**2.2 Histone modifications in breast cancer 2.2.1 Histone acetylation in breast cancer**

Histone acetylation is a dynamic process directed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Normally, Transcription factors recruit coactivators with HAT activity to regulatory DNA sites, whereas transcriptional repressors recruit corepressors with HDAC activity (Sun et al., 2001). A summary of known HAT proteins is presented in Table 1 (Sterner et al., 2000; Yang, 2004; Kimura et al., 2005).

Many HATs have also be showed to be involved in breast cancer. Among of them, p300/CBP and NCOAs are the most important and well-characterised HAT proteins associated with breast cancer.

#### **2.2.1.1 p300/CBP**

p300 and its close homolog CBP (CREB-binding protein) are often referred to as a single entity. p300 and CBP share several conserved domains: (1) the bromodomain (Br), which is frequently found in mammalian HATs; (2) three cysteine-histidine (CH)-rich domains (CH1, CH2 and CH3); (3) a KIX domain; and (4) an ADA2-homology domain, which shows extensive similarity to Ada2p, a yeast transcriptional co-activator. The N- and C-terminal domains of p300/CBP can act as transactivation domains, and the CH1, CH3 and the KIX domains are likely to be important in mediating protein-protein interactions, and a number of cellular and viral proteins bind to these regions. The acetyl-transferase domain is located in the central region of the protein, and the Br domain could function in recognising different acetylated motifs (Fig 3A, B) (Chan et al., 2001). p300/CBP contribute to acetylation of H3-K56 and promotes the subsequent assembly of newly-synthesized DNA into chromatin (Das et al., 2009). It is a non-DNA-binding transcriptional coactivator which stimulates transcription of target genes by interacting, either directly or through cofactors, with numerous promoter-binding transcription factors such as CREB, nuclear hormone receptors, and oncoprotein-related activators such as c-Fos, c-Jun, c-Myb and AML1 (Fig 3C) (Kitabayashi et al., 1998; Sterner et al., 2000).

Histone Modification and Breast Cancer 325

Fig. 3. Organisation of p300/CBP proteins. (A) Comparison of p300 and CBP. The dark regions indicate the areas of highest homology; (B) The functional domains in p300; (C) One

p300/CBP is a ubiquitously expressed, global transcriptional coactivator that is involved in most important cellular programs, such as cell cycle control, differentiation, and apoptosis. Mice nullizygous for p300 or double heterozygous for p300 and CBP showed defects in neurulation and heart development, and then exhibited embryonic lethality, and mutations in p300 and CBP are associated with certain human disease processes (Giles et al., 1998; Yao et al., 1998; Giordano et al., 1999). A role for p300 in tumor suppression has been proposed by the fact that disturbance of p300 function by viral oncoproteins is essential for the transformation of rodent primary cells and, consistent with this hypothesis, mutations of p300 have been identied in certain types of human cancers, including breast carcinomas

It showed that both the localization of p300 and the recruitment to aggresomes differ between breast cancers and normal mammary glands. The expression level of p300 in breast cancer epithelia is higher than that in normal mammary gland. Cytoplasmic localization of p300 was also observed in tumor epithelia whereas nuclear localization was found in normal mammary glands in both animal models and in non-malignant adjacent areas of human breast cancer specimens. Proteasomal inhibition induced p300 redistribution to aggresomes in tumor but not

The regulation of gene expression by nuclear receptors (NRs) controls the phenotypic properties and diverse biologies of target cells. In breast cancer cells, estrogen receptor alpha (ERα) is a master regulator of transcriptional stimulation and repression (Frasor et al., 2003). Upon E2 treatment, gene transcription is widely impacted, creating highly complex regulatory networks whose ultimate goal is the stimulation or suppression of specic biological processes. p300/CBP can function as a transcriptional cofactor of ERs and other

in normal mammary gland-derived cells (Fermento et al., 2010).

of the potential model for the action of p300/CBP in the transcriptional regulation

(Kitabayashi et al., 1998; Sterner et al., 2000).

(Gayther et al., 2000).


Table 1. Summary of major human HATs

Basic functions

H3/H4 Nuclear receptor coactivators

Hat1 H4 Histone deposition, chromatin

Tip60 H2A/H3/ H4 Transcriptional co-regulator, DNA

MORF H2A/H3/ H4 Transcriptional coactivator (strong

HBO1 H3/H4 DNA replication, transcriptional

Gcn5 H3/H4 Transcriptional coactivator PCAF H3/H4 Transcriptional coactivator

MOZ H3 Transcriptional coactivator

TAFII250 H3/H4 TBP-associated factor, transcription

TFIIIC H3/H4 RNA polymerase III transcription

initiation TFIIIC220

ATF-2 H4/H2B Transcriptional activator CIITA H4 Transcriptional coactivator

CDY H4 Histone-to-protamine transition

TFIIIC110 TFIIIC90

Table 1. Summary of major human HATs

Global transcriptional coactivator

(transcriptional response to hormone

assembly and gene silencing

repair and apoptosis

homology to MOZ)

during spermatogenesis

initiation, kinase and ubiquitin ligase

corepressor

specificity

3/H4

signals) NCOA1

Family Members Histone

(SRC-1) NCOA2 (SRC-2) NCOA3 (SRC-3)

Nuclear receptor coactivators (p160, SRC)

GNAT

MYST

P300/CBP H2A/H2B/H

Fig. 3. Organisation of p300/CBP proteins. (A) Comparison of p300 and CBP. The dark regions indicate the areas of highest homology; (B) The functional domains in p300; (C) One of the potential model for the action of p300/CBP in the transcriptional regulation (Kitabayashi et al., 1998; Sterner et al., 2000).

p300/CBP is a ubiquitously expressed, global transcriptional coactivator that is involved in most important cellular programs, such as cell cycle control, differentiation, and apoptosis. Mice nullizygous for p300 or double heterozygous for p300 and CBP showed defects in neurulation and heart development, and then exhibited embryonic lethality, and mutations in p300 and CBP are associated with certain human disease processes (Giles et al., 1998; Yao et al., 1998; Giordano et al., 1999). A role for p300 in tumor suppression has been proposed by the fact that disturbance of p300 function by viral oncoproteins is essential for the transformation of rodent primary cells and, consistent with this hypothesis, mutations of p300 have been identied in certain types of human cancers, including breast carcinomas (Gayther et al., 2000).

It showed that both the localization of p300 and the recruitment to aggresomes differ between breast cancers and normal mammary glands. The expression level of p300 in breast cancer epithelia is higher than that in normal mammary gland. Cytoplasmic localization of p300 was also observed in tumor epithelia whereas nuclear localization was found in normal mammary glands in both animal models and in non-malignant adjacent areas of human breast cancer specimens. Proteasomal inhibition induced p300 redistribution to aggresomes in tumor but not in normal mammary gland-derived cells (Fermento et al., 2010).

The regulation of gene expression by nuclear receptors (NRs) controls the phenotypic properties and diverse biologies of target cells. In breast cancer cells, estrogen receptor alpha (ERα) is a master regulator of transcriptional stimulation and repression (Frasor et al., 2003). Upon E2 treatment, gene transcription is widely impacted, creating highly complex regulatory networks whose ultimate goal is the stimulation or suppression of specic biological processes. p300/CBP can function as a transcriptional cofactor of ERs and other

Histone Modification and Breast Cancer 327

or TIF2) and NOCA3 (SRC-3, p/CIP, RAC3, ACTR, AIB1 or TRAM-1). These three members have an overall sequence similarity of 50–55% and sequence identity of 43–48%. They contain three structural domains. The N-terminal basic helix-loop-helix-Per/ARNT/ Sim (bHLH-PAS) domain is the most conserved region and is required for interact with several transcription factors (such as myogenin, MEF-2C and TEF, but not be obligator for NRs) and then enhance the transcription (Onate et al., 1995; Belandia et al., 2000). The central region contains three LXXLL (L, leucine; X, any amino acid) motifs, which form an amphipathic αhelix and are responsible for interacting with NRs (Heery et al., 1997; Darimont et al., 1998). The C-terminus contains two intrinsic transcriptional activation domains (AD1 and AD2). The AD1 region binds p300/CBP (but not interact with NRs), and this recruitment of p300/CBP to the chromatin is essential for NCOA-mediated transcriptional activation (Yao et al., 1996). The AD2 domain interacts with histone methyltransferases, coactivatorassociated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferases (PRMT1) (Koh et al., 2001). Based on such molecular features, NCOAs interact with ligandbound nuclear receptors and recruit histone acetyltransferases and methyltransferases to specific enhancer/promotor regions, which in turn results in chromatin remodeling, assembly of general transcription factors and recruitment of RNA Polymerase II for transcriptional activation (Fig 5) (Zhang et al., 2004; Xu et al., 2009). Furthermore, The Ctermini of NCOAs itself also contain HAT activity domains (Chen et al., 1997; Spencer et al., 1997), and the poly Q encoding sequence in the C-terminal of NCOA3 gene is genetically unstable and is an easy target for somatic mutations in cancer cells (Wong et al., 2006).

Fig. 5. Molecular structure of NCOAs and their functional mechanisms in steroid hormoneinduced gene expression. Abbreviations: H, hormone; NRID, NR interaction domain; TBP, the TATA binding protein; TAFIIs, TBP-associated general transcription factors (GTFs).

Except of NRs, NCOAs also serve as coactivators for many other transcription factors associated with breast cancer, such as HIF1, NF-κB, E2F1, p53, RB and MRTFs (Zhang et al., 2004; Xu et al., 2009). By regulating a broad range of gene expression controlled by NRs and non-NR transcription factors, NCOAs regulate diverse events in the development of breast cancer. Either NCOA1 or NCOA2 deficiency can reduce ductal side branching and alveologenesis in the mammary gland (Xu et al., 1998; Mukherjee et al., 2006), and NCOA3−/− mice show growth retardation, delayed puberty, reduced female reproductive

In normal human breast, the levels of the three NCOA proteins in epithelial cells are usually low or undetectable (Hudelist et al., 2003). NCOA1 is overexpressed in 19% to 29% of breast cancers and plays important roles in cell proliferation, lymph node metastasis, disease recurrence and poor disease-free survival (DFS) (Fleming et al., 2004). Therefore, elevated

function and blunted mammary gland development (Xu et al., 2000).

nuclear hormone receptors (Hanstein et al., 1996). Compared to CBP, NRIP1 and NCOAs, which play more gene-specic roles in the ER-dependent transcription, p300 seemed to be the only cofactor that appeared to be recruited at all the target genes of ER and plays a central role in both transcriptional activation and repression. After E2 treatment, ERα recruits coactivator complexes including of p300 and initiates transient stimulation of transcription via binds to ERα binding sites of target genes. If it could offer a more stable nucleation site for coactivator proteins (i.e. SRC-3), leading to histone acetylation and engagement of RNA polymerase II (Pol II), the transcriptional activation status would be maintained. Alternatively, ERα can cause transcriptional repression by recruiting, via p300, CtBP1-containing repressor complexes which lead to RNA polymerase II dismissal and histone deacetylation (Fig 4) (Stossi et al., 2009). In addition, the breast cancer susceptibility gene BRCA1 can strongly inhibits the transcriptional activity of ERα in human breast and prostate cancer cell lines, and this event is correlates with its down-regulation of p300 (but not CBP) (Fan et al., 2002). p300 also plays roles in the regulation of CYP19 I.3/II (aromatase), the key enzyme in estrogen biosynthesis and an important target in breast cancer (Subbaramaiah et al., 2008).

Fig. 4. Proposed model for ERα-mediated activation or repression of target genes via p300 (Stossi et al., 2009).

Another important role of p300 in breast cancer is the regulation of p53, a famous tumor suppressor. p53 can be acetylated by p300 in response to DNA damage to regulate its DNAbinding and transcriptional functions (Yuan et al., 1999). What's more, the N terminus of p300/CBP exhibits the ubiquitin ligase E3/E4 activity and is required for physiologic p53 polyubiquitination and degradation. Depletion of CBP or p300 could enhance the stabilization of p53 (Grossman et al., 2003; Shi et al., 2009).

Furthermore, p300/CBP has also been identified as a coactivator of HIF1α (hypoxiainducible factor 1 alpha), and thus plays a role in the stimulation of hypoxia-induced genes (such as VEGF, GLUT1, etc) and development of glycolysis, which is the most important metabolic marker of cancer (Ruas et al., 2005).

#### **2.2.1.2 Nuclear receptor coactivators**

The Nuclear receptor coactivator family (NCOA), also named as p160 or steroid receptor coactivator, contains three homologous members: NCOA1 (SRC-1), NCOA2 (SRC-2, GRIP1

nuclear hormone receptors (Hanstein et al., 1996). Compared to CBP, NRIP1 and NCOAs, which play more gene-specic roles in the ER-dependent transcription, p300 seemed to be the only cofactor that appeared to be recruited at all the target genes of ER and plays a central role in both transcriptional activation and repression. After E2 treatment, ERα recruits coactivator complexes including of p300 and initiates transient stimulation of transcription via binds to ERα binding sites of target genes. If it could offer a more stable nucleation site for coactivator proteins (i.e. SRC-3), leading to histone acetylation and engagement of RNA polymerase II (Pol II), the transcriptional activation status would be maintained. Alternatively, ERα can cause transcriptional repression by recruiting, via p300, CtBP1-containing repressor complexes which lead to RNA polymerase II dismissal and histone deacetylation (Fig 4) (Stossi et al., 2009). In addition, the breast cancer susceptibility gene BRCA1 can strongly inhibits the transcriptional activity of ERα in human breast and prostate cancer cell lines, and this event is correlates with its down-regulation of p300 (but not CBP) (Fan et al., 2002). p300 also plays roles in the regulation of CYP19 I.3/II (aromatase), the key enzyme in estrogen biosynthesis and an important target in breast

Fig. 4. Proposed model for ERα-mediated activation or repression of target genes via p300

Another important role of p300 in breast cancer is the regulation of p53, a famous tumor suppressor. p53 can be acetylated by p300 in response to DNA damage to regulate its DNAbinding and transcriptional functions (Yuan et al., 1999). What's more, the N terminus of p300/CBP exhibits the ubiquitin ligase E3/E4 activity and is required for physiologic p53 polyubiquitination and degradation. Depletion of CBP or p300 could enhance the

Furthermore, p300/CBP has also been identified as a coactivator of HIF1α (hypoxiainducible factor 1 alpha), and thus plays a role in the stimulation of hypoxia-induced genes (such as VEGF, GLUT1, etc) and development of glycolysis, which is the most important

The Nuclear receptor coactivator family (NCOA), also named as p160 or steroid receptor coactivator, contains three homologous members: NCOA1 (SRC-1), NCOA2 (SRC-2, GRIP1

stabilization of p53 (Grossman et al., 2003; Shi et al., 2009).

metabolic marker of cancer (Ruas et al., 2005).

**2.2.1.2 Nuclear receptor coactivators** 

cancer (Subbaramaiah et al., 2008).

(Stossi et al., 2009).

or TIF2) and NOCA3 (SRC-3, p/CIP, RAC3, ACTR, AIB1 or TRAM-1). These three members have an overall sequence similarity of 50–55% and sequence identity of 43–48%. They contain three structural domains. The N-terminal basic helix-loop-helix-Per/ARNT/ Sim (bHLH-PAS) domain is the most conserved region and is required for interact with several transcription factors (such as myogenin, MEF-2C and TEF, but not be obligator for NRs) and then enhance the transcription (Onate et al., 1995; Belandia et al., 2000). The central region contains three LXXLL (L, leucine; X, any amino acid) motifs, which form an amphipathic αhelix and are responsible for interacting with NRs (Heery et al., 1997; Darimont et al., 1998). The C-terminus contains two intrinsic transcriptional activation domains (AD1 and AD2). The AD1 region binds p300/CBP (but not interact with NRs), and this recruitment of p300/CBP to the chromatin is essential for NCOA-mediated transcriptional activation (Yao et al., 1996). The AD2 domain interacts with histone methyltransferases, coactivatorassociated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferases (PRMT1) (Koh et al., 2001). Based on such molecular features, NCOAs interact with ligandbound nuclear receptors and recruit histone acetyltransferases and methyltransferases to specific enhancer/promotor regions, which in turn results in chromatin remodeling, assembly of general transcription factors and recruitment of RNA Polymerase II for transcriptional activation (Fig 5) (Zhang et al., 2004; Xu et al., 2009). Furthermore, The Ctermini of NCOAs itself also contain HAT activity domains (Chen et al., 1997; Spencer et al., 1997), and the poly Q encoding sequence in the C-terminal of NCOA3 gene is genetically unstable and is an easy target for somatic mutations in cancer cells (Wong et al., 2006).

Fig. 5. Molecular structure of NCOAs and their functional mechanisms in steroid hormoneinduced gene expression. Abbreviations: H, hormone; NRID, NR interaction domain; TBP, the TATA binding protein; TAFIIs, TBP-associated general transcription factors (GTFs).

Except of NRs, NCOAs also serve as coactivators for many other transcription factors associated with breast cancer, such as HIF1, NF-κB, E2F1, p53, RB and MRTFs (Zhang et al., 2004; Xu et al., 2009). By regulating a broad range of gene expression controlled by NRs and non-NR transcription factors, NCOAs regulate diverse events in the development of breast cancer. Either NCOA1 or NCOA2 deficiency can reduce ductal side branching and alveologenesis in the mammary gland (Xu et al., 1998; Mukherjee et al., 2006), and NCOA3−/− mice show growth retardation, delayed puberty, reduced female reproductive function and blunted mammary gland development (Xu et al., 2000).

In normal human breast, the levels of the three NCOA proteins in epithelial cells are usually low or undetectable (Hudelist et al., 2003). NCOA1 is overexpressed in 19% to 29% of breast cancers and plays important roles in cell proliferation, lymph node metastasis, disease recurrence and poor disease-free survival (DFS) (Fleming et al., 2004). Therefore, elevated

Histone Modification and Breast Cancer 329

methylation at H3K4 or H3K36, mono- methylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 is associated with transcriptional activation, whereas trimethylations of H3K27, H3K9 H3K79, and H4K20 are linked to transcriptional repression (Rea et al., 2000; Kouzarides, 2007; Wang et al., 2007). Many HKMTs have been isolated and characterized (Tab 2). Up to now, except of Dot1, all the HKMTs contains a conserved SET [Su(var), Enhancer of zeste, trithorax] domain that is responsible for catalysis and binding of cofactor S-adenosyl-l- methionine (AdoMet), and many of them has been shown to play roles in the

NSD3 is amplied in human breast cancer cell lines and primary tumors and identied at the breakpoint of t(8;11)(p11.2;p15), resulting in a fusion of the NUP98 and NSD genes

SMYD3 is a novel SET-domain-containing lysine histone methyltransferase which has been regarded as an important factor in carcinogenesis. Formed a complex with RNA polymerase II through an interaction with the RNA helicase HELZ, SMYD3 specically methylates H3K4 and activates the transcription of a set of downstream genes (including of Nkx2.8, hTERT, WNT10B, VEGFR1, c-Met, etc) containing a ''5′ - CCCTCC - 3′" or "5′ - GGAGGG - 3" sequence in the promoter region (Fig 6) (Hamamoto et al., 2004; Hamamoto et al., 2006; Kunizaki et al., 2007; Zou et al., 2009). It seems that the N-terminal region of SMYD3 plays an important role for the regulation of its methyltransferase activity, and the cleavage of 34 amino acids in the N-terminal region or interaction with heat shock protein 90 alpha (HSP90α) may enhance the histone methyltransferase (HMTase) activity compared to the full-length protein (Silva et al., 2008). Enhanced expression of SMYD3 is essential for the growth of many cancer cells (such as breast cancer, colorectal carcinoma, hepatocellular carcinoma, etc), and it also could stimulate cell adhesion and migration, whereas suppression of SMYD3 by RNAi or other reagents induces apoptosis and inhibits cell proliferation and migration (Hamamoto et al., 2004; Hamamoto et al., 2006; Luo et al., 2007; Wang et al., 2008; Luo et al., 2009; Zou et al., 2009; Luo et al., 2010). SMYD3 may be an important coactivator of estrogen receptor (ER) in the estrogen signal pathway. It can directly interact with the ligand binding domain of ER, in turn augments ER target gene

Fig. 6. SMYD3-mediated histone H3-K4 methylation and transcriptional regulation. (Sims et

EZH2 overexpression has been found in breast cancer, its elevation is associated with poor prognosis. It seems that EZH2 might be associated with the regulation of pRB–E2F pathway and genes involved in homologous recombination pathway of DNA repair (Zeidler et al., 2005). However, the detailed mechanism of EZH2 in cancer is not yet clear. Another study has shown that EZH2 is also overexpressed in preneoplastic breast lesions and morphologically normal breast epithelium adjacent to the pre-invasive and invasive lesions, indicating that it might be a marker of epithelium at higher risk for neoplastic

breast cancer.

al., 2004)

transformation (Ding et al., 2006).

(Angrand et al., 2001; Rosati et al., 2002).

expression via histone H3-K4 methylation (Kim 2009).

NCOA1 has been regarded as an independent predictor of breast cancer recurrence following therapy (Redmond et al., 2009). Although the evidence were not very sufficient, NCOA2 overexpression might also promote proliferation and invasion of breast cancer cells (Kishimoto et al., 2005). The amplification (in less than 10%) and elevated expression (in over 30%) of NCOA3 were be detected in breast cancer, and its overexpression in breast cancer usually correlates with the expression of ERBB2 , matrix metalloproteinase 2 (MMP2), MMP9 and PEA3 and with larger tumor size, higher tumor grade, and/or poor DFS (Anzick et al., 1997; Hudelist et al., 2003; Harigopal et al., 2009; Xu et al., 2009). What's more, elevated NCOA3 is able to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between NCOA3 and E2F1, but not ER (Louie et al., 2004).

In addition, NCOAs play important roles in the chemotherapy resistance of breast cancer. Increased expression levels of the ER-NCOA3 complex were found in tamoxifen-resistant cells, and such overexpression of NCOA3 could enhance the agonist activity of tamoxifen and therefore, reduces its antitumor activity in patients with breast cancer (Smith et al., 1997; Zhao et al., 2009).

#### **2.2.1.3 HDACs**

The 18 HDACs identied so far can be categorized into four classes: class I (HDAC1–3, HDAC8), class II (HDAC4–7, 9–10), class III (Sirtuin1–7) and class IV (HDAC11). Class I, II, and IV HDACs share homology in both sequence and structure and all require a zinc ion for catalytic activity. In contrast, class III HDACs shares no similarities in their sequence or structure with class I, II, or IV HDACs and requires nicotinamide adenine dinucleotide (NAD+) for catalytic activity (Ellis et al., 2009; Mottet et al., 2010). HDACs remove the acetyl groups from histone lysine tails and are thought to facilitate transcriptional repression by decreasing the level of histone acetylation. Like HATs, HDACs also have non-histone targets (Bolden et al., 2006; Wang et al., 2007).

Several HDACs have been found to be involved in breast cancer. In ER-positive breast cancer MCF-7 cells, expression of HDAC6 was increased after being treated by estradiol, and the elevated HDAC6 could deacetylate alpha-tubulin and increase cell motility. While the ER antagonist tamoxifen (TAM) or ICI 182,780 could prevent estradiol-induced HDAC6 upregulation, and then reduce cell motility. The *in vivo* assays showed that the patients with high levels of HDAC6 mRNA tended to be more responsive to endocrine treatment than those with low levels, indicating that the levels of HDAC6 expression might be used as both as a marker of endocrine responsiveness and also as a prognostic indicator in breast cancer (Zhang et al., 2004; Saji et al., 2005). Besides, HDAC1, Sirtuin3 (SIRT3), SIRT7 are all overexpressed in breast cancer (Zhang et al., 2005; Michan et al., 2007; Saunders et al., 2007). HDAC4 overexpression and mutations have also been found in breast cancer samples (Sjoblom et al., 2006).

#### **2.2.2 Histone methylation in breast cancer**

Histones can be mono-, di-, or tri-methylated at lysine or arginine residues by histone methyltransferases (HMTs). Many HMTs, including both lysine-specific HMTs (eg. SMYD3) and arginine-specific HMTs (eg. PRMT1 and CARM1), have been shown to act as ER coactivators and be involved in breast cancer.

#### **2.2.2.1 Histone lysine methyltransferase (HKMTs)**

Histone lysine methylation occurs on histone H3 at ε-amino group of lysines 4, 9, 14, 27, 36, and 79 and on histone H4 at lysines 20 and 59 (Strahl et al., 2000; Lee et al., 2005). In general,

NCOA1 has been regarded as an independent predictor of breast cancer recurrence following therapy (Redmond et al., 2009). Although the evidence were not very sufficient, NCOA2 overexpression might also promote proliferation and invasion of breast cancer cells (Kishimoto et al., 2005). The amplification (in less than 10%) and elevated expression (in over 30%) of NCOA3 were be detected in breast cancer, and its overexpression in breast cancer usually correlates with the expression of ERBB2 , matrix metalloproteinase 2 (MMP2), MMP9 and PEA3 and with larger tumor size, higher tumor grade, and/or poor DFS (Anzick et al., 1997; Hudelist et al., 2003; Harigopal et al., 2009; Xu et al., 2009). What's more, elevated NCOA3 is able to promote estrogen-independent cell proliferation depends on the function of E2F1 and

In addition, NCOAs play important roles in the chemotherapy resistance of breast cancer. Increased expression levels of the ER-NCOA3 complex were found in tamoxifen-resistant cells, and such overexpression of NCOA3 could enhance the agonist activity of tamoxifen and therefore, reduces its antitumor activity in patients with breast cancer (Smith et al., 1997;

The 18 HDACs identied so far can be categorized into four classes: class I (HDAC1–3, HDAC8), class II (HDAC4–7, 9–10), class III (Sirtuin1–7) and class IV (HDAC11). Class I, II, and IV HDACs share homology in both sequence and structure and all require a zinc ion for catalytic activity. In contrast, class III HDACs shares no similarities in their sequence or structure with class I, II, or IV HDACs and requires nicotinamide adenine dinucleotide (NAD+) for catalytic activity (Ellis et al., 2009; Mottet et al., 2010). HDACs remove the acetyl groups from histone lysine tails and are thought to facilitate transcriptional repression by decreasing the level of histone acetylation. Like HATs, HDACs also have non-histone

Several HDACs have been found to be involved in breast cancer. In ER-positive breast cancer MCF-7 cells, expression of HDAC6 was increased after being treated by estradiol, and the elevated HDAC6 could deacetylate alpha-tubulin and increase cell motility. While the ER antagonist tamoxifen (TAM) or ICI 182,780 could prevent estradiol-induced HDAC6 upregulation, and then reduce cell motility. The *in vivo* assays showed that the patients with high levels of HDAC6 mRNA tended to be more responsive to endocrine treatment than those with low levels, indicating that the levels of HDAC6 expression might be used as both as a marker of endocrine responsiveness and also as a prognostic indicator in breast cancer (Zhang et al., 2004; Saji et al., 2005). Besides, HDAC1, Sirtuin3 (SIRT3), SIRT7 are all overexpressed in breast cancer (Zhang et al., 2005; Michan et al., 2007; Saunders et al., 2007). HDAC4 overexpression and mutations have also been found in breast cancer samples

Histones can be mono-, di-, or tri-methylated at lysine or arginine residues by histone methyltransferases (HMTs). Many HMTs, including both lysine-specific HMTs (eg. SMYD3) and arginine-specific HMTs (eg. PRMT1 and CARM1), have been shown to act as ER

Histone lysine methylation occurs on histone H3 at ε-amino group of lysines 4, 9, 14, 27, 36, and 79 and on histone H4 at lysines 20 and 59 (Strahl et al., 2000; Lee et al., 2005). In general,

the association between NCOA3 and E2F1, but not ER (Louie et al., 2004).

Zhao et al., 2009). **2.2.1.3 HDACs** 

(Sjoblom et al., 2006).

targets (Bolden et al., 2006; Wang et al., 2007).

**2.2.2 Histone methylation in breast cancer** 

coactivators and be involved in breast cancer.

**2.2.2.1 Histone lysine methyltransferase (HKMTs)** 

methylation at H3K4 or H3K36, mono- methylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 is associated with transcriptional activation, whereas trimethylations of H3K27, H3K9 H3K79, and H4K20 are linked to transcriptional repression (Rea et al., 2000; Kouzarides, 2007; Wang et al., 2007). Many HKMTs have been isolated and characterized (Tab 2). Up to now, except of Dot1, all the HKMTs contains a conserved SET [Su(var), Enhancer of zeste, trithorax] domain that is responsible for catalysis and binding of cofactor S-adenosyl-l- methionine (AdoMet), and many of them has been shown to play roles in the breast cancer.

NSD3 is amplied in human breast cancer cell lines and primary tumors and identied at the breakpoint of t(8;11)(p11.2;p15), resulting in a fusion of the NUP98 and NSD genes (Angrand et al., 2001; Rosati et al., 2002).

SMYD3 is a novel SET-domain-containing lysine histone methyltransferase which has been regarded as an important factor in carcinogenesis. Formed a complex with RNA polymerase II through an interaction with the RNA helicase HELZ, SMYD3 specically methylates H3K4 and activates the transcription of a set of downstream genes (including of Nkx2.8, hTERT, WNT10B, VEGFR1, c-Met, etc) containing a ''5′ - CCCTCC - 3′" or "5′ - GGAGGG - 3" sequence in the promoter region (Fig 6) (Hamamoto et al., 2004; Hamamoto et al., 2006; Kunizaki et al., 2007; Zou et al., 2009). It seems that the N-terminal region of SMYD3 plays an important role for the regulation of its methyltransferase activity, and the cleavage of 34 amino acids in the N-terminal region or interaction with heat shock protein 90 alpha (HSP90α) may enhance the histone methyltransferase (HMTase) activity compared to the full-length protein (Silva et al., 2008). Enhanced expression of SMYD3 is essential for the growth of many cancer cells (such as breast cancer, colorectal carcinoma, hepatocellular carcinoma, etc), and it also could stimulate cell adhesion and migration, whereas suppression of SMYD3 by RNAi or other reagents induces apoptosis and inhibits cell proliferation and migration (Hamamoto et al., 2004; Hamamoto et al., 2006; Luo et al., 2007; Wang et al., 2008; Luo et al., 2009; Zou et al., 2009; Luo et al., 2010). SMYD3 may be an important coactivator of estrogen receptor (ER) in the estrogen signal pathway. It can directly interact with the ligand binding domain of ER, in turn augments ER target gene expression via histone H3-K4 methylation (Kim 2009).

Fig. 6. SMYD3-mediated histone H3-K4 methylation and transcriptional regulation. (Sims et al., 2004)

EZH2 overexpression has been found in breast cancer, its elevation is associated with poor prognosis. It seems that EZH2 might be associated with the regulation of pRB–E2F pathway and genes involved in homologous recombination pathway of DNA repair (Zeidler et al., 2005). However, the detailed mechanism of EZH2 in cancer is not yet clear. Another study has shown that EZH2 is also overexpressed in preneoplastic breast lesions and morphologically normal breast epithelium adjacent to the pre-invasive and invasive lesions, indicating that it might be a marker of epithelium at higher risk for neoplastic transformation (Ding et al., 2006).

Histone Modification and Breast Cancer 331

PRDM2 (RIZ1) was originally identied as a pRb-binding protein, and its inactivation and underexpression via mutations or promoter hypermethylation had been found in a number of tumors including breast, colon, liver and lung cancers, as well as neuroblastoma, melanoma and osteosarcomas (Kim et al., 2003; Wang et al., 2007). Overexpression of PRDM2 induces G2/M cell-cycle arrest and apoptosis in tumor cell lines, while PRDM2-/ mice are prone to developing B cell lymphoma and stomach cancer (Steele-Perkins et al.,

The protein arginine methyltransferase (PRMT) family is the major HRMTs up to now. The PRMTs are classified into four groups depending on the type of methylarginine they generate: Type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6 and PRMT8) catalyze the formation of ω-NG, monomethylarginines (MMA) and ω-NG, NG-asymmetric dimethylarginines (aDMA); Type II PRMTs (PRMT5, PRMT7 and PRMT9) catalyze the formation of MMA and ω-NG, N'G-symmetric dimethylarginines (sDMA); Type III PRMTs (remained unclear) catalyze only the monomethylation of arginine residues in proteins; Type IV PRMTs (only be found in *Saccharomyces cerevisiae* up to date) catalyze the methylation at delta (Δ) nitrogen atom of arginine residues (Niewmierzycka et al., 1999;

Compared to HKMTs, The evidence for the involvement of HRMTs in human cancers is not as solid. However, underexpression of PRMT1 has been observed in breast cancer (Scorilas et al., 2000). PRMT4, also known as coactivator-associated arginine methyltransferase-1 (CARM1), is a coactivator for nuclear receptors and is oversexpressed in prostate and breast cancers (El et al., 2006). PRMT4 plays an important role in estrogeninduced cell cycle progression in the MCF-7 breast cancer cell line. Upon estrogen stimulation, the E2F1 promoter is subject to PRMT4-dependent dimethylation on H3R17, and this recruitment of PRMT4 by ERα are dependent on the presence of the NCOA3

It used to be considered that histone methylation was a permanent and irreversible histone modification. However, in recent decade, many enzymes have been identified with the ability to demethylate methylated histone lysine/arginine residues via amine oxidation, hydroxylation or deimination (Cloos et al., 2008). The histone demethylases could be divided into three distinct classes. The rst class (petidylarginine deiminase 4, PADI4) converts a methyl-lysine to citrulline. The second class (lysine-specic demethylase 1, LSD1) reverses histone H3K4 and H3K9 modications by an oxidative demethylation reaction. The third class of demethylases is the family of Jumonji C (JmjC)-domain containing histone demethylases (JHDMs). Contrast to LSD1, JHDMs can demethylate all three methylated states (mono- di- and tri-methylated lysine). Up to now, JHDMs have been found to demethylate H3K36 (JHDM1), H3K9 (JHDM2A) and H3K9/K27 (JHDM3 and JMJD2A-D)

Histone demethylase JARID1B (PLU-1) is shown to be overexpressed in breast cancers but low expressed in normal adult tissues, and it is essential for the proliferation of the MCF-7 breast cancer cell line and for the tumor growth of mammary carcinoma cells in nude mice. Several target genes of JARID1B have also been identified to be associated with breast cancer proliferation, such as 14–3–3σ, BRCA1, CAV1, and HOXA5 (Lu et al., 1999; Yamane

2001; Gibbons, 2005).

(Frietze et al., 2008).

**2.2.2.3 Histone demethylase** 

(Klose et al., 2006; Miremadi et al., 2007).

Boisvert et al., 2005; Bachand, 2007).

**2.2.2.2 Histone arginine methyltransferase (HRMTs)** 


Table 2. Summary of major human HKMTs (Pan et al., 2010)

NDS2 H4K20 Transcriptional

SETD2 H3K36 Transcriptional

SMYD2 H3K36 Transcriptional

SMYD4 Unclear Transcriptional

SETD8 H4K20 Transcriptional

SETDB1 H3K9 Transcriptional

H3K27

SMYD3 H3K4 Mainly be

SMYD5 Unclear Unclear EZ EZH2 H3K27 Transcriptional

SUV4~20 SUV4~20H1, SUV4~20H2 H4K20 Heterochromatin PRDM2 H3K9 Transcriptional

Others SET7/9 H3K4 Transcriptional

Dot1 Dot1L H3K79 Transcriptional

EHMT1 H3K9,

Table 2. Summary of major human HKMTs (Pan et al., 2010)

Non-SET domain-containing proteins

SMYD SMYD1 H3K4 Transcriptional

SET1 MLL1, MLL2, MLL3 H3K4 Transcriptional

specificity

H4K20

H3K27

Basic functions

H3K9 Transcriptional

repression

activation

activation

Mainly be transcriptional repression

activation

repression

activation

repression

repression

activation

activation

repression

repression

repression

Transcriptional repression

transcriptional activation

Transcriptional activation

Family Members Histone

SET2 NSD1 H3K36,

NSD3 H3K4,

SUV39 SUV39H1, SUV39H2, SULT1E1, G9A,

**SET domain-containing proteins** 

CLLL8

PRDM2 (RIZ1) was originally identied as a pRb-binding protein, and its inactivation and underexpression via mutations or promoter hypermethylation had been found in a number of tumors including breast, colon, liver and lung cancers, as well as neuroblastoma, melanoma and osteosarcomas (Kim et al., 2003; Wang et al., 2007). Overexpression of PRDM2 induces G2/M cell-cycle arrest and apoptosis in tumor cell lines, while PRDM2-/ mice are prone to developing B cell lymphoma and stomach cancer (Steele-Perkins et al., 2001; Gibbons, 2005).
