zhang, R., 2015. Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1Amutated cancers. Nat. Med. 21, 231e238. http://dx.doi.org/ 10.1038/nm.3799. Borley, J., Brown, R., 2015. Epigenetic mechanisms and therapeutic targets of chemotherapy resistance in epithelial ovarian cancer. Ann. Med. 47, 359e369. http://dx.doi.org/ 10.3109/07853890.2015.1043140. Bradley, W.D., Arora, S., Busby, J., Balasubramanian, S., Gehling, V.S., Nasveschuk, C.G., Vaswani, R.G., Yuan, C.-C., ndez, J., Hatton, C., Zhao, F., Williamson, K.E., Iyer, P., Me Campbell, R., Cantone, N., Garapaty-Rao, S., Audia, J.E., Cook, A.S., Dakin, L.A., Albrecht, B.K., Harmange, J.-C., Daniels, D.L., Cummings, R.T., Bryant, B.M., Normant, E., Trojer, P., 2014. EZH2 inhibitor efficacy in non-Hodgkin’s lymphoma does not require suppression of H3K27 monomethylation. Chem. Biol. 21, 1463e1475. http:// dx.doi.org/10.1016/j.chembiol.2014.09.017. Brien, G.L., Valerio, D.G., Armstrong, S.A., 2016. Exploiting the epigenome to control cancer-promoting gene-expression programs. Cancer Cell 29, 464e476. http://dx.doi.org/10.1016/ j.ccell.2016.03.007.

1508


Cancer Genome Atlas Network, 2012. Comprehensive molecular portraits of human breast tumours. Nature 490, 61e70. http:// dx.doi.org/10.1038/nature11412. Cao, F., Townsend, E.C., Karatas, H., Xu, J., Li, L., Lee, S., Liu, L., Chen, Y., Ouillette, P., Zhu, J., Hess, J.L., Atadja, P., Lei, M., Qin, Z.S., Malek, S., Wang, S., Dou, Y., 2014. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 53, 247e261. http://dx.doi.org/10.1016/ j.molcel.2013.12.001. Carofino, B.L., Ayanga, B., Justice, M.J., 2013. A mouse model for inducible overexpression of Prdm14 results in rapid-onset and highly penetrant T-cell acute lymphoblastic leukemia (T-ALL). Dis. Model Mech. 6, 1494e1506. http://dx.doi.org/10.1242/ dmm.012575. Casciello, F., Windloch, K., Gannon, F., Lee, J.S., 2015. Functional role of G9a histone methyltransferase in cancer. Front Immunol. 6, 487. http://dx.doi.org/10.3389/fimmu.2015.00487. Chan-Penebre, E., Kuplast, K.G., Majer, C.R., Boriack-Sjodin, P.A., Wigle, T.J., Johnston, L.D., Rioux, N., Munchhof, M.J., Jin, L., Jacques, S.L., West, K.A., Lingaraj, T., Stickland, K., Ribich, S.A., Raimondi, A., Scott, M.P., Waters, N.J., Pollock, R.M., Smith, J.J., Barbash, O., Pappalardi, M., Ho, T.F., Nurse, K., Oza, K.P., Gallagher, K.T., Kruger, R., Moyer, M.P., Copeland, R.A., Chesworth, R., Duncan, K.W., 2015. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Nat. Chem. Biol. 11, 432e437. http://dx.doi.org/10.1038/ nchembio.1810. Chang, Y., Sun, L., Kokura, K., Horton, J.R., Fukuda, M., Espejo, A., Izumi, V., Koomen, J.M., Bedford, M.T., Zhang, X., Shinkai, Y., Fang, J., Cheng, X., 2011. MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a. Nat. Commun. 2, 533. http:// dx.doi.org/10.1038/ncomms1549. Chapman-Rothe, N., Curry, E., Zeller, C., Liber, D., Stronach, E., Gabra, H., Ghaem-Maghami, S., Brown, R., 2013. Chromatin H3K27me3/H3K4me3 histone marks define gene sets in highgrade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells. Oncogene 32, 4586e4592. http://dx.doi.org/10.1038/ onc.2012.477. Chen, J., Luo, Q., Yuan, Y., Huang, X., Cai, W., Li, C., Wei, T., Zhang, L., Yang, M., Liu, Q., Ye, G., Dai, X., Li, B., 2010. Pygo2 associates with MLL2 histone methyltransferase and GCN5 histone acetyltransferase complexes to augment Wnt target gene expression and breast cancer stem-like cell expansion. Mol. Cell. Biol. 30, 5621e5635. http://dx.doi.org/10.1128/ MCB.00465-10. Chen, Y., McGee, J., Chen, X., Doman, T.N., Gong, X., Zhang, Y., Hamm, N., Ma, X., Higgs, R.E., Bhagwat, S.V., Buchanan, S., Peng, S.-B., Staschke, K.A., Yadav, V., Yue, Y., Kouros-Mehr, H., 2014. Identification of druggable cancer driver genes amplified across TCGA datasets. PLoS One 9, e98293. http://dx.doi.org/ 10.1371/journal.pone.0098293. Cho, M.-H., Park, J.-H., Choi, H.-J., Park, M.-K., Won, H.-Y., Park, Y.-J., Lee, C.H., Oh, S.-H., Song, Y.-S., Kim, H.S., Oh, Y.-H., Lee, J.-Y., Kong, G., 2015. DOT1L cooperates with the c-Mycp300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression. Nat. Commun. 6, 7821. http://dx.doi.org/10.1038/ncomms8821. Ciriello, G., Gatza, M.L., Beck, A.H., Wilkerson, M.D., Rhie, S.K., Pastore, A., Zhang, H., McLellan, M., Yau, C., Kandoth, C., Bowlby, R., Shen, H., Hayat, S., Fieldhouse, R., Lester, S.C., Tse, G.M.K., Factor, R.E., Collins, L.C., Allison, K.H., Chen, Y.-Y., Jensen, K., Johnson, N.B., Oesterreich, S., Mills, G.B., Cherniack, A.D., Robertson, G., Benz, C., Sander, C., Laird, P.W., Hoadley, K.A., King, T.A., TCGA Research Network, Perou, C.M., 2015. Comprehensive molecular portraits of

invasive lobular breast cancer. Cell 163, 506e519. http:// dx.doi.org/10.1016/j.cell.2015.09.033. Collett, K., Eide, G.E., Arnes, J., Stefansson, I.M., Eide, J., Braaten, A., Aas, T., Otte, A.P., Akslen, L.A., 2006. Expression of enhancer of zeste homologue 2 is significantly associated with increased tumor cell proliferation and is a marker of aggressive breast cancer. Clin. Cancer Res. 12, 1168e1174. http://dx.doi.org/10.1158/1078-0432.CCR-05-1533. Crea, F., Fornaro, L., Paolicchi, E., Masi, G., Frumento, P., Loupakis, F., Salvatore, L., Cremolini, C., Schirripa, M., Graziano, F., Ronzoni, M., Ricci, V., Farrar, W.L., Falcone, A., Danesi, R., 2012. An EZH2 polymorphism is associated with clinical outcome in metastatic colorectal cancer patients. Ann. Oncol. 23, 1207e1213. http://dx.doi.org/10.1093/annonc/ mdr387. Curtis, C., Shah, S.P., Chin, S.-F., Turashvili, G., Rueda, O.M., Dunning, M.J., Speed, D., Lynch, A.G., Samarajiwa, S., Yuan, Y., € f, S., Ha, G., Haffari, G., Bashashati, A., Russell, R., Gra McKinney, S., METABRIC Group, Langerød, A., Green, A., Provenzano, E., Wishart, G., Pinder, S., Watson, P., Markowetz, F., Murphy, L., Ellis, I., Purushotham, A., Børresen, S., Caldas, C., Aparicio, S., Dale, A.-L., Brenton, J.D., Tavare 2012. The genomic and transcriptomic architecture of 2000 breast tumours reveals novel subgroups. Nature 486, 346e352. http://dx.doi.org/10.1038/nature10983. Daigle, S.R., Olhava, E.J., Therkelsen, C.A., Basavapathruni, A., Jin, L., Boriack-Sjodin, P.A., Allain, C.J., Klaus, C.R., Raimondi, A., Scott, M.P., Waters, N.J., Chesworth, R., Moyer, M.P., Copeland, R.A., Richon, V.M., Pollock, R.M., 2013. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood 122, 1017e1025. http://dx.doi.org/10.1182/ blood-2013-04-497644. de Vries, N.A., Hulsman, D., Akhtar, W., de Jong, J., Miles, D.C., Blom, M., van Tellingen, O., Jonkers, J., van Lohuizen, M., 2015. Prolonged Ezh2 depletion in glioblastoma causes a robust switch in cell fate resulting in tumor progression. Cell Rep. 10, 383e397. http://dx.doi.org/10.1016/j.celrep.2014.12.028. Deng, Q., Huang, S., 2004. PRDM5 is silenced in human cancers and has growth suppressive activities. Oncogene 23, 4903e4910. http://dx.doi.org/10.1038/sj.onc.1207615. Ding, L., Kleer, C.G., 2006. Enhancer of Zeste 2 as a marker of preneoplastic progression in the breast. Cancer Res. 66, 9352e9355. http://dx.doi.org/10.1158/0008-5472.CAN-06-2384. Dong, C., Wu, Y., Yao, J., Wang, Y., Yu, Y., Rychahou, P.G., Evers, B.M., Zhou, B.P., 2012. G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer. J. Clin. Invest. 122, 1469e1486. http://dx.doi.org/ 10.1172/JCI57349. Dowhan, D.H., Harrison, M.J., Eriksson, N.A., Bailey, P., Pearen, M.A., Fuller, P.J., Funder, J.W., Simpson, E.R., Leedman, P.J., Tilley, W.D., Brown, M.A., Clarke, C.L., Muscat, G.E.O., 2012. Protein arginine methyltransferase 6dependent gene expression and splicing: association with breast cancer outcomes. Endocr. Relat. Cancer 19, 509e526. http://dx.doi.org/10.1530/ERC-12-0100. Du, J., Johnson, L.M., Jacobsen, S.E., Patel, D.J., 2015. DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16, 519e532. http:// dx.doi.org/10.1038/nrm4043. Du, Y., Carling, T., Fang, W., Piao, Z., Sheu, J.C., Huang, S., 2001. Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily. Cancer Res. 61, 8094e8099. Edmunds, J.W., Mahadevan, L.C., Clayton, A.L., 2008. Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation. EMBO J. 27, 406e420. http://dx.doi.org/10.1038/sj.emboj.7601967.


Ezhkova, E., Lien, W.-H., Stokes, N., Pasolli, H.A., Silva, J.M., Fuchs, E., 2011. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 25, 485e498. http://dx.doi.org/ 10.1101/gad.2019811. Falahi, F., Huisman, C., Kazemier, H.G., van der Vlies, P., Kok, K., Hospers, G.A.P., Rots, M.G., 2013. Towards sustained silencing of HER2/neu in cancer by epigenetic editing. Mol. Cancer Res. 11, 1029e1039. http://dx.doi.org/10.1158/1541-7786.MCR-120567. Falandry, C., Fourel, G., Galy, V., Ristriani, T., Horard, B., Bensimon, E., Salles, G., Gilson, E., Magdinier, F., 2010. CLLD8/ KMT1F is a lysine methyltransferase that is important for chromosome segregation. J. Biol. Chem. 285, 20234e20241. http://dx.doi.org/10.1074/jbc.M109.052399. ^a, N.C.R., de Andrade, C., de Angelis Faria, J.A.Q.A., Corre Campos, A.C., Santos Samuel de Almeida, Dos, R., Rodrigues, T.S., de Goes, A.M., Gomes, D.A., Silva, F.P., 2013. Set domain-containing protein 4 (SETD4) is a newly identified cytosolic and nuclear lysine methyltransferase involved in breast cancer cell proliferation. J. Cancer Sci. Ther. 5, 58e65. Fraga, M.F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G., Bonaldi, T., Haydon, C., Ropero, S., rez-Rosado, A., Calvo, E., Lopez, J.A., Petrie, K., Iyer, N.G., Pe Cano, A., Calasanz, M.J., Colomer, D., Piris, M.A., Ahn, N., Imhof, A., Caldas, C., Jenuwein, T., Esteller, M., 2005. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37, 391e400. http://dx.doi.org/10.1038/ng1531. Gao, Y., Zhao, Y., Zhang, J., Lu, Y., Liu, X., Geng, P., Huang, B., Zhang, Y., Lu, J., 2016. The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1. Sci. Rep. 6, 19874. http://dx.doi.org/10.1038/srep19874. Gazzerro, P., Abbondanza, C., D’Arcangelo, A., Rossi, M., Medici, N., Moncharmont, B., Puca, G.A., 2006. Modulation of RIZ gene expression is associated to estradiol control of MCF-7 breast cancer cell proliferation. Exp. Cell Res. 312, 340e349. http://dx.doi.org/10.1016/j.yexcr.2005.11.002. Gonzalez, M.E., Li, X., Toy, K., DuPrie, M., Ventura, A.C., Banerjee, M., Ljungman, M., Merajver, S.D., Kleer, C.G., 2009. Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1. Oncogene 28, 843e853. http://dx.doi.org/10.1038/ onc.2008.433. Gonzalez, M.E., Moore, H.M., Li, X., Toy, K.A., Huang, W., Sabel, M.S., Kidwell, K.M., Kleer, C.G., 2014. EZH2 expands breast stem cells through activation of NOTCH1 signaling. Proc. Natl. Acad. Sci. U.S.A 111, 3098e3103. http://dx.doi.org/ 10.1073/pnas.1308953111. ^ te , J., 2007. Alternative Goulet, I., Gauvin, G., Boisvenue, S., Co splicing yields protein arginine methyltransferase 1 isoforms with distinct activity, substrate specificity, and subcellular localization. J. Biol. Chem. 282, 33009e33021. http://dx.doi.org/ 10.1074/jbc.M704349200. Greer, E.L., Shi, Y., 2012. Histone methylation: a dynamic mark in health, disease and inheritance. Nat. Rev. Genet. 13, 343e357. http://dx.doi.org/10.1038/nrg3173. Guccione, E., Bassi, C., Casadio, F., Martinato, F., Cesaroni, M., € scher, B., Amati, B., 2007. Methylation of Schuchlautz, H., Lu histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 449, 933e937. http://dx.doi.org/ 10.1038/nature06166. r, G., Seto, E., Wright, K.L., 2004. PRDI-BF1 Gyory, I., Wu, J., Feje recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat. Immunol. 5, 299e308. http:// dx.doi.org/10.1038/ni1046.

1509

Habashy, H.O., Rakha, E.A., Ellis, I.O., Powe, D.G., 2013. The oestrogen receptor coactivator CARM1 has an oncogenic effect and is associated with poor prognosis in breast cancer. Breast Cancer Res. Treat. 140, 307e316. http://dx.doi.org/10.1007/ s10549-013-2614-y. Hamamoto, R., Silva, F.P., Tsuge, M., Nishidate, T., Katagiri, T., Nakamura, Y., Furukawa, Y., 2006. Enhanced SMYD3 expression is essential for the growth of breast cancer cells. Cancer Sci. 97, 113e118. http://dx.doi.org/10.1111/j.13497006.2006.00146.x. He, L., Yu, J.X., Liu, L., Buyse, I.M., Wang, M.S., Yang, Q.C., Nakagawara, A., Brodeur, G.M., Shi, Y.E., Huang, S., 1998. RIZ1, but not the alternative RIZ2 product of the same gene, is underexpressed in breast cancer, and forced RIZ1 expression causes G2-M cell cycle arrest and/or apoptosis. Cancer Res. 58, 4238e4244. Heintzman, N.D., Stuart, R.K., Hon, G., Fu, Y., Ching, C.W., Hawkins, R.D., Barrera, L.O., Van Calcar, S., Qu, C., Ching, K.A., Wang, W., Weng, Z., Green, R.D., Crawford, G.E., Ren, B., 2007. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311e318. http://dx.doi.org/10.1038/ ng1966. Hidalgo, I., Herrera-Merchan, A., Ligos, J.M., Carramolino, L., ~ ez, J., Martinez, F., Dominguez, O., Torres, M., Gonzalez, S., Nun 2012. Ezh1 is required for hematopoietic stem cell maintenance and prevents senescence-like cell cycle arrest. Cell Stem Cell 11, 649e662. http://dx.doi.org/10.1016/ j.stem.2012.08.001. € vgren, K., Aradottir, S., GruvbergerHolm, K., Grabau, D., Lo Saal, S., Howlin, J., Saal, L.H., Ethier, S.P., Bendahl, P.-O., € m, P., Ferno € , M., Ryde n, L., Hegardt, C., St al, O., Malmstro r, M., 2012. Global H3K27 trimethylation and Borg, A., Ringne EZH2 abundance in breast tumor subtypes. Mol. Oncol. 6, 494e506. http://dx.doi.org/10.1016/j.molonc.2012.06.002. Hsu, J.-M., Chen, C.-T., Chou, C.-K., Kuo, H.-P., Li, L.-Y., Lin, C.-Y., Lee, H.-J., Wang, Y.-N., Liu, M., Liao, H.-W., Shi, B., Lai, C.-C., Bedford, M.T., Tsai, C.-H., Hung, M.-C., 2011. Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation. Nat. Cell Biol. 13, 174e181. http://dx.doi.org/10.1038/ncb2158. Hu, D., Gur, M., Zhou, Z., Gamper, A., Hung, M.-C., Fujita, N., Lan, L., Bahar, I., Wan, Y., 2015. Interplay between arginine methylation and ubiquitylation regulates KLF4-mediated genome stability and carcinogenesis. Nat. Commun. 6, 8419. http://dx.doi.org/10.1038/ncomms9419. Hu, L., Zhu, Y.T., Qi, C., Zhu, Y.-J., 2009. Identification of Smyd4 as a potential tumor suppressor gene involved in breast cancer development. Cancer Res. 69, 4067e4072. http://dx.doi.org/ 10.1158/0008-5472.CAN-08-4097. Hua, K.-T., Wang, M.-Y., Chen, M.-W., Wei, L.-H., Chen, C.-K., Ko, C.-H., Jeng, Y.-M., Sung, P.-L., Jan, Y.-H., Hsiao, M., Kuo, M.L., Yen, M.-L., 2014. The H3K9 methyltransferase G9a is a marker of aggressive ovarian cancer that promotes peritoneal metastasis. Mol. Cancer 13, 189. http://dx.doi.org/10.1186/ 1476-4598-13-189. rez-Burgos, L., Zhang, X., Huang, J., Dorsey, J., Chuikov, S., Pe Jenuwein, T., Reinberg, D., Berger, S.L., 2010. G9a and Glp methylate lysine 373 in the tumor suppressor p53. J. Biol. Chem. 285, 9636e9641. http://dx.doi.org/10.1074/ jbc.M109.062588. rez-Burgos, L., Placek, B.J., Sengupta, R., Richter, M., Huang, J., Pe Dorsey, J.A., Kubicek, S., Opravil, S., Jenuwein, T., Berger, S.L., 2006. Repression of p53 activity by Smyd2-mediated methylation. Nature 444, 629e632. http://dx.doi.org/10.1038/ nature05287. Jansson, M., Durant, S.T., Cho, E.-C., Sheahan, S., Edelmann, M., Kessler, B., La Thangue, N.B., 2008. Arginine methylation

1510


regulates the p53 response. Nat. Cell Biol. 10, 1431e1439. http://dx.doi.org/10.1038/ncb1802. raljai, R., Vilkova, A.V., Khramtsova, G.F., Jene-Sanz, A., Va Khramtsov, A.I., Olopade, O.I., Lopez-Bigas, N., Benevolenskaya, E.V., 2013. Expression of polycomb targets predicts breast cancer prognosis. Mol. Cell. Biol. 33, 3951e3961. http://dx.doi.org/10.1128/MCB.00426-13. Jones, B., Su, H., Bhat, A., Lei, H., Bajko, J., Hevi, S., Baltus, G.A., Kadam, S., Zhai, H., Valdez, R., Gonzalo, S., Zhang, Y., Li, E., Chen, T., 2008. The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet. 4, e1000190. http://dx.doi.org/10.1371/ journal.pgen.1000190. Jørgensen, S., Schotta, G., Sørensen, C.S., 2013. Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Res. 41, 2797e2806. http://dx.doi.org/ 10.1093/nar/gkt012. Kamangar, F., Dores, G.M., Anderson, W.F., 2006. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 24, 2137e2150. http://dx.doi.org/10.1200/ JCO.2005.05.2308. Kanchi, K.L., Johnson, K.J., Lu, C., McLellan, M.D., Leiserson, M.D.M., Wendl, M.C., Zhang, Q., Koboldt, D.C., Xie, M., Kandoth, C., McMichael, J.F., Wyczalkowski, M.A., Larson, D.E., Schmidt, H.K., Miller, C.A., Fulton, R.S., Spellman, P.T., Mardis, E.R., Druley, T.E., Graubert, T.A., Goodfellow, P.J., Raphael, B.J., Wilson, R.K., Ding, L., 2014. Integrated analysis of germline and somatic variants in ovarian cancer. Nat. Commun. 5, 3156. http://dx.doi.org/ 10.1038/ncomms4156. Kandoth, C., McLellan, M.D., Vandin, F., Ye, K., Niu, B., Lu, C., Xie, M., Zhang, Q., McMichael, J.F., Wyczalkowski, M.A., Leiserson, M.D.M., Miller, C.A., Welch, J.S., Walter, M.J., Wendl, M.C., Ley, T.J., Wilson, R.K., Raphael, B.J., Ding, L., 2013. Mutational landscape and significance across 12 major cancer types. Nature 502, 333e339. http://dx.doi.org/10.1038/ nature12634. € Szewczyk, M.M., Yu, Z., Eram, M.S., Yang, X., Kaniskan, H.U., Schmidt, K., Luo, X., Dai, M., He, F., Zang, I., Lin, Y., Kennedy, S., Li, F., Dobrovetsky, E., Dong, A., Smil, D., Min, S.J., Landon, M., Lin-Jones, J., Huang, X.-P., Roth, B.L., Schapira, M., Atadja, P., Barsyte-Lovejoy, D., Arrowsmith, C.H., Brown, P.J., Zhao, K., Jin, J., Vedadi, M., 2015. A potent, selective and cell-active allosteric inhibitor of protein arginine methyltransferase 3 (PRMT3). Angew. Chem. Int. Ed. Engl. 54, 5166e5170. http://dx.doi.org/10.1002/ anie.201412154. Kassambara, A., Klein, B., Moreaux, J., 2009. MMSET is overexpressed in cancers: link with tumor aggressiveness. Biochem. Biophys. Res. Commun. 379, 840e845. http:// dx.doi.org/10.1016/j.bbrc.2008.12.093. Katoh, M., 2016. Mutation spectra of histone methyltransferases with canonical SET domains and EZH2-targeted therapy. Epigenomics 8, 285e305. http://dx.doi.org/10.2217/epi.15.89. Keniry, A., Gearing, L.J., Jansz, N., Liu, J., Holik, A.Z., Hickey, P.F., Kinkel, S.A., Moore, D.L., Breslin, K., Chen, K., Liu, R., Phillips, C., Pakusch, M., Biben, C., Sheridan, J.M., Kile, B.T., Carmichael, C., Ritchie, M.E., Hilton, D.J., Blewitt, M.E., 2016. Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing. Epigenetics Chromatin 9, 16. http://dx.doi.org/10.1186/s13072016-0064-6. Kim, H., Heo, K., Kim, J.H., Kim, K., Choi, J., An, W., 2009. Requirement of histone methyltransferase SMYD3 for estrogen receptor-mediated transcription. J. Biol. Chem. 284, 19867e19877. http://dx.doi.org/10.1074/jbc.M109.021485.

Kim, J.-H., Sharma, A., Dhar, S.S., Lee, S.-H., Gu, B., Chan, C.-H., Lin, H.-K., Lee, M.G., 2014. UTX and MLL4 coordinately regulate transcriptional programs for cell proliferation and invasiveness in breast cancer cells. Cancer Res. 74, 1705e1717. http://dx.doi.org/10.1158/0008-5472.CAN-13-1896. Kim, K.H., Kim, W., Howard, T.P., Vazquez, F., Tsherniak, A., Wu, J.N., Wang, W., Haswell, J.R., Walensky, L.D., Hahn, W.C., Orkin, S.H., Roberts, C.W.M., 2015. SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nat. Med. 21, 1491e1496. http://dx.doi.org/10.1038/nm.3968. Kim, K.H., Roberts, C.W.M., 2016. Targeting EZH2 in cancer. Nat. Med. 22, 128e134. http://dx.doi.org/10.1038/nm.4036. Kirmizis, A., Santos-Rosa, H., Penkett, C.J., Singer, M.A., € hler, J., Green, R.D., Vermeulen, M., Mann, M., Ba Kouzarides, T., 2007. Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature 449, 928e932. http://dx.doi.org/10.1038/nature06160. Kleer, C.G., Cao, Q., Varambally, S., Shen, R., Ota, I., Tomlins, S.A., Ghosh, D., Sewalt, R.G.A.B., Otte, A.P., Hayes, D.F., Sabel, M.S., Livant, D., Weiss, S.J., Rubin, M.A., Chinnaiyan, A.M., 2003. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc. Natl. Acad. Sci. U.S.A 100, 11606e11611. http://dx.doi.org/10.1073/ pnas.1933744100. Knutson, S.K., Kawano, S., Minoshima, Y., Warholic, N.M., Huang, K.-C., Xiao, Y., Kadowaki, T., Uesugi, M., Kuznetsov, G., Kumar, N., Wigle, T.J., Klaus, C.R., Allain, C.J., Raimondi, A., Waters, N.J., Smith, J.J., Porter-Scott, M., Chesworth, R., Moyer, M.P., Copeland, R.A., Richon, V.M., Uenaka, T., Pollock, R.M., Kuntz, K.W., Yokoi, A., Keilhack, H., 2014. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol. Cancer Ther. 13, 842e854. http://dx.doi.org/10.1158/15357163.MCT-13-0773. Knutson, S.K., Warholic, N.M., Wigle, T.J., Klaus, C.R., Allain, C.J., Raimondi, A., Porter-Scott, M., Chesworth, R., Moyer, M.P., Copeland, R.A., Richon, V.M., Pollock, R.M., Kuntz, K.W., Keilhack, H., 2013. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc. Natl. Acad. Sci. U.S.A 110, 7922e7927. http://dx.doi.org/10.1073/pnas.1303800110. Koh, C.M., Bezzi, M., Guccione, E., 2015. The where and the how of PRMT5. Curr. Mol. Biol. Rep. 1, 19e28. http://dx.doi.org/ 10.1007/s40610-015-0003-5. Koppens, M., Van Lohuizen, M., 2016. Context-dependent actions of polycomb repressors in cancer. Oncogene 35, 1341e1352. http://dx.doi.org/10.1038/onc.2015.195. Krause, C.D., Yang, Z.-H., Kim, Y.-S., Lee, J.-H., Cook, J.R., Pestka, S., 2007. Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential. Pharmacol. Ther. 113, 50e87. http:// dx.doi.org/10.1016/j.pharmthera.2006.06.007. Kudithipudi, S., Jeltsch, A., 2014. Role of somatic cancer mutations in human protein lysine methyltransferases. Biochim. Biophys. Acta 1846, 366e379. http://dx.doi.org/ 10.1016/j.bbcan.2014.08.002. Kuo, A.J., Cheung, P., Chen, K., Zee, B.M., Kioi, M., Lauring, J., Xi, Y., Park, B.H., Shi, X., Garcia, B.A., Li, W., Gozani, O., 2011. NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming. Mol. Cell 44, 609e620. http:// dx.doi.org/10.1016/j.molcel.2011.08.042. guelin, W., Koche, R., Teater, M., Spitzer, B., LaFave, L.M., Be Chramiec, A., Papalexi, E., Keller, M.D., Hricik, T., Konstantinoff, K., Micol, J.-B., Durham, B., Knutson, S.K., Campbell, J.E., Blum, G., Shi, X., Doud, E.H., Krivtsov, A.V., Chung, Y.R., Khodos, I., de Stanchina, E., Ouerfelli, O., Adusumilli, P.S., Thomas, P.M., Kelleher, N.L., Luo, M., Keilhack, H., Abdel-Wahab, O., Melnick, A., Armstrong, S.A.,


Levine, R.L., 2015. Loss of BAP1 function leads to EZH2dependent transformation. Nat. Med. 21, 1344e1349. http:// dx.doi.org/10.1038/nm.3947. Laible, G., Wolf, A., Dorn, R., Reuter, G., Nislow, C., Lebersorger, A., Popkin, D., Pillus, L., Jenuwein, T., 1997. Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J. 16, 3219e3232. http://dx.doi.org/ 10.1093/emboj/16.11.3219. Le Romancer, M., Treilleux, I., Leconte, N., Robin-Lespinasse, Y., Sentis, S., Bouchekioua-Bouzaghou, K., Goddard, S., GobertGosse, S., Corbo, L., 2008. Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol. Cell 31, 212e221. http://dx.doi.org/10.1016/ j.molcel.2008.05.025. Li, K.-C., Hua, K.-T., Lin, Y.-S., Su, C.-Y., Ko, J.-Y., Hsiao, M., Kuo, M.-L., Tan, C.-T., 2014. Inhibition of G9a induces DUSP4dependent autophagic cell death in head and neck squamous cell carcinoma. Mol. Cancer 13, 172. http://dx.doi.org/10.1186/ 1476-4598-13-172. Li, X., Gonzalez, M.E., Toy, K., Filzen, T., Merajver, S.D., Kleer, C.G., 2009. Targeted overexpression of EZH2 in the mammary gland disrupts ductal morphogenesis and causes epithelial hyperplasia. Am. J. Pathol. 175, 1246e1254. http://dx.doi.org/ 10.2353/ajpath.2009.090042. Lim, E., Vaillant, F., Wu, D., Forrest, N.C., Pal, B., Hart, A.H., Asselin-Labat, M.-L., Gyorki, D.E., Ward, T., Partanen, A., Feleppa, F., Huschtscha, L.I., Thorne, H.J., ConFab, k, Fox, S.B., Yan, M., French, J.D., Brown, M.A., Smyth, G.K., Visvader, J.E., Lindeman, G.J., 2009. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat. Med. 15, 907e913. http:// dx.doi.org/10.1038/nm.2000. Liu, B., Zhang, X., Song, F., Liu, Q., Dai, H., Zheng, H., Cui, P., Zhang, L., Zhang, W., Chen, K., 2016a. A functional single nucleotide polymorphism of SET8 is prognostic for breast cancer. Oncotarget 7, 34277e34287. http://dx.doi.org/10.18632/ oncotarget.9099. Liu, B., Zhang, X., Song, F., Zheng, H., Zhao, Y., Li, H., Zhang, L., Yang, M., Zhang, W., Chen, K., 2016b. MiR-502/SET8 regulatory circuit in pathobiology of breast cancer. Cancer Lett. 376, 259e267. http://dx.doi.org/10.1016/j.canlet.2016.04.008. Liu, L., Kimball, S., Liu, H., Holowatyj, A., Yang, Z.-Q., 2015. Genetic alterations of histone lysine methyltransferases and their significance in breast cancer. Oncotarget 6, 2466e2482. http://dx.doi.org/10.18632/oncotarget.2967. Liu, S., Brind’Amour, J., Karimi, M.M., Shirane, K., Bogutz, A., Lefebvre, L., Sasaki, H., Shinkai, Y., Lorincz, M.C., 2014. Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells. Genes Dev. 28, 2041e2055. http://dx.doi.org/ 10.1101/gad.244848.114. vry, N., Lupien, M., 2012. Chromatin Magnani, L., Brunelle, M., Ge landscape and endocrine response in breast cancer. Epigenomics 4, 675e683. http://dx.doi.org/10.2217/epi.12.64. Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C.L., Dynlacht, B.D., Reinberg, D., 2008. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol. Cell 32, 503e518. http://dx.doi.org/10.1016/ j.molcel.2008.11.004. Martin, C., Zhang, Y., 2005. The diverse functions of histone lysine methylation. Nat. Rev. Mol. Cell Biol. 6, 838e849. http:// dx.doi.org/10.1038/nrm1761. Mathioudaki, K., Scorilas, A., Ardavanis, A., Lymberi, P., Tsiambas, E., Devetzi, M., Apostolaki, A., Talieri, M., 2011. Clinical evaluation of PRMT1 gene expression in breast cancer. Tumour Biol. 32, 575e582. http://dx.doi.org/10.1007/s13277010-0153-2.

1511

Matkar, S., Sharma, P., Gao, S., Gurung, B., Katona, B.W., Liao, J., Muhammad, A.B., Kong, X.-C., Wang, L., Jin, G., Dang, C.V., Hua, X., 2015. An epigenetic pathway regulates sensitivity of breast cancer cells to HER2 inhibition via FOXO/c-Myc axis. Cancer Cell 28, 472e485. http://dx.doi.org/10.1016/ j.ccell.2015.09.005. Mazur, P.K., Reynoird, N., Khatri, P., Jansen, P.W.T.C., Wilkinson, A.W., Liu, S., Barbash, O., Van Aller, G.S., Huddleston, M., Dhanak, D., Tummino, P.J., Kruger, R.G., Garcia, B.A., Butte, A.J., Vermeulen, M., Sage, J., Gozani, O., 2014. SMYD3 links lysine methylation of MAP3K2 to Rasdriven cancer. Nature 510, 283e287. http://dx.doi.org/10.1038/ nature13320. McCabe, M.T., Ott, H.M., Ganji, G., Korenchuk, S., Thompson, C., Van Aller, G.S., Liu, Y., Graves, A.P., Pietra Della, A., Diaz, E., LaFrance, L.V., Mellinger, M., Duquenne, C., Tian, X., Kruger, R.G., McHugh, C.F., Brandt, M., Miller, W.H., Dhanak, D., Verma, S.K., Tummino, P.J., Creasy, C.L., 2012. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108e112. http:// dx.doi.org/10.1038/nature11606. Messaoudi, El, S., Fabbrizio, E., Rodriguez, C., Chuchana, P., Fauquier, L., Cheng, D., Theillet, C., Vandel, L., Bedford, M.T., Sardet, C., 2006. Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene. Proc. Natl. Acad. Sci. U.S.A 103, 13351e13356. http://dx.doi.org/10.1073/pnas.0605692103. Michalak, E.M., Nacerddine, K., Pietersen, A., Beuger, V., Pawlitzky, I., Cornelissen-Steijger, P., Wientjens, E., Tanger, E., Seibler, J., Van Lohuizen, M., Jonkers, J., 2013. Polycomb group gene Ezh2 regulates mammary gland morphogenesis and maintains the luminal progenitor pool. Stem Cells 31, 1910e1920. Mitchell, L.H., Boriack-Sjodin, P.A., Smith, S., Thomenius, M., Rioux, N., Munchhof, M., Mills, J.E., Klaus, C., Totman, J., Riera, T.V., Raimondi, A., Jacques, S.L., West, K., Foley, M., Waters, N.J., Kuntz, K.W., Wigle, T.J., Scott, M.P., Copeland, R.A., Smith, J.J., Chesworth, R., 2016. Novel oxindole sulfonamides and sulfamides: EPZ031686, the first orally bioavailable small molecule SMYD3 inhibitor. ACS Med. Chem. Lett. 7, 134e138. http://dx.doi.org/10.1021/ acsmedchemlett.5b00272. Mitchell, L.H., Drew, A.E., Ribich, S.A., Rioux, N., Swinger, K.K., Jacques, S.L., Lingaraj, T., Boriack-Sjodin, P.A., Waters, N.J., Wigle, T.J., Moradei, O., Jin, L., Riera, T., Porter-Scott, M., Moyer, M.P., Smith, J.J., Chesworth, R., Copeland, R.A., 2015. Aryl pyrazoles as potent inhibitors of arginine methyltransferases: identification of the first PRMT6 Tool compound. ACS Med. Chem. Lett. 6, 655e659. http:// dx.doi.org/10.1021/acsmedchemlett.5b00071. Moelans, C.B., de Weger, R.A., Monsuur, H.N., Maes, A.H.J., van Diest, P.J., 2010a. Molecular differences between ductal carcinoma in situ and adjacent invasive breast carcinoma: a multiplex ligation-dependent probe amplification study. Anal Cell Pathol (Amst) 33, 165e173. http://dx.doi.org/10.3233/ACPCLO-2010-0546. Moelans, C.B., de Weger, R.A., Monsuur, H.N., Vijzelaar, R., van Diest, P.J., 2010b. Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes. Mod. Pathol. 23, 1029e1039. http://dx.doi.org/10.1038/ modpathol.2010.84. Mounir, Z., Korn, J.M., Westerling, T., Lin, F., Kirby, C.A., Schirle, M., McAllister, G., Hoffman, G., Ramadan, N., Hartung, A., Feng, Y., Kipp, D.R., Quinn, C., Fodor, M., Baird, J., Schoumacher, M., Meyer, R., Deeds, J., Buchwalter, G., Stams, T., Keen, N., Sellers, W.R., Brown, M., Pagliarini, R.A., 2016. ERG signaling in prostate cancer is driven through

1512


PRMT5-dependent methylation of the Androgen Receptor. Elife 5. http://dx.doi.org/10.7554/eLife.13964. Mzoughi, S., Tan, Y.X., Low, D., Guccione, E., 2016. The role of PRDMs in cancer: one family, two sides. Curr. Opin. Genet. Dev. 36, 83e91. http://dx.doi.org/10.1016/j.gde.2016.03.009. Neault, M., Mallette, F.A., Vogel, G., Michaud-Levesque, J., Richard, S., 2012. Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor. Nucleic Acids Res. 40, 9513e9521. http:// dx.doi.org/10.1093/nar/gks764. Nguyen, A.T., Zhang, Y., 2011. The diverse functions of Dot1 and H3K79 methylation. Genes Dev. 25, 1345e1358. http:// dx.doi.org/10.1101/gad.2057811. Nguyen, H., Allali-Hassani, A., Antonysamy, S., Chang, S., Chen, L.H., Curtis, C., Emtage, S., Fan, L., Gheyi, T., Li, F., Liu, S., Martin, J.R., Mendel, D., Olsen, J.B., Pelletier, L., Shatseva, T., Wu, S., Zhang, F.F., Arrowsmith, C.H., Brown, P.J., Campbell, R.M., Garcia, B.A., Barsyte-Lovejoy, D., Mader, M., Vedadi, M., 2015. LLY-507, a cell-active, potent, and selective inhibitor of protein-lysine methyltransferase SMYD2. J. Biol. Chem. 290, 13641e13653. http://dx.doi.org/10.1074/ jbc.M114.626861. Nik-Zainal, S., Davies, H., Staaf, J., Ramakrishna, M., Glodzik, D., Zou, X., Martincorena, I., Alexandrov, L.B., Martin, S., Wedge, D.C., Van Loo, P., Ju, Y.S., Smid, M., Brinkman, A.B., Morganella, S., Aure, M.R., Lingjærde, O.C., Langerød, A., r, M., Ahn, S.-M., Boyault, S., Brock, J.E., Broeks, A., Ringne Butler, A., Desmedt, C., Dirix, L., Dronov, S., Fatima, A., Foekens, J.A., Gerstung, M., Hooijer, G.K.J., Jang, S.J., Jones, D.R., Kim, H.-Y., King, T.A., Krishnamurthy, S., Lee, H.J., Lee, J.-Y., Li, Y., McLaren, S., Menzies, A., Mustonen, V., , I., Pivot, X., Purdie, C.A., Raine, K., O’Meara, S., Pauporte lez, F.G., Romieu, G., Ramakrishnan, K., Rodrıguez-Gonza Sieuwerts, A.M., Simpson, P.T., Shepherd, R., Stebbings, L., Stefansson, O.A., Teague, J., Tommasi, S., Treilleux, I., Van den Eynden, G.G., Vermeulen, P., Vincent-Salomon, A., Yates, L., Caldas, C., van’t Veer, L., Tutt, A., Knappskog, S., Tan, B.K.T., Jonkers, J., Borg, A., Ueno, N.T., Sotiriou, C., Viari, A., Futreal, P.A., Campbell, P.J., Span, P.N., Van Laere, S., Lakhani, S.R., Eyfjord, J.E., Thompson, A.M., Birney, E., Stunnenberg, H.G., van de Vijver, M.J., Martens, J.W.M., Børresen-Dale, A.-L., Richardson, A.L., Kong, G., Thomas, G., Stratton, M.R., 2016. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 534, 47e54. http://dx.doi.org/10.1038/nature17676. Nimura, K., Ura, K., Shiratori, H., Ikawa, M., Okabe, M., Schwartz, R.J., Kaneda, Y., 2009. A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to WolfeHirschhorn syndrome. Nature 460, 287e291. http://dx.doi.org/10.1038/ nature08086. Nishikawa, N., Toyota, M., Suzuki, H., Honma, T., Fujikane, T., Ohmura, T., Nishidate, T., Ohe-Toyota, M., Maruyama, R., Sonoda, T., Sasaki, Y., Urano, T., Imai, K., Hirata, K., Tokino, T., 2007. Gene amplification and overexpression of PRDM14 in breast cancers. Cancer Res. 67, 9649e9657. http://dx.doi.org/ 10.1158/0008-5472.CAN-06-4111. O’Carroll, D., Erhardt, S., Pagani, M., Barton, S.C., Surani, M.A., Jenuwein, T., 2001. The polycomb-group gene Ezh2 is required for early mouse development. Mol. Cell. Biol. 21, 4330e4336. http://dx.doi.org/10.1128/MCB.21.13.4330-4336.2001. O’Neill, D.J., Williamson, S.C., Alkharaif, D., Monteiro, I.C.M., Goudreault, M., Gaughan, L., Robson, C.N., Gingras, A.-C., Binda, O., 2014. SETD6 controls the expression of estrogenresponsive genes and proliferation of breast carcinoma cells. Epigenetics 9, 942e950. http://dx.doi.org/10.4161/epi.28864. Oh, T.G., Bailey, P., Dray, E., Smith, A.G., Goode, J., Eriksson, N., Funder, J.W., Fuller, P.J., Simpson, E.R., Tilley, W.D., Leedman, P.J., Clarke, C.L., Grimmond, S., Dowhan, D.H.,

Muscat, G.E.O., 2014. PRMT2 and RORg expression are associated with breast cancer survival outcomes. Mol. Endocrinol. 28, 1166e1185. http://dx.doi.org/10.1210/me.20131403. Okada, Y., Feng, Q., Lin, Y., Jiang, Q., Li, Y., Coffield, V.M., Su, L., Xu, G., Zhang, Y., 2005. hDOT1L links histone methylation to leukemogenesis. Cell 121, 167e178. http://dx.doi.org/10.1016/ j.cell.2005.02.020. Onder, T.T., Kara, N., Cherry, A., Sinha, A.U., Zhu, N., Bernt, K.M., Cahan, P., Marcarci, B.O., Unternaehrer, J., Gupta, P.B., Lander, E.S., Armstrong, S.A., Daley, G.Q., 2012. Chromatinmodifying enzymes as modulators of reprogramming. Nature 483, 598e602. http://dx.doi.org/10.1038/nature10953. Pal, B., Bouras, T., Shi, W., Vaillant, F., Sheridan, J.M., Fu, N., Breslin, K., Jiang, K., Ritchie, M.E., Young, M., Lindeman, G.J., Smyth, G.K., Visvader, J.E., 2013. Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2. Cell Rep 3, 411e426. http://dx.doi.org/ 10.1016/j.celrep.2012.12.020. Pasqualucci, L., Compagno, M., Houldsworth, J., Monti, S., Grunn, A., Nandula, S.V., Aster, J.C., Murty, V.V., Shipp, M.A., Dalla-Favera, R., 2006. Inactivation of the PRDM1/BLIMP1 gene in diffuse large B cell lymphoma. J. Exp. Med. 203, 311e317. http://dx.doi.org/10.1084/jem.20052204. Patani, N., Jiang, W.G., Newbold, R.F., Mokbel, K., 2011. Histonemodifier gene expression profiles are associated with pathological and clinical outcomes in human breast cancer. Anticancer Res. 31, 4115e4125. Patel, J.B., Appaiah, H.N., Burnett, R.M., Bhat-Nakshatri, P., Wang, G., Mehta, R., Badve, S., Thomson, M.J., Hammond, S., Steeg, P., Liu, Y., Nakshatri, H., 2011. Control of EVI-1 oncogene expression in metastatic breast cancer cells through microRNA miR-22. Oncogene 30, 1290e1301. http://dx.doi.org/ 10.1038/onc.2010.510. Perou, C.M., Sørlie, T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Rees, C.A., Pollack, J.R., Ross, D.T., Johnsen, H., Akslen, L.A., Fluge, O., Pergamenschikov, A., Williams, C., Zhu, S.X., Lønning, P.E., Børresen-Dale, A.L., Brown, P.O., Botstein, D., 2000. Molecular portraits of human breast tumours. Nature 406, 747e752. http://dx.doi.org/10.1038/35021093. Peserico, A., Germani, A., Sanese, P., Barbosa, A.J., di Virgilio, V., Fittipaldi, R., Fabini, E., Bertucci, C., Varchi, G., Moyer, M.P., Caretti, G., del Rio, A., Simone, C., 2015. A SMYD3 smallmolecule inhibitor impairing cancer cell growth. J. Cell. Physiol. 230, 2447e2460. http://dx.doi.org/10.1002/jcp.24975. Peters, A.H., O’Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., € fer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Scho Kohlmaier, A., Opravil, S., Doyle, M., Sibilia, M., Jenuwein, T., 2001. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323e337. Phalke, S., Mzoughi, S., Bezzi, M., Jennifer, N., Mok, W.C., Low, D.H.P., Thike, A.A., Kuznetsov, V.A., Tan, P.H., Voorhoeve, P.M., Guccione, E., 2012. p53-Independent regulation of p21Waf1/Cip1 expression and senescence by PRMT6. Nucleic Acids Res. 40, 9534e9542. http://dx.doi.org/ 10.1093/nar/gks858. Polstein, L.R., Perez-Pinera, P., Kocak, D.D., Vockley, C.M., Bledsoe, P., Song, L., Safi, A., Crawford, G.E., Reddy, T.E., Gersbach, C.A., 2015. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158e1169. http://dx.doi.org/10.1101/gr.179044.114. Powers, M.A., Fay, M.M., Factor, R.E., Welm, A.L., Ullman, K.S., 2011. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 71, 5579e5587. http:// dx.doi.org/10.1158/0008-5472.CAN-11-0458.


Puppe, J., Drost, R., Liu, X., Joosse, S.A., Evers, B., CornelissenSteijger, P., Nederlof, P., Yu, Q., Jonkers, J., van Lohuizen, M., Pietersen, A.M., 2009. BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A. Breast Cancer Res. 11, R63. http://dx.doi.org/10.1186/bcr2354. Raaphorst, F.M., Meijer, C.J.L.M., Fieret, E., Blokzijl, T., Mommers, E., Buerger, H., Packeisen, J., Sewalt, R.A.B., Otte, A.P., van Diest, P.J., 2003. Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene. Neoplasia 5, 481e488. Rao, B., Shibata, Y., Strahl, B.D., Lieb, J.D., 2005. Dimethylation of histone H3 at lysine 36 demarcates regulatory and nonregulatory chromatin genome-wide. Mol. Cell. Biol. 25, 9447e9459. http://dx.doi.org/10.1128/MCB.25.21.94479459.2005. Rao, R.C., Dou, Y., 2015. Hijacked in cancer: the KMT2 (MLL) family of methyltransferases. Nat. Rev. Cancer 15, 334e346. http:// dx.doi.org/10.1038/nrc3929. Rathert, P., Dhayalan, A., Murakami, M., Zhang, X., Tamas, R., Jurkowska, R., Komatsu, Y., Shinkai, Y., Cheng, X., Jeltsch, A., 2008. Protein lysine methyltransferase G9a acts on nonhistone targets. Nat. Chem. Biol. 4, 344e346. http://dx.doi.org/ 10.1038/nchembio.88. Rayasam, G.V., Wendling, O., Angrand, P.-O., Mark, M., Niederreither, K., Song, L., Lerouge, T., Hager, G.L., Chambon, P., Losson, R., 2003. NSD1 is essential for early postimplantation development and has a catalytically active SET domain. EMBO J. 22, 3153e3163. http://dx.doi.org/10.1093/ emboj/cdg288. Reijm, E.A., Jansen, M.P.H.M., Ruigrok-Ritstier, K., van Staveren, I.L., Look, M.P., van Gelder, M.E.M., Sieuwerts, A.M., Sleijfer, S., Foekens, J.A., Berns, E.M.J.J., 2011. Decreased expression of EZH2 is associated with upregulation of ER and favorable outcome to tamoxifen in advanced breast cancer. Breast Cancer Res. Treat. 125, 387e394. http://dx.doi.org/ 10.1007/s10549-010-0836-9. Ross, J.S., Badve, S., Wang, K., Sheehan, C.E., Boguniewicz, A.B., Otto, G.A., Yelensky, R., Lipson, D., Ali, S., Morosini, D., Chliemlecki, J., Elvin, J.A., Miller, V.A., Stephens, P.J., 2015. Genomic profiling of advanced-stage, metaplastic breast carcinoma by next-generation sequencing reveals frequent, targetable genomic abnormalities and potential new treatment options. Arch. Pathol. Lab. Med. 139, 642e649. http://dx.doi.org/10.5858/ arpa.2014-0200-OA. Saddic, L.A., West, L.E., Aslanian, A., Yates, J.R., Rubin, S.M., Gozani, O., Sage, J., 2010. Methylation of the retinoblastoma tumor suppressor by SMYD2. J. Biol. Chem. 285, 37733e37740. http://dx.doi.org/10.1074/jbc.M110.137612. Salz, T., Deng, C., Pampo, C., Siemann, D., Qiu, Y., Brown, K., Huang, S., 2015. Histone methyltransferase hSETD1A is a novel regulator of metastasis in breast cancer. Mol. Cancer Res. 13, 461e469. http://dx.doi.org/10.1158/1541-7786.MCR-140389. Schotta, G., Lachner, M., Sarma, K., Ebert, A., Sengupta, R., Reuter, G., Reinberg, D., Jenuwein, T., 2004. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251e1262. http://dx.doi.org/10.1101/gad.300704. Schoumacher, M., Le Corre, S., Houy, A., Mulugeta, E., Stern, M.H., Roman-Roman, S., Margueron, R., 2016. Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status. Nat. Med. 22, 577e578. http://dx.doi.org/10.1038/nm.4098. € ler, A., Baggiolini, A., Schwarz, D., Varum, S., Zemke, M., Scho € beler, D., Sommer, L., 2014. Draganova, K., Koseki, H., Schu Ezh2 is required for neural crest-derived cartilage and bone

1513

formation. Development 141, 867e877. http://dx.doi.org/ 10.1242/dev.094342. Seligson, D.B., Horvath, S., Shi, T., Yu, H., Tze, S., Grunstein, M., Kurdistani, S.K., 2005. Global histone modification patterns predict risk of prostate cancer recurrence. Nature 435, 1262e1266. http://dx.doi.org/10.1038/nature03672. Shema, E., Jones, D., Shoresh, N., Donohue, L., Ram, O., Bernstein, B.E., 2016. Single-molecule decoding of combinatorially modified nucleosomes. Science 352, 717e721. http://dx.doi.org/10.1126/science.aad7701. Shen, X., Liu, Y., Hsu, Y.-J., Fujiwara, Y., Kim, J., Mao, X., Yuan, G.C., Orkin, S.H., 2008. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491e502. http://dx.doi.org/10.1016/j.molcel.2008.10.016. Shinkai, Y., Tachibana, M., 2011. H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 25, 781e788. http:// dx.doi.org/10.1101/gad.2027411. Si, W., Huang, W., Zheng, Y., Yang, Y., Liu, X., Shan, L., Zhou, X., Wang, Y., Su, D., Gao, J., Yan, R., Han, X., Li, W., He, L., Shi, L., Xuan, C., Liang, J., Sun, L., Wang, Y., Shang, Y., 2015. Dysfunction of the reciprocal feedback loop between GATA3and ZEB2-nucleated repression programs contributes to breast cancer metastasis. Cancer Cell 27, 822e836. http://dx.doi.org/ 10.1016/j.ccell.2015.04.011. Song, X., Gao, T., Wang, N., Feng, Q., You, X., Ye, T., Lei, Q., Zhu, Y., Xiong, M., Xia, Y., Yang, F., Shi, Y., Wei, Y., Zhang, L., Yu, L., 2016. Selective inhibition of EZH2 by ZLD1039 blocks H3K27 methylation and leads to potent anti-tumor activity in breast cancer. Sci. Rep. 6, 20864. http://dx.doi.org/10.1038/ srep20864. Souroullas, G.P., Jeck, W.R., Parker, J.S., Simon, J.M., Liu, J.-Y., Paulk, J., Xiong, J., Clark, K.S., Fedoriw, Y., Qi, J., Burd, C.E., Bradner, J.E., Sharpless, N.E., 2016. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632e640. http://dx.doi.org/10.1038/nm.4092. Squazzo, S.L., O’Geen, H., Komashko, V.M., Krig, S.R., Jin, V.X., Jang, S.-W., Margueron, R., Reinberg, D., Green, R., Farnham, P.J., 2006. Suz12 binds to silenced regions of the genome in a cell-type-specific manner. Genome Res. 16, 890e900. http://dx.doi.org/10.1101/gr.5306606. € tzold, R.R., Bauer, U.-M., 2012. € thnick, D., No Stein, C., Riedl, S., Ru The arginine methyltransferase PRMT6 regulates cell proliferation and senescence through transcriptional repression of tumor suppressor genes. Nucleic Acids Res. 40, 9522e9533. http://dx.doi.org/10.1093/nar/gks767. € tzer, A., Bodega, B., Stojic, L., Jasencakova, Z., Prezioso, C., Stu Pasini, D., Klingberg, R., Mozzetta, C., Margueron, R., Puri, P.L., Schwarzer, D., Helin, K., Fischle, W., Orlando, V., 2011. Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells. Epigenetics Chromatin 4, 16. http://dx.doi.org/10.1186/17568935-4-16. Stolzenburg, S., Beltran, A.S., Swift-Scanlan, T., Rivenbark, A.G., Rashwan, R., Blancafort, P., 2015. Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene 34, 5427e5435. http:// dx.doi.org/10.1038/onc.2014.470. Sørlie, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Thorsen, T., Quist, H., Matese, J.C., Brown, P.O., Botstein, D., Lønning, P.E., Børresen-Dale, A.L., 2001. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. U.S.A 98, 10869e10874. http://dx.doi.org/10.1073/ pnas.191367098.

1514


Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H., Shinkai, Y., 2002. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779e1791. http://dx.doi.org/10.1101/gad.989402. Tan, J., Ong, C.K., Lim, W.K., Ng, C.C.Y., Thike, A.A., Ng, L.M., Rajasegaran, V., Myint, S.S., Nagarajan, S., Thangaraju, S., Dey, S., Nasir, N.D.M., Wijaya, G.C., Lim, J.Q., Huang, D., Li, Z., Wong, B.H., Chan, J.Y.S., McPherson, J.R., Cutcutache, I., Poore, G., Tay, S.T., Tan, W.J., Putti, T.C., Ahmad, B.S., Iau, P., Chan, C.W., Tang, A.P.H., Yong, W.S., Madhukumar, P., Ho, G.H., Tan, V.K.M., Wong, C.Y., Hartman, M., Ong, K.W., Tan, B.K.T., Rozen, S.G., Tan, P., Tan, P.H., Teh, B.T., 2015. Genomic landscapes of breast fibroepithelial tumors. Nat. Genet. 47, 1341e1345. http://dx.doi.org/10.1038/ng.3409. Tee, W.-W., Pardo, M., Theunissen, T.W., Yu, L., Choudhary, J.S., Hajkova, P., Surani, M.A., 2010. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev. 24, 2772e2777. http://dx.doi.org/ 10.1101/gad.606110. Teyssier, C., Le Romancer, M., Sentis, S., Jalaguier, S., Corbo, L., s, V., 2010. Protein arginine methylation in estrogen Cavaille signaling and estrogen-related cancers. Trends Endocrinol. Metab. 21, 181e189. http://dx.doi.org/10.1016/ j.tem.2009.11.002. Thakore, P.I., Black, J.B., Hilton, I.B., Gersbach, C.A., 2016. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat. Methods 13, 127e137. http:// dx.doi.org/10.1038/nmeth.3733. Thakore, P.I., D’Ippolito, A.M., Song, L., Safi, A., Shivakumar, N.K., Kabadi, A.M., Reddy, T.E., Crawford, G.E., Gersbach, C.A., 2015. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143e1149. http://dx.doi.org/10.1038/nmeth.3630. Thomassen, M., Tan, Q., Kruse, T.A., 2009. Gene expression metaanalysis identifies chromosomal regions and candidate genes involved in breast cancer metastasis. Breast Cancer Res. Treat. 113, 239e249. http://dx.doi.org/10.1007/s10549-008-9927-2. Vakoc, C.R., Sachdeva, M.M., Wang, H., Blobel, G.A., 2006. Profile of histone lysine methylation across transcribed mammalian chromatin. Mol. Cell. Biol. 26, 9185e9195. http://dx.doi.org/ 10.1128/MCB.01529-06. van ’t Veer, L.J., Dai, H., van de Vijver, M.J., He, Y.D., Hart, A.A.M., Mao, M., Peterse, H.L., van der Kooy, K., Marton, M.J., Witteveen, A.T., Schreiber, G.J., Kerkhoven, R.M., Roberts, C., Linsley, P.S., Bernards, R., Friend, S.H., 2002. Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530e536. http://dx.doi.org/10.1038/415530a. Van Aller, G.S., Graves, A.P., Elkins, P.A., Bonnette, W.G., McDevitt, P.J., Zappacosta, F., Annan, R.S., Dean, T.W., Su, D.S., Carpenter, C.L., Mohammad, H.P., Kruger, R.G., 2016. Structure-based design of a novel SMYD3 inhibitor that bridges the SAM-and MEKK2-binding pockets. Structure 24, 774e781. http://dx.doi.org/10.1016/j.str.2016.03.010. Vedadi, M., Barsyte-Lovejoy, D., Liu, F., Rival-Gervier, S., AllaliHassani, A., Labrie, V., Wigle, T.J., Dimaggio, P.A., Wasney, G.A., Siarheyeva, A., Dong, A., Tempel, W., Wang, S.C., Chen, X., Chau, I., Mangano, T.J., Huang, X.-P., Simpson, C.D., Pattenden, S.G., Norris, J.L., Kireev, D.B., Tripathy, A., Edwards, A., Roth, B.L., Janzen, W.P., Garcia, B.A., Petronis, A., Ellis, J., Brown, P.J., Frye, S.V., Arrowsmith, C.H., Jin, J., 2011. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat. Chem. Biol. 7, 566e574. http://dx.doi.org/10.1038/nchembio.599. Vougiouklakis, T., Hamamoto, R., Nakamura, Y., Saloura, V., 2015. The NSD family of protein methyltransferases in human

cancer. Epigenomics 7, 863e874. http://dx.doi.org/10.2217/ epi.15.32. Wagner, E.J., Carpenter, P.B., 2012. Understanding the language of Lys36 methylation at histone H3. Nat. Rev. Mol. Cell Biol. 13, 115e126. http://dx.doi.org/10.1038/nrm3274. Wang, C., Guo, Z., Wu, C., Li, Y., Kang, S., 2012. A polymorphism at the miR-502 binding site in the 30 untranslated region of the SET8 gene is associated with the risk of epithelial ovarian cancer. Cancer Genet. 205, 373e376. http://dx.doi.org/10.1016/ j.cancergen.2012.04.010. Wang, F., Qi, L.S., 2016. Applications of CRISPR genome engineering in cell biology. Trends Cell Biol. http://dx.doi.org/ 10.1016/j.tcb.2016.08.004 in press. Wang, J., Duan, Z., Nugent, Z., Zou, J.X., Borowsky, A.D., Zhang, Y., Tepper, C.G., Li, J.J., Fiehn, O., Xu, J., Kung, H.-J., Murphy, L.C., Chen, H.-W., 2016. Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes. Cancer Lett. 378, 69e79. http://dx.doi.org/10.1016/ j.canlet.2016.05.004. Wang, L., Jin, Q., Lee, J.-E., Su, I.-H., Ge, K., 2010. Histone H3K27 methyltransferase Ezh2 represses Wnt genes to facilitate adipogenesis. Proc. Natl. Acad. Sci. U.S.A. 107, 7317e7322. http://dx.doi.org/10.1073/pnas.1000031107. nchez-Morgan, N., Wang, X., Belguise, K., O’Neill, C.F., Sa Romagnoli, M., Eddy, S.F., Mineva, N.D., Yu, Z., Min, C., Trinkaus-Randall, V., Chalbos, D., Sonenshein, G.E., 2009. RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1. Mol. Cell. Biol. 29, 3832e3844. http://dx.doi.org/10.1128/MCB.00032-09. Wei, H., Mundade, R., Lange, K.C., Lu, T., 2014. Protein arginine methylation of non-histone proteins and its role in diseases. Cell Cycle 13, 32e41. http://dx.doi.org/10.4161/cc.27353. Wei, Y., Xia, W., Zhang, Z., Liu, J., Wang, H., Adsay, N.V., Albarracin, C., Yu, D., Abbruzzese, J.L., Mills, G.B., Bast, R.C., Hortobagyi, G.N., Hung, M.-C., 2008. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol. Carcinog. 47, 701e706. http://dx.doi.org/10.1002/mc.20413. Wozniak, R.J., Klimecki, W.T., Lau, S.S., Feinstein, Y., Futscher, B.W., 2007. 5-Aza-20 -deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation. Oncogene 26, 77e90. http://dx.doi.org/10.1038/ sj.onc.1209763. Wray, J., Williamson, E.A., Royce, M., Shaheen, M., Beck, B.D., Lee, S.-H., Nickoloff, J.A., Hromas, R., 2009. Metnase mediates resistance to topoisomerase II inhibitors in breast cancer cells. PLoS One 4, e5323. http://dx.doi.org/10.1371/ journal.pone.0005323. Xu, J., Shao, Z., Li, D., Xie, H., Kim, W., Huang, J., Taylor, J.E., Pinello, L., Glass, K., Jaffe, J.D., Yuan, G.-C., Orkin, S.H., 2015. Developmental control of polycomb subunit composition by GATA factors mediates a switch to non-canonical functions. Mol. Cell 57, 304e316. http://dx.doi.org/10.1016/ j.molcel.2014.12.009. Yang, S., Zhang, Y., Meng, F., Liu, Y., Xia, B., Xiao, M., Xu, Y., Ning, X., Li, H., Lou, G., 2013. Overexpression of multiple myeloma SET domain (MMSET) is associated with advanced tumor aggressiveness and poor prognosis in serous ovarian carcinoma. Biomarkers 18, 257e263. http://dx.doi.org/10.3109/ 1354750X.2013.773082. Yang, Y., Bedford, M.T., 2013. Protein arginine methyltransferases and cancer. Nat. Rev. Cancer 13, 37e50. http://dx.doi.org/ 10.1038/nrc3409. Yang, Z.-Q., Liu, G., Bollig-Fischer, A., Giroux, C.N., Ethier, S.P., 2010. Transforming properties of 8p11-12 amplified genes in


human breast cancer. Cancer Res. 70, 8487e8497. http:// dx.doi.org/10.1158/0008-5472.CAN-10-1013. Yao, R., Jiang, H., Ma, Y., Wang, L., Wang, L., Du, J., Hou, P., Gao, Y., Zhao, L., Wang, G., Zhang, Y., Liu, D.-X., Huang, B., Lu, J., 2014. PRMT7 induces epithelial-to-mesenchymal transition and promotes metastasis in breast cancer. Cancer Res. 74, 5656e5667. http://dx.doi.org/10.1158/0008-5472.CAN-14-0800. Ye, K., Wang, J., Jayasinghe, R., Lameijer, E.-W., McMichael, J.F., Ning, J., McLellan, M.D., Xie, M., Cao, S., Yellapantula, V., Huang, K.-L., Scott, A., Foltz, S., Niu, B., Johnson, K.J., Moed, M., Slagboom, P.E., Chen, F., Wendl, M.C., Ding, L., 2016. Systematic discovery of complex insertions and deletions in human cancers. Nat. Med. 22, 97e104. http://dx.doi.org/ 10.1038/nm.4002. Yokoyama, Y., Matsumoto, A., Hieda, M., Shinchi, Y., Ogihara, E., Hamada, M., Nishioka, Y., Kimura, H., Yoshidome, K., Tsujimoto, M., Matsuura, N., 2014. Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity. Breast Cancer Res. 16, R66. http://dx.doi.org/10.1186/bcr3681. Yoon, K.-A., Gil, H.J., Han, J., Park, J., Lee, J.S., 2010. Genetic polymorphisms in the polycomb group gene EZH2 and the risk of lung cancer. J. Thorac. Oncol. 5, 10e16. http://dx.doi.org/ 10.1097/JTO.0b013e3181c422d9. Yu, J., Angelin-Duclos, C., Greenwood, J., Liao, J., Calame, K., 2000. Transcriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol. Cell. Biol. 20, 2592e2603. bert, J., Li, E., Richard, S., 2009. A mouse PRMT1 Yu, Z., Chen, T., He null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation. Mol. Cell. Biol. 29, 2982e2996. http://dx.doi.org/10.1128/MCB.00042-09. Zack, T.I., Schumacher, S.E., Carter, S.L., Cherniack, A.D., Saksena, G., Tabak, B., Lawrence, M.S., Zhsng, C.-Z., Wala, J., Mermel, C.H., Sougnez, C., Gabriel, S.B., Hernandez, B., Shen, H., Laird, P.W., Getz, G., Meyerson, M., Beroukhim, R., 2013. Pan-cancer patterns of somatic copy number alteration. Nat. Genet. 45, 1134e1140. http://dx.doi.org/10.1038/ng.2760. Zalatan, J.G., Lee, M.E., Almeida, R., Gilbert, L.A., Whitehead, E.H., La Russa, M., Tsai, J.C., Weissman, J.S., Dueber, J.E., Qi, L.S., Lim, W.A., 2015. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339e350. http://dx.doi.org/10.1016/j.cell.2014.11.052. Zhang, L., Deng, L., Chen, F., Yao, Y., Wu, B., Wei, L., Mo, Q., Song, Y., 2014. Inhibition of histone H3K79 methylation

1515

selectively inhibits proliferation, self-renewal and metastatic potential of breast cancer. Oncotarget 5, 10665e10677. http:// dx.doi.org/10.18632/oncotarget.2496. Zhang, T., Cooper, S., Brockdorff, N., 2015. The interplay of histone modifications e writers that read. EMBO Rep. 16, 1467e1481. http://dx.doi.org/10.15252/embr.201540945. Zhang, X., Peng, D., Xi, Y., Yuan, C., Sagum, C.A., Klein, B.J., Tanaka, K., Wen, H., Kutateladze, T.G., Li, W., Bedford, M.T., Shi, X., 2016. G9a-mediated methylation of ERa links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression. Nat. Commun. 7, 10810. http://dx.doi.org/ 10.1038/ncomms10810. Zhang, X., Tanaka, K., Yan, J., Li, J., Peng, D., Jiang, Y., Yang, Z., Barton, M.C., Wen, H., Shi, X., 2013. Regulation of estrogen receptor a by histone methyltransferase SMYD2-mediated protein methylation. Proc. Natl. Acad. Sci. U.S.A 110, 17284e17289. http://dx.doi.org/10.1073/pnas.1307959110. Zhao, Q., Rank, G., Tan, Y.T., Li, H., Moritz, R.L., Simpson, R.J., Cerruti, L., Curtis, D.J., Patel, D.J., Allis, C.D., Cunningham, J.M., Jane, S.M., 2009. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing. Nat. Struct. Mol. Biol. 16, 304e311. http://dx.doi.org/10.1038/nsmb.1568. Zhong, J., Cao, R.-X., Liu, J.-H., Liu, Y.-B., Wang, J., Liu, L.-P., Chen, Y.-J., Yang, J., Zhang, Q.-H., Wu, Y., Ding, W.-J., Hong, T., Xiao, X.-H., Zu, X.-Y., Wen, G.-B., 2014. Nuclear loss of protein arginine N-methyltransferase 2 in breast carcinoma is associated with tumor grade and overexpression of cyclin D1 protein. Oncogene 33, 5546e5558. http://dx.doi.org/10.1038/ onc.2013.500. Zhong, J., Cao, R.-X., Zu, X.-Y., Hong, T., Yang, J., Liu, L., Xiao, X.H., Ding, W.-J., Zhao, Q., Liu, J.-H., Wen, G.-B., 2012. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. 279, 316e335. http:// dx.doi.org/10.1111/j.1742-4658.2011.08426.x. Zingg, D., Debbache, J., Schaefer, S.M., Tuncer, E., Frommel, S.C., Cheng, P., Arenas-Ramirez, N., Haeusel, J., Zhang, Y., Bonalli, M., McCabe, M.T., Creasy, C.L., Levesque, M.P., Boyman, O., Santoro, R., Shakhova, O., Dummer, R., Sommer, L., 2015. The epigenetic modifier EZH2 controls melanoma growth and metastasis through silencing of distinct tumour suppressors. Nat. Commun. 6, 6051. http:// dx.doi.org/10.1038/ncomms7051.

[New targeted therapies in breast cancer].

Triple-negative breast cancer: new perspectives for targeted therapies.

New targeted therapies for breast cancer: A focus on tumor microenvironmental signals and chemoresistant breast cancers.

New targeted therapies in pancreatic cancer.

Targeted Therapies in Breast Cancer: Implications for Advanced Oncology Practice.

HER2- metastatic breast cancer.

Targeted Therapies for Brain Metastases from Breast Cancer.

Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer.

Targeted Therapies for Prostate Cancer.

Histone lysine methyltransferases as anti-cancer targets for drug discovery.

Targeted therapies: TH3RESA trial, overcoming hurdles in breast cancer.

Lapatinib: new opportunities for management of breast cancer.

Targeted Therapies in HER2-Positive Breast Cancer - a Systematic Review.

Targeted therapies in breast cancer: New challenges to fight against resistance.

Genetic alterations of histone lysine methyltransferases and their significance in breast cancer.

[Bone targeted therapies: new agents].

Targeted therapies in gastroesophageal cancer.

Pancreatic cancer: optimizing treatment options, new, and emerging targeted therapies.

Targeted therapies for metastatic bladder cancer.

KLK-targeted Therapies for Prostate Cancer.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

Hypoxic tumor microenvironment: Opportunities to develop targeted therapies.

miRNA dysregulation in breast cancer.

Changes in BAI1 and nestin expression are prognostic indicators for survival and metastases in breast cancer and provide opportunities for dual targeted therapies.