Rongxi Yang
email: r.yang@dkfz.de

Michelle Dick

Frederik Marme

Andreas Schneeweiss

Anne Langheinz

Kari Hemminki

Christian Sutter

Peter Bugert

Barbara Wappenschmidt

Raymonda Varon

Sarah Schott

Bernhard H F Weber

Dieter Niederacher

Norbert Arnold

Alfons Meindl

Claus R Bartram

Rita K Schmutzler

Heiko Müller

Volker Arndt

Hermann Brenner

Christof Sohn

Barbara Burwinkel

Genetic variants within miR-126 and miR-335 are not associated with breast cancer risk

MicroRNAs (miRNAs) are 20~22 nt non-coding RNAs which promote the degradation of target mRNAs or repression of the translation of mRNAs by sequence specific targeting.

Many miRNAs are considered as oncogenes or tumor suppressors. play roles in the suppression of breast cancer metastasis by inhibiting tumor growth, proliferation and cell invasion. The effects of SNPs within the two miRNAs are still unknown.

In our study, we analysed two SNPs, rs4636297 within miR-126 and rs41272366 within miR-335, in three study populations for a putative association with breast cancer risk. We compared the genotype and allele frequencies of rs4636297and rs41272366 in 2854 cases versus 3188 controls of the three study populations independently and combined. None of the performed analyses showed statistically significant results. In conclusion, our data suggest that the two genetic variants within miR-126 and miR-335 are not associated with breast cancer risk.

Introduction

MicroRNAs are a cluster of small endogenous non-coding RNAs (20~22 nt) that can bind to the 3' untranslated region of their target messenger RNAs (mRNAs) and lead to degradation or translation suppression of mRNAs [START_REF] Baek | The impact of microRNAs on protein output[END_REF][START_REF] Zeng | MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms[END_REF]. A single miRNA can repress the expression of hundreds of proteins [START_REF] Selbach | Widespread changes in protein synthesis induced by microRNAs[END_REF]. MiRNAs are involved in the regulation of several cellular pathways including pathways important for cancer development such as immune system regulation, haematopoiesis, angiogenesis, cell proliferation, differentiation and apoptosis [START_REF] Baltimore | MicroRNAs: new regulators of immune cell development and function[END_REF][START_REF] Brennecke | bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila[END_REF][START_REF] Chang | Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis[END_REF][START_REF] Calin | MiR-15a and miR-16-1 cluster functions in human leukemia[END_REF][START_REF] Dews | Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster[END_REF][START_REF] Xiao | MicroRNA control in the immune system: basic principles[END_REF]. Elevated or decreased expression of miRNAs has been found in various tumor types including breast cancer [START_REF] Croce | Causes and consequences of microRNA dysregulation in cancer[END_REF][START_REF] Khoshnaw | MicroRNA involvement in the pathogenesis and management of breast cancer[END_REF]. Some miRNAs thereby have been considered as tumor suppressors or oncogenes [START_REF] Johnson | RAS is regulated by the let-7 microRNA family[END_REF][13][START_REF] O'donnell | c-Myc-regulated microRNAs modulate E2F1 expression[END_REF]. They have been shown to be involved in cancer initiation and progression by suppressing the expression of cancer-related genes [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF]. Notably, miRNA processing defects also increase the risk of tumorigenesis [START_REF] Kumar | Impaired microRNA processing enhances cellular transformation and tumorigenesis[END_REF]. Furthermore, miRNA expression profiles can be molecular markers for cancer diagnosis and therapy [START_REF] Calin | MicroRNA signatures in human cancers[END_REF][START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF].

Breast cancer accounts for one fourth of all female cancers, making it by far the most common cancer in women worldwide, and after lung cancer the second most frequent cancer (10.4 % of all cancers) in the world [START_REF] Key | Epidemiology of breast cancer[END_REF]. Taking the U.S. female population as an example, approximately one in eight women will develop breast cancer at some time in their lives [START_REF] Belkora | Assessing risk communication in breast cancer: are continuous measures of patient knowledge better than categorical?[END_REF].

Unfavourable combinations of polymorphic genetic variants in low-penetrance susceptibility genes contribute to breast cancer risk. Most of these susceptibility genes have not been discovered yet [START_REF] Ponder | Cancer genetics[END_REF][START_REF] Pharoah | Polygenic susceptibility to breast cancer and implications for prevention[END_REF].

Tavazoie and co-workers reported miR-126 and miR-335 as metastasis suppressor genes.

They found that restoring the expression of miR-126 and miR-335 in malignant breast cancer cells can suppress the metastasis to lung and bone [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. MiR-126 inhibits the tumour growth and proliferation, whereas miR-335 suppresses metastatic cell invasion [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. Mutations and single nucleotide polymorphisms (SNPs) in miRNAs or miRNA target sites have been shown to be associated with the risk of various cancers [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF][START_REF] Duan | Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA[END_REF][START_REF] Jazdzewski | Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma[END_REF][START_REF] Shen | A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis[END_REF][START_REF] Yang | A genetic variant in the pre-miR-27a oncogene is associated with a reduced familial breast cancer risk[END_REF][START_REF] Tchatchou | A variant affecting a putative miRNA target site in estrogen receptor (ESR) 1 is associated with breast cancer risk in premenopausal women[END_REF]. Some SNPs located in the miRNAs or their flanking regions are reported to have potential impact on the maturation of miRNAs [START_REF] Wu | Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs[END_REF]. In order to study the potential association of SNPs within miR-126 and miR-335 with breast cancer risk, we sequenced the miR-126 and miR-335 and their flanking regions ( ± 200 bp) and verified the presence of three SNPs, rs4636297, rs3807348 and rs41272366. In our previous study, rs3807348 was genotyped, whereas rs4636297 and rs41272366 were not investigated as they could not be analysed by the TaqMan genotyping assay [START_REF] Yang | A genetic variant in the pre-miR-27a oncogene is associated with a reduced familial breast cancer risk[END_REF]. Here, we have investigated rs4636297 and rs41272366 by the MALDI-TOF mass spectrometry (Sequenom, CA) in three different study populations for an association with breast cancer risk.

Materials and methods

Study populations

The familial breast cancer study cohort of the German Consortium of Hereditary Breast and Ovarian Cancer (GC-HBOC):

Genotyping was performed on genomic DNA of BRCA1/2 mutation-negative index patients from 1217 German breast cancer families, comprising a subset of 115 bilateral breast cancer cases, versus 1422 unrelated healthy German women. BRCA1/2 mutation-negativity in the unrelated breast cancer cases had been tested by applying mutational screening of all exons by denaturing high performance liquid chromatography (DHPLC), and followed by direct sequencing of conspicuous exons [START_REF] Meindl | Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population[END_REF]. Breast cancer samples were collected during the years 1997-2007 by seven centres of the GC-HBOC (centres of Heidelberg, Wuerzburg, Cologne, Kiel, Duesseldorf, Munich and Berlin, see authors' affiliations). German index patients were first diagnosed with breast cancer and then referred to a family registry. All the samples from the breast cancer patients were obtained around the diagnosis. The informed consent for the study was given to all breast cancer patients.

The control population included unrelated healthy female blood donors collected by the German Red Cross Blood Service of Baden-Wuerttemberg-Hessia and the Institute of Transfusion Medicine and Immunology (Mannheim), sharing the same ethnic background with the breast cancer patients (Caucasian/German population). All control individuals were healthy when donating their blood. None of the control individuals had a reported family history of breast cancer. Age distributions in controls and breast cancer cases were similar (controls: mean age 45.8 years old, median age 49 years old, range from 18 to 68 years old; cases: mean age 46.2 years old, median age 46 years old, range from 19 to 87 years old). The controls were randomly selected during the years 2004-2007 for this study and no further inclusion criteria were applied during recruitment. The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany). According to the German guidelines for blood donation, all blood donors were examined by a standard questionnaire.

The informed consent for the study was given to all participants.

The Breast Cancer study of the University Clinic Heidelberg (BSUCH):

Genotyping was performed on genomic DNA of from 1115 German breast cancer patients

The ESTHER/VERDI Study:

Genotyping was performed on genomic DNA of from 513 German breast cancer patients, recruited in the German Federal state of Saarland between 1997 and 2003, versus 520 unrelated healthy German female controls recruited from the Saarland's general population in 2000-2002. Recruitment of cases was performed by all hospitals providing primary treatment for breast cancer patients in Saarland as a part of the VERDI study [START_REF] Arndt | Provider delay among patients with breast cancer in Germany: a population-based study[END_REF] and the ESTHER II study [START_REF] Widschwendter | Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study[END_REF]. 94 % of the samples from the breast cancer patients were obtained after the surgery. Controls were recruited by general practitioners in Saarland during a general health screening examination in the context of the ESTHER I study [START_REF] Gao | Association between chronic atrophic gastritis and serum antibodies to 15 Helicobacter pylori proteins measured by multiplex serology[END_REF]. For this analysis, 520 women, frequency matched by age to the cases, were randomly selected from ESTHER I. The studies were approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany) and of the Medical Association of Saarland (Saarbruecken, Germany). Informed consent was obtained in each case. Age distributions in controls and breast cancer cases were similar (controls: mean age 62.3 years old, median age 63 years old, range from 49 to 75 years old; cases: mean age 60.5 years old, median age 62 years old, range from 30 to 79 years old).

SNP verification and sequencing

In order to verify annotated SNPs from the SNP database (NCBI) and to identify potential new SNPs, miR126 and miR335 genes, including pre-miRNAs and about ± 200 bp flanking regions, were amplified by standard PCR (in order to unfold the secondary structure of miRNAs, 5 % DMSO was added to the PCR buffer). PCR products were purified with the ExoSAP-IT purification kit (USB Corp.) and were sequenced in one of the directions by the 3130XL Genetic Analyzer (Applied Biosystems). Sequencing results were analyzed by the Sequencing Analysis 5.2 software (Applied Biosystems). As a result, three SNPs, rs4636297, rs3807348 and rs41272366, were verified in these segments.

Genotyping by MALDI-TOF mass spectrometry

We investigated the genotype frequencies of rs4636297 and rs41272366 in this study by the MALDI-TOF mass spectrometry (Sequenom MassArray, San Diego, CA). rs3807348 has already been examined in our previous study by a TaqMan genotyping assay.

Assays were designed by the Sequenom Assay Design software v2.0.7.0. The whole process followed the protocol of multiplexed genotyping reactions using iPLEX Gold for MassArray.

At the primary PCR step, DNA was amplified under following conditions: 95 ºC for 2 min, then 45 cycles of (95 ºC for 30 s, 56 ºC for 30 s, and 72 ºC for 1 min), and a further extension at 72 ºC for 5 min before storage at 4 ºC. After purification by the SAP kit, the PCR products were treated by the extend reaction with iPLEX Extend Cocktail (following the instruction of Sequenom). The microplate was thermocycled as follows: 94 ºC for 30 s, then 50 cycles of (94 ºC for 5 s then 4 cycles of ( 52 ºC for 5 s, 80 ºC for 5 s )), a final extension at 72 ºC for 3 min and stored at 4 ºC. After cleaned by CLEAN Resin, the samples were dispensed to a 384 SpectroCHIP by the Nanodispenser. The chip was analyzed by a Bruker Autoflex Mass Spectrometer system. Data were collected by the SpectroACQUIRE v3.

Statistical analysis

The Hardy-Weinberg equilibrium [START_REF] Widschwendter | Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study[END_REF] test was applied using the chi-square 'goodness-of-fit' test offered as a tool by the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Genotype-specific odds ratios (OR), 95 % confidence intervals (CI) and P values were computed by unconditional logistic regression with SAS version 9.1 (SAS Institute Inc., Cary, NC). Age, treated as a continuous variable was included in the regression as a covariate. Study population, treated as a discrete variable was included in the regression as a covariate. P values were calculated using twosided chi-square test. The meta analysis was done by STATA 9.0. Power was calculated using the power and sample size calculation software PS version 2.1.31

(http://www.mc.vanderbilt.edu/prevmed/ps/index.htm) [START_REF] Dupont | Power and sample size calculations for studies involving linear regression[END_REF].

Results

We sequenced the DNA segments including the pre-miR-126, pre-miR-335 and their respective flanking regions (± 200 bp) in more than 100 randomly chosen familial breast cancer cases. Using this approach three SNPs, rs4636297, rs3807348 and rs41272366, were verified in these segments. We were able to localize rs4636297 12 bp downstream of pre-miR-126 and rs41272366 20 bp downstream of pre-miR-335. Neither these two SNPs themselves nor their linkage SNPs (r 2 >= 0. 1), showing no association with breast cancer. The associations of the two SNPs with breast cancer were also analysed after stratification by age (age < 50 years old and >= 50 years old). However, none of the subgroups showed any significant association with breast cancer risk. The associations of the two SNPs with breast cancer were also analysed by single study population separately, but no significant association was observed. Meta-analysis did not indicate any between-study heterogeneity (for rs4636297, P heterogeneity = 0.528; for rs41272366, P heterogeneity = 0.275, Figure 1).

Discussion

More and more miRNAs are reported to be involved in the process of cancer [START_REF] Calin | MicroRNA signatures in human cancers[END_REF]. MiR-126 and miR-335 have been reported as tumor suppressor genes [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. They can inhibit tumor development and metastasis through negatively regulating a set of oncogenes. MiR-126 is involved in the angiogenesis. It binds to the 3' untranslated region of VEGF and subsequently negatively regulates the VEGF pathway [START_REF] Fish | miR-126 regulates angiogenic signaling and vascular integrity[END_REF][START_REF] Liu | MiR-126 restoration downregulate VEGF and inhibit the growth of lung cancer cell lines in vitro and in vivo[END_REF]. MiR-335 suppresses metastasis and migration through targeting the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF]. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumors from patients who relapse, and the loss of expression of either miRNA is associated with poor distal metastasis-free survival [START_REF] Tavazoie | Endogenous human microRNAs that suppress breast cancer metastasis[END_REF].

Our work focused on the SNPs in the breast cancer related tumor suppressor genes, miR-126 and miR-335. As it has been reported that a mutation or a SNP in a miRNA gene can affect the transcription and/or procession of the pri-and pre-miRNAs [START_REF] Wu | Genetic variations of microRNAs in human cancer and their effects on the expression of miRNAs[END_REF], we have studied not only the SNPs in the pre-miRNAs but also vicinal SNPs in miRNA flanking regions (about ± 200 bp). Given our sample size, we had a power of 80 % (α = 0.05) to detect an OR of 1.18 for SNP rs4636297 and an OR of 0.66 for SNP rs41272366.

Both SNP rs4636297 and rs41272366 are located in the flanking region of pre-miRNAs, their effects on miRNA secondary structures and free energy is uncertain. We used the RNAfold program [START_REF] Markham | DINAMelt web server for nucleic acid melting prediction[END_REF] Unfortunately, these data is not available for us from all study cohorts.

In conclusion, by investigating three study populations, we have not found an association between the SNPs rs4636297 and rs41272366 and breast cancer risk. All the three different studies consistently showed negative results. However, it would be interesting to investigate the association between these SNPs and breast cancer prognosis in the future.

  recruited at the Department of Obstetrics and Gynecology of the University Hospital in Heidelberg, Germany, between 2006 and 2009. All patients gave informed written consent. The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany) and all patients gave informed written consent. The control population included 1236 unrelated healthy female blood donors collected by the German Red Cross Blood Service of Baden-Wuerttemberg-Hessia and Institute of Transfusion Medicine and Immunology (Mannheim), as described above. The controls were randomly selected during the years 2006-2008 for this study. Age distributions in controls and breast cancer cases were similar (controls: mean age 52.5 years old, median age 56 years old, range from 30 to 69 years old; cases: mean age 54.8 years old, median age 56 years old, range from 21 to 88 years old).

  3.1.3 software and visualised by the MassArray Typer v4.0 TyperAnalyzer software. Sequences of primers are available upon request.

Figure

  Figure

Figure 1 .

 1 Figure 1. ORs (log scale) of rs4636297 and rs41272355 for variant genotypes compared with the wild type genotypes in dominant genetic model.

  The results were adjusted by age and studies with the SAS software version 9.1. Allele and genotype frequencies of rs4636297 and rs41272366 were comparable in breast cancer cases and controls. rs4636297: ([A] vs [G], OR = 1.01, 95 % CI 0.94-1.10, P =0.73; [GA] vs [GG], OR = 1.01, 95 % CI 0.89-1.13, P = 0.93; [AA] vs [GG], OR = 1.04, 95 % CI 0.87-1.23, P =

	0.69; P trend = 0.73, Table 1) and rs41272366: ([A] vs [T], OR = 0.90, 95 % CI 0.69-1.17, P =
	0.43; [AT] vs [TT], OR = 0.88, 95 % CI 0.68-1.15, P = 0.35; P trend = 0.43, Table
	126 (not validated SNPs in dbSNP), rs34051017 and rs7030829, could not be verified in our
	sample set possibly due to wrong annotations or very low frequency.
	Genotype analysis of the two SNPs was performed on genomic DNA of three breast cancer
	study populations (GC-HBOC, BSUCH, ESTHER/VERDI, see materials and methods). All
	of the samples previously genotyped by sequencing were re-genotyped by the MALDI-TOF
	mass spectrometry assay and attained a concordance rate of 100 %. The MALDI-TOF mass

8) had been analysed, so far, by the Cancer Genetic Markers of Susceptibility genome wide association study (CGEMS) (https://caintegrator.nci.nih.gov/cgems/browseSetup.do). The CGEMS is a whole-genome association study conducted by the National Cancer Institute (NCI) enterprise to identify breast cancer susceptibility genes using Illumina HumanHap550 assays on approximately 1200 breast cancer patients and 1200 controls. The additional two annotated SNP in pre-miRspectrometry genotyping results were validated by re-genotyping 5 % of all the samples attaining concordance rates of 100 % for all the investigated SNPs. Genotype distributions in controls and cases were consistent with the Hardy-Weinberg equilibrium

[START_REF] Widschwendter | Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study[END_REF]

.

  (http://www.bioinfo.rpi.edu/applications/hybrid/) to predict the most stable secondary structure and free energy of both wild-types and variants. Comparing to the wildtype [G] allele, the [A] variant of rs4636297 has no effects on the secondary structure of pre-miR-126 itself, but effects the secondary structure of flanking region. Meanwhile, the free energy ΔG is increased from -52.7 kcal/mol of the wild-type to -51.2 kcal/mol of the[A] allele variant. The effect of [A] allele in rs41272366 is similar and has no impact on the secondary structure of pre-miR-335. The [A] allele increases the free energy ΔG from -54.6 kcal/mol of the wild-type to -54.2 kcal/mol of the variant. However, neither of the two SNPs affects the secondary structure of pre-miRNAs. Therefore, it is very likely that neither rs4636297 nor rs41272366 has effect on the miR-126 and miR-335 maturation and subsequently are not associated with breast cancer risk.Recently, some polymorphisms in miRNAs have been reported to be associated with metastasis or survival of cancer patients[START_REF] Christensen | Mature microRNA sequence polymorphism in MIR196A2 is associated with risk and prognosis of head and neck cancer[END_REF][START_REF] Lee | Prognostic impact of microRNA-related gene[END_REF][40]. Since miR-126 and miR-335 have been reported to be involved in tumor development, angiogenesis and distant metastasis, it is possible that these SNPs affect breast cancer prognosis such as metastasis and overall survival.

  Dou TH, Geng L, Zhou FG, Gu X, Wang H, Gao CF (2010) Association of a variant in MIR 196A2 with susceptibility to hepatocellular carcinoma in male Chinese patients with chronic hepatitis B virus infection. Hum Immunol 71:621-626.

		polymorphisms on survival of patients with colorectal cancer. J Cancer Res Clin
		Oncol 136:1073-1078.
	40.	Qi P,

Table 1 . Genotype frequencies of rs4636297 and rs41272366 in the GC-HBOC, BSUCH and ESTHER study populations

 1 adjusted for age and study populations; all analyses done with SAS Version 9.1 Proc Logistic.

	Table
	SNP	Genotypes	Case (%)	Control (%)	OR a	95 % CI	P b
		GG	957 (39.5)	1093 (39.9)	1.000
		GA	1136 (46.9)	1287 (47.0)	1.005	0.893-1.132	0.9278
	miR-126	AA	327 (13.5)	361 (13.2)	1.036	0.871-1.232	0.6925
	rs4636297	[A]vs[G]			1.014	0.936-1.099	0.7327
							P trend = 0.7315
		TT	2283 (95.7)	2580 (95.2)	1.000
		AT	102 (4.3)	130 (4.8)	0.880	0.675-1.149	0.3478
	miR-335 rs41272366	AA	1 (0)	0 (0)	-	-	0.9665
		[A]vs[T]			0.901	0.694-1.170	0.4342
							P trend = 0.4307

a chi-square test for trend. b

Acknowledgments

This study was supported by the Dietmar-Hopp Foundation, the Helmholtz society and the German Cancer Research Center (DKFZ). The German breast cancer samples were collected within a project funded by the Deutsche Krebshilfe (Grant number: 107054). The VERDI study was supported by the Deutsche Krebshilfe (Grant number: M24/95/BRI). The ESTHER study was supported by a grant from the Baden-Württemberg Ministry of Science, Research and Arts.


Annette Hayden

Peter W M Johnson

Graham Packham

Dr Simon J Crabb
email: s.j.crabb@southampton.ac.uk

Graham Packha

S-adenosylhomocysteine hydrolase inhi bi tion by 3-deazanepl anocin A anal ogues induces anti-cancer effects in breast cancer cell lines and synergy with both histone deacetyl ase and HER2 inhi bition

Keywords: S-adenosylhomocysteine hydrolase, 3-Deazaneplanocin A, histone deacetylase inhibitor, breast cancer, trastuzumab, enhancer of zeste homolog 2

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In addition to abnormalit ies of DNA sequence, epigenetic changes are also recognised to underpin breast cancer development. Such abnormalities are attractive for therapeutic intervention in that, unlike DNA sequence changes, they are potentially open to direct modulation through inhibition of en zy matic mediators [START_REF] Esteller | Ep igenetics in cancer[END_REF][START_REF] Simon | Roles of the EZH2 histone methyltransferase in cancer epigenetics[END_REF].

Abnormal ep igenetic marks in breast cancer are described at the level o f DNA methylat ion and post -translational modifications to histones. Histone acetylation is generally functions as a transcriptionally activating mark wh ilst DNA methylation at gene promoters produces repression. In contrast, histone methylation marks may be either repressive or activating dependent on the relevant lysine/arginine methylation site within the h istone tail [START_REF] Esteller | Ep igenetics in cancer[END_REF]. In addition to DNA methylation and histone acetylation, there is increasing evidence to implicate histone methylation as a potential therapeutic target for cancer. The main med iator of histone methylation studied to date in breast cancer is EZH2, a member of PRC2 which also includes embryonic ectoderm develop ment (EED) and suppressor of zeste 12 ho molog (SUZ12). EZH2 has a role in regulation of transcriptional regulation and chromatin state. PRC2 induces silencing in itiation whilst a further co mplex, PRC1, with more variable composition including BMI1 and RING1b, allows for gene silencing maintenance. EZH2 is a histone methyltransferase which induces transcriptionally repressive trimethylat ion marks at histone H3 lysine 27 (H3K27me3) [START_REF] Laible | Mammalian ho mologues of the Polycomb-group gene Enhancer of zeste med iate gene silencing in Drosophila heterochromatin and at S. cerev isiae telo meres[END_REF][START_REF] Satijn | Po lycomb group protein co mplexes: do different co mplexes regulate distinct target genes?[END_REF]. Histone lysine methyltransferases such as EZH2 require methyl g roup donation fro m the cofactor S-adenosyl-L-methionine (AdoMet). EED and SUZ12 are required in co mplex for fu ll functional activity. In addit ion, EZH2 induces transcriptional effects distinct from its histone methylation and methyltransferase activity, for examp le transcriptionally repressive or activatin g effects mediated through oestrogen and Wnt signalling pathways [START_REF] Hwang | EZH2 regulates the transcription of estrogen-responsive genes through association with REA , an estrogen receptor corepressor[END_REF]30,[START_REF] Ih | Polyco mb Group Protein Ezh2 Controls Actin Poly merization and Cell Signaling[END_REF][START_REF] Tonini | Ezh 2 re duces the ability of HDAC1dependent pRb2//p130 t ranscriptional repression of cyclin A[END_REF]. EZH2/PRC2 activ ity occurs in concert with other transcriptionally repressive epigenetic processes. Thus histone methyltransferase ac tivity of EZH2 is potentiated by the recruitment of HDAC activity through interaction with EED [START_REF] Van Der Vlag | Transcriptional repression mediated by the human polyco mb -group protein EED involves histone deacetylation[END_REF]. Furthermore, DNA methyltransferase binding to EZH2 repressed genes depends on the presence and activity of EZH2 wh ich is required for DNA methylation of EZH2 target promoters [START_REF] Vire | The Polyco mb group protein EZH2 direct ly controls DNA methylat ion[END_REF].

EZH2 is over-expressed in breast cancer compared with normal breast epithelia and expression is associated with invasiveness, increased proliferation, markers of an aggressive phenotype (basal or HER2 positive) and reduced survival outcomes [START_REF] Bach Mann | EZH2 Exp ression Is Associated With High Pro liferation Rate and Aggressive Tumor Subgroups in Cutaneous Melanoma and Cancers of the Endometriu m, Prostate, and Breast[END_REF][START_REF] Bracken | EZH2 is downstream o f the pRB-E2F pathway, essential for p roliferat ion and amp lified in cancer[END_REF][START_REF] Collett | Exp ression of Enhancer of Zeste Ho mologue 2 Is Significantly Associated with Increased Tumor Cell Proliferation and Is a Marker of Aggressive Breast Cancer[END_REF][START_REF] Kleer | EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformat ion of breast epithelial cells[END_REF][START_REF] Pietersen | EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer[END_REF]. Overexpression of EZH2 in immo rtalized mammary epithelial cell lines promotes anchorage independent growth and cell invasion and EZH2 over-exp ressing cells are tu morigenic when injected into mammary fat pads of nude mice [START_REF] Cha | Akt -Mediated Phosphorylation of EZH2 Suppresses Methylation of Lysine 27 in Histone H3[END_REF][START_REF] Kleer | EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformat ion of breast epithelial cells[END_REF]. Furthermore EZH2 is required for embryonic stem cell survival and deletion is embryonic lethal [START_REF] O'carroll | The polyco mb -group gene Ezh2 is required for early mouse development[END_REF]. EZH2 knockdown in oestrogen receptor negative cell line models decreased proliferation, inhibited G 2 /M cell cycle transition and suppressed xenograft growth [START_REF] Gon | Do wnregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcino ma and requires BRCA 1[END_REF].

Therefore evidence for the role of EZH2 in breast cancer indicates a potential therapeutic target for the man ipulation of epigenetic processes in breast cancer at the level of histone methylation. Furthermore, abnormal expression has been shown in breast cancer for other histone methyltransferases (e.g. NSD3 amplificat ion, SM YD3 over-expression, PRMT1 depletion) along with over-expression of the histone demethylase JARID1B [START_REF] Miremad I | Cancer genetics of epigenetic genes[END_REF]. These are less well researched regarding relevance to breast cancer pathogenesis or potential therapeutic targets.

Recent evidence indicates that the carbocyclic adenosine analogue DZNep has anti-tumour activity which stems, at least in part, fro m PRC2 inhibit ion through decreased expression and activity of EZH2. DZNep inhib it s AdoHcy hydrolase, preventing hydrolysis of AdoHcy to homocysteine and methionin e. Subsequent cellular accumulat ion of AdoHcy results in upstream inhibit ion of AdoMet metabolis m (to AdoHcy). AdoMet metabolism represents a key cellular mechanis m for methyl group donation for a variety of dependant metabolic processes which are therefore d isrupted, including production of methyltransferases such as EZH2.

DZNep and other AHI analogues have been previously investigated as potential anti viral agents [START_REF] Clercq | John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral act ivity[END_REF]. In an initial report describing anticancer effects, DZNep induced depletion of EZH2, EED and SUZ12, and reversal of repressive H3K27me3 and H4K20me3 marks along with apoptosis in breast cancer (MCF7) and colorectal cancer cell line models. DZNep induced re-exp ression of a gene set with partial overlap to those re-expressed by siRNA deplet ion of PRC2 co mponents. Precise mechanis ms of EZH2 depletion by DZNep remain unclear but appear to be in part a function of proteasomal degradation and linked to dependency of PRC2 members on each other for stability and function [START_REF] Fiskus | Co mbined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells[END_REF][START_REF] Pasini | Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity[END_REF][START_REF] Tan | Pharmacologic d isruption of Polyco mb-repressive comp lex 2-mediated gene repression selectively induces apoptosis in cancer cells[END_REF]. Subsequent work has shown DZNep is in fact likely to have broader inhibitory effect on methyltransferases beyond EZH2. Indeed removal of both repressive (H3K27me3, H4K20me3) and activating (H3K4me3) but not all (H3K9me3, H3K36me3) h istone methylation marks was demonstrated [START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF]. Thus DZNep likely acts as a broad methyltransferase inhibitor with effects on both inhibitory and activating histone methylation marks that might allow resetting of the epigenetic 'ground state' [START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF].

Differences have been seen in the patterns of activating trimethylation marks at H3K4 in response to DZNep which were depleted in HT29, SW480 (colorectal) and M CF7 (breast) cancer cell lines but increased in OCI -AML3 and HCL-60 (acute myeloid leukaemia, AM L) cells [START_REF] Fiskus | Co mbined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells[END_REF][START_REF] Jiang | DA CT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modificat ions[END_REF][START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF][START_REF] Tan | Pharmacologic d isruption of Polyco mb-repressive comp lex 2-mediated gene repression selectively induces apoptosis in cancer cells[END_REF]. Thus effects by this agent may be cancer and/or context specific.

Here we describe bio logical anti cancer effects of DZNep in mu lt iple breast cancer cell lines and extend findings to other AHI structural analogues for the first time. In addition we have investigated interactions seen with potentially co mp lementary epigenetic and targeted therapeutic approaches for breast cancer.

Methods

Cell

Immunoblotting

Cells were plated at 1.5x10 5 per 35 mm well. After 24 hours, cells were treated by replacement of culture med iu m supplemented with the indicated compound or equivalent concentration of DMSO solvent. Samp les were harvested as previously described for histone modifications [START_REF] Crabb | Characterisation of the in vitro activ ity of the depsipeptide histone deacetylase inhibitor spiruchostatin A[END_REF] and for other proteins using detergent lysates [START_REF] Packham | Mammalian cells express two differently localized Bag -1 isoforms generated by alternative translation init iation[END_REF]. Antibodies used were mouse monoclonal anti-EZH2 (3147) fro m New Eng land Bio Labs (UK) Ltd (Hitchin, UK), mouse monoclonal anti-HSC70 (sc-7298) and rabbit polyclonal anti-HER2 (sc-284) fro m Santa Cru z Biotechnology (Santa Cruz, CA) and rabbit polyclonal anti-H3K27me3 and rabbit polyclonal antiacetylated histone H4 at lysine 8 (acH4K8) fro m M illipore (U.K.) Limited (Watford, UK). HSC70 exp ression was used as a protein loading control. Results are representative fro m a min imu m of two separate experiments.

Cell Pro liferat ion Assays

Cells (1000/well in a 96 well plate) were incubated for 6 days in various concentrations of compound or solvent control, prior to cell nu mber analysis using MTS reagent (Pro mega, Southampton, UK) accord ing to the manufacturer's instructions. 5 μL MTS reagent was added per well containing 100 μL of cu lture mediu m for 1.5 hours prior to colorimet ric analysis on a Varioskan Flash spectral plate reader (Thermo Scientific, Basingstoke, UK). Each data point was derived fro m triplicate determinations and results are expressed as mean values as percentage MTS conversion with respect to samples exposed to DMSO solvent alone. (DMSO d id not alter MTS conversion compared to untreated cells in any experiment, data not shown). IC 50 values were derived following non linear reg ression using GraphPad Prism software (GraphPad So ftware, La Jo lla, CA) and are presented as mean values ± SEM fro m a minimu m of three separate experiments in each cell line for each co mpound.

Synergism in cell proliferation assays, as a result of co mbination exposure to compounds, was assessed using fractional effect analyses using CalcuSyn software (version 2.0, Biosoft, Cambridge, UK) according to the manufacturer's instructions. This software produces a combination index (CI) value where a value of less than 1.0 indicates a synergistic interaction [START_REF] Chou | Drug comb ination studies and their synergy quantification using the Chou -Talalay method[END_REF].

Cell Cycle Analysis

Cells were plated at 1.5x10 5 per 35 mm well. At 24 hours, cells were treated by replacement of cu lture mediu m supplemented with the relevant AHI co mpound or equivalent concentration of DMSO solvent for the indicated durations. Cells were harvested and stained with propidiu m iod ide (PI) for analysis of cell cycle phase and sub -G 1 fraction content using flow cytometry as previously described [START_REF] Crabb | BA G-1 inhibits PPA Rgamma -induced cell death, but not PPARgamma -induced transcription, cell cycle arrest or differentiation in breast cancer cells[END_REF]. Data were analysed as the percentage of cells in the indicated cell cycle phase of total cells in cell cycle and for analyses of cells in the sub-G 0 fraction as the percentage of total cells. Presented data are a representative experiment fro m a min imu m of 2 separate experiments in each indicated cell line. A ll experiments were performed with trip licate determinations for each data point.

Analysis of Apoptosis

Ce lls were plated at 1.5x10 5 per 35 mm well. At 24 hours, cells were treated by replacement of cu lture mediu m supplemented with the indicated compound or equivalent concentration of DMSO. Cells were harvested after the durations indicated and flow cyto metry was undertaken as previously described [START_REF] Crabb | BA G-1 inhibits PPA Rgamma -induced cell death, but not PPARgamma -induced transcription, cell cycle arrest or differentiation in breast cancer cells[END_REF]. Cells were stained by resuspending in 500 μL o f binding buffer containing 2.5 μL of annexin V-FITC, 1.25 μL of PI or both per sample. Presented data are a representative experiment fro m a min imu m of 2 separate experiments in each indicated cell line. A ll experiments were performed with trip licate determinations for each data point. Data are presented as the percentage of total cells that are either live (unstained), early apoptotic (annexin V positive) or late apoptotic (annexin V/PI positive). Differentiation analysis 1.5x10 5 cells per 35 mm well were plated on pre-sterilised microscope slides. After 24 hours cells were t reated with DZNep. Nile red staining for analysis of differentiation (of cytoplasmic lipid droplet accu mulat ion) according to the methods of Greenspan et al [START_REF] Greenspan | Nile red: a selective fluorescent stain for intracellular lipid droplets[END_REF], was carried out as previously described [START_REF] Crabb | BA G-1 inhibits PPA Rgamma -induced cell death, but not PPARgamma -induced transcription, cell cycle arrest or differentiation in breast cancer cells[END_REF]. All data are representative from a minimu m of 2 separate experiments. Presented data are a representative experiment fro m a minimu m of 2 separate experiments in each indicated cell line. Lipid d roplet quantification was undertaken in three separate reperasentative microspcope fields and a mean value per field of v iew ± SEM was determined.

Statistics

Determinations of statistically different mean values were performed using Students t -test.

Results

AHI analogues inhibit cell p roliferat ion in breast cancer cells

Although previous reports show that DZNep can induce apoptosis in breast and other cancer cell lines [START_REF] Tan | Pharmacologic d isruption of Polyco mb-repressive comp lex 2-mediated gene repression selectively induces apoptosis in cancer cells[END_REF], the structural analogues Nep A and DZA remain untested regarding their anticancer activity. We co mpared each in cell proliferation assays, revealing significant and consistent differences in potencies with respect to growth inhibit ion in each of three breast cancer cell lines (Fig. 1b-e preferentially inhibit ing cancer cell gro wth (Fig. 1f).

AdoHcy hydrolase inhibition induces G 2 /M cell cycle arrest and apoptosis of breast cancer cell lines

Having shown that AHI analogues inhibit proliferation of b reast cancer cells we investigated if this was due to cell cycle arrest and/or cell death. Time course experiments to determine cell cycle effects of DZNep were performed at a dose of 10µM as this was found to produce robust inhibition of cell pro liferation (Fig. 2; Supplementary Tab le S1). DZNep induced cell cycle arrest and significant increase in the G 2 /M cell co mponent after 72 hours in M CF7 and MDA-MB-231 cells (Fig. 2b,c; Supplementary Table S1). SKBr3 cells remained relatively resistant to DZNep until 144 hours undergoing a mo re modest but statistically significant G 2 /M arrest (Fig. 3d, Supplementary Table S1). DZNep also induced a significant increase in the sub -G 0 cell co mponent (indicative of cell death) after 144 hours in each cell line (Fig. 2e, Supplementary Table S1). We also tested the effects of DZA on cell cycle progression and again found G 2 /M cell cycle arrest and cell death after 72 hours in MCF7 cells (Fig. 2f, g, Supplementary Tab le S2).

Experiments to determine if cell death was occurring through induction of apoptosis revealed a significant increase in annexin V and annexin V/PI positive cells after 144 hours exposure to 10 μM DZNep in all three breast cancer cell lines (Fig. 3) ind icating induction of apoptosis in response to AdoHcy hydrolase inhibition.

AdoHcy hydrolase inhibition induces cytoplasmic lip id accumu latio n in breast cancer cells

After 72 hours exposure to DZNep, M CF7 and MDA-MB-231 cells underwent morphological changes, increasing in size with a reduced nuclear to cytoplasmic rat io and lacking the regular uniform order o f untreated cells. Changes in SKBr3 cells were less marked (Fig. 4a). Accu mulation of cytoplasmic lipid droplets is validated as an experimental marker of cellu lar d ifferentiation in breast cancer cells using methods described by Greenspan et al [START_REF] Crabb | BA G-1 inhibits PPA Rgamma -induced cell death, but not PPARgamma -induced transcription, cell cycle arrest or differentiation in breast cancer cells[END_REF][START_REF] Crabb | Characterisation of the in vitro activ ity of the depsipeptide histone deacetylase inhibitor spiruchostatin A[END_REF][START_REF] Greenspan | Nile red: a selective fluorescent stain for intracellular lipid droplets[END_REF][START_REF] Munster | The h istone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of hu man breast cancer cells[END_REF]. We therefore undertook Nile red staining of cytoplasmic lipid droplets following DZNep exposure. This revealed significantly increased droplet numbers in all three cell lines although SKBr3 cells have higher background staining and a less marked increase compared to untreated cells (Fig. 4b,c ). These results are consistent with a differentiat ion response to AdoHcy inhibition in breast cancer cells.

Co mbination AdoHcy hydrolase/HDAC inhibit ion has synergistic anti breast cancer effects

Co mbination epigenetic approaches are being investigated clinically and previous reports indicate synergistic interactions between HDAC inhib ition and DZNep in AM L and colorectal cancer models [START_REF] Fiskus | Co mbined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells[END_REF][START_REF] Jiang | DA CT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modificat ions[END_REF][START_REF] Nolan | W ill h istone deacetylase inhibitors require co mbination with other agents to fulfil their therapeutic potential?[END_REF]. We therefore investigated for interactions between either DZNep or DZA and the pan zinc dependant (class I, II and IV) HDA C inhibitor TSA in breast cancer cell lines in growth inh ibition assays. We found synergistic interaction with respect to growth inhibit ion between DZNep and TSA in M CF7 and MDA -MB-231 cells (Fig. 5a ). At doses of 100 nM and 25 nM respectively for examp le, the individual drugs inhibited gro wth by less than 10% in MCF7 cells, co mpared to over 40% when the drugs are comb ined. In M CF7 and M DA -MB-231, cells evidence for a synergistic interaction was supported by detection of a CI value of less than 1.0. By contrast, DZNe p and TSA were found to be additive (CI ≈ 1) rather than synergistic in SKBr3 cells. EZH2 expression and acH4K8 were assessed in cell lines treated with DZNep, TSA or both in all three cell lines (Fig. 5b). In each, EZH2 depletion and H4K8 hyperacetylation was detected in response to each agent individually wh ich was greater with dual administration.

Co mbination growth inhibit ion assays were fu rther exp lored using 25 nM DZA and 25 nM TSA (Fig. 5c ).

Significantly increased growth inhibit ion was observed in all cell lines with the combination treat ment co mpared to either agent alone. Evidence for a synergistic interaction was supported by a CI value < 1.0 for each cell line.

Immunoblot analysis demonstrated depletion of both EZH2 exp ression and H3K27 trimethylation to be enhanced in DZA and TSA dual treated cells (Fig. 5d).

Co mbined AdoHcy hydrolase/HER2 inhib ition synergizes in HER2 over-exp ressing breast cancer cells

Our experiments found AHIs to be less potent against SKBr3 cells which are a model for HER2 over expressing breast cancer. We therefore hypothesized that combination therapy with the HER2 directed therapeutic monocolonal antibody trastuzumab might 'unlock' sensitivity to inhib itors of AdoHcy hydrolase. Comb ination growth inhibit ion assays were perfo rmed for AdoHcy hydrolase inhibition and trastuzumab in SKBr3 cells (Fig. 6a). More marked growth inhib ition was observed with the comb ination treatment support ed by a CI o f 0.05 indicating strong synergism. Immunoblot analysis revealed a decrease in HER2 exp ression after either DZA or trastuzumab wh ich was augmented by combination treat ment (Fig. 6b).

Discussion

Recent data have shown EZH2 depletion and mod ification of histone methylation status by DZNep in M CF7 breast cancer cells [START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF][START_REF] Tan | Pharmacologic d isruption of Polyco mb-repressive comp lex 2-mediated gene repression selectively induces apoptosis in cancer cells[END_REF]. We sought to characterise the biological effects of this and other AHI co mpounds in model systems for this disease. Our results confirm the ability of DZNep to reduce expression of EZH2 in MCF7 cells but also now other breast cancer cell lines and to inhibit cell proliferation with nanomolar potency.

Importantly, this anti-proliferative effect was not seen in two non-malignant cell line models. Thus, analogous to HDA C inhibit ion, AdoHcy hydrolase inhibition may provide an opportunity to target a therapeutic window between malignant and non-malignant cells by exp loiting differing epigenetic profiles. Ho wever, the difference in potency ratio between malignant and non malignant models is markedly greater for AHIs than fo r HDAC inhibitors where ratios of 4-30 fold in potency are described [START_REF] Crabb | Characterisation of the in vitro activ ity of the depsipeptide histone deacetylase inhibitor spiruchostatin A[END_REF][START_REF] Huang | Suberoylanilide hydro xamic acid as a potential therapeutic agent for human breast cancer treatment[END_REF]. Our work therefore provides a rat ionale to target histone methylation dynamics in breast cancer.

For the first time, we have also demons trated anti breast cancer effects for other AHIs using the DZNep structural analogues DZA and Nep A. Over 30 carbocyclic and acyclic adenosine analogue AHIs have been investigated for antiviral act ivity since the 1980s. Most exhib it broad ant iviral act ivity wh ich correlates to in vitro potency as AdoHcy hydrolase inhibitors [START_REF] Clercq | John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral act ivity[END_REF]. As the mechanis m of act ivity is through secondary inhibition of methyl donors from AdoMet, it is reasonable to assume that AdoMet dependant methyltransferase inhibit ion, and thus anticancer activity, represents a class effect. Differences in anti breast cancer cell potency occurred amongst the compounds we investigated (DZA>DZNep >Nep A). In contrast, fro m the antiviral literature, differing relative antiv iral potencies (inhibit ion of cytopathogenicity in primary rabbit kidney cells against vaccinia or vesicular stomatitis virus es) were seen for these compounds (DZNep≈NepA>DZA) [START_REF] Clercq | John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral act ivity[END_REF]. Factors underpinning potency differences as anti-cancer agents such as cellular uptake, intra cellular drug metabolis m, AdoHcy hydrolase affin ity, target specificity and drug efflu x remain to be determined. Further investigation of the anti-cancer activity of this group of compounds is warranted and raises opportunities to begin to dissect structure/function relationships. Beyond this agent class, specific inhibitors of EZH2 and other methyltransferase enzy mes would be of great interest. This would allow assessment of specificity of action on anti cancer activ ity in co mparative studies to AHIs which appear likely to be indirect broad methyltransferase inhibitors [START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF]. As with HDA C inhibit ion, it remains unclear whether target selectivity (e.g. for EZH2) will be valuable when developing a therapeutic approach based around histone methyltransferase activity.

Having demonstrated potent anti breast cancer effects of AHIs, we sought to describe the biological processes underpinning this. We have shown that both DZNep and DZA have mult ifactorial anticancer effects includ ing induction of G 2 /M cell cycle arrest, apoptosis and differentiat ion. Thus inhibition of AdoMet dependent methyltransferases by AHIs to reset epigenetic abnormalities in breast cancer mirrors biological effects produced by HDAC inhibition [START_REF] Crabb | Characterisation of the in vitro activ ity of the depsipeptide histone deacetylase inhibitor spiruchostatin A[END_REF][START_REF] Huang | Suberoylanilide hydro xamic acid as a potential therapeutic agent for human breast cancer treatment[END_REF]. We found such effects generally required 72 to 144 hours to become apparent at concentrations around the IC 50 value for growth inhib ition in contrast to HDAC inhibit ion which in our experience is more rapid in its biological action (typically 24 to 72 hours ) [START_REF] Crabb | Characterisation of the in vitro activ ity of the depsipeptide histone deacetylase inhibitor spiruchostatin A[END_REF]. Th is may have imp licat ions if such compounds were developed towards clinical applications in terms of duration of exposure required for biological effect. This prolonged delay to biological outcome is also no table in light of recent data indicating that the epigenetic 'ground state' may rap idly reset (at least at the level of gene expression) following removal of DZNep [START_REF] Miranda | DZNep is a global h istone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation[END_REF].

We have also demonstrated synergistic interactions between AHIs and HDAC inhibition mirroring recent description of similar interactions in acute myeloid leukaemia and colorectal cancer models [START_REF] Fiskus | Co mbined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells[END_REF][START_REF] Jiang | DA CT3 is an epigenetic regulator of Wnt/beta-catenin signaling in colorectal cancer and is a therapeutic target of histone modificat ions[END_REF]. Clinical evidence for efficacy of HDAC inhib ition as a single agent approach in breast cancer has been modest to date and we and others have argued that HDAC inhib ition will require a co mb ination therapeutic strategy for optimal outcomes [START_REF] Luu | A Phase II Trial o f Vo rinostat (Suberoylanilide Hydro xamic Acid) in Metastatic Breast Cancer: A Californ ia Cancer Consortium Study[END_REF][START_REF] Munster | Clinical and bio logical effects of valpro ic acid as a h istone deacetylase inhibitor on tumor and surrogate tissues: phase I/II trial of valpro ic acid and epirubicin/FEC[END_REF][START_REF] Nolan | W ill h istone deacetylase inhibitors require co mbination with other agents to fulfil their therapeutic potential?[END_REF]. Our results are consistent with a model whereby optimal anti breast cancer effect is dependent on a mo re profound reset of underlying epigenetic abnormalit ies requiring both HDAC and histone methyltransferase inhibit ion. PRC2 recruit ment of HDA Cs by EED to aug ment transcriptional repression might potentially underpin the mechanis m by wh ich such synergy occurs [START_REF] Van Der Vlag | Transcriptional repression mediated by the human polyco mb -group protein EED involves histone deacetylation[END_REF]. Such co mbination strategies should therefore be investigated further for this disease as well as comb inations with DNA methyltransferase inhibit ion in view of the recruit ment of DNA methyltransferase activity to H3K27me3 repressive marks by EZH2 [START_REF] Vire | The Polyco mb group protein EZH2 direct ly controls DNA methylat ion[END_REF].

In addition, we have demonstrated synergistic interaction between AHIs and trastuzumab in a HER2 over expressing breast cancer model. This was acco mpanied by a reduction in HER2 protein expression which was maximised by co-ad min istration. Mechanisms for HER2 depletion remain to be elucid ated but might include effects on transcription through chromatin remodelling or indirectly for example by interacting with chaperone (e.g. HSP90) function as is seen with HDA C inhibit ion [START_REF] Bali | Activity of suberoylanilide hydro xamic Acid against human breast cancer cells with amp lification of her-2[END_REF]. Therapeutic targeting of histone methylation status in co mbination with established anti HER2 targeted therapies may therefore be of value in breast cancer subtypes dependant on this pathway. The mechanistic interaction between these two therapeutic approaches warrants further investigation.

In conclusion, we have demonstrated that the anti-breast cancer effects of AdoHcy hydrolase inhibition are underpinned by G 2 /M cell cycle arrest, apoptosis and cellular differentiat ion and that these are class effects for AHI analogues. Furthermore, A HIs produce synergistic interactions with HDAC inhibition and HER2 targeted therapy in breast cancer models. Our findings provide a basis for the development of therapeutic strategies to target histone methylation profiles in b reast cancer.

  Lines and Reagents Reagents were fro m Sig ma (Poole, UK) unless stated. MDA-MB-231, SKBr3, MCF10A and M CF7 cells (fro m ECA CC, Salisbury, UK) were maintained in Du lbecco's modified Eagle's med iu m (Lon za, Wokingham, UK) with 10% (v/v) fetal calf seru m (PAA Laboratories, Pasching, Austria), 2mM L-Glutamine and 5000 U/ ml penicillin/streptomycin (Invitrogen, Paisley, UK) at 37°C with 10% (v/v) CO 2 . Normal human dermal fibroblasts (NHDF) were maintained in Fibroblast Basal Mediu m with 10% (v/v) fetal calf serum and 0.5 mg/ ml hydrocortisone (Cambrex, Wokingham, UK) at 37°C in 5% (v/v) CO 2 . DZNep (NSC 606385), DZA (NSC 167897), and Nep A (NSC 316458) were obtained fro m the NCI/ DTP Open Chemical Repository (http://dtp.cancer.gov) and kept as stock solutions in dimethyl sulfo xide (DM SO) at -20°C. Chemical structures are shown in Figure 1a . Trastuzumab was a kind gift fro m Southampton University Hospitals NHS Trust Pharmacy Depart ment.

  ). Using MCF7 cells as a reference, DZA is ~4 fold more potent then DZNep (p≤0.01) with both being nanomolar inhibitors of proliferation, and ~200 fold mo re potent compared to Nep A (p≤0.05). IC 50 values for growth inhib ition in M CF7 cells were DZA 55 ± 13 nM; DZNep 194 ± 20 nM; Nep A 11895 ± 2012 nM. Similar IC 50 values and potencies were seen in MDA-MB-231 cells (DZA 57 ± 15 nM; DZNep 235 ± 26 nM; Nep A 11920 ± 1711 nM). SKBr3 cells exh ibited relative resistance by ~2-4 fo ld for IC 50 values of each compound compared to MCF7 cells (DZA 97 ± 11 nM; DZNep 821 ± 251 nM; Nep A 21225 ± 275 nM). DZNep did not induce a growth inhibitory effect in normal cells in concentrations up to 100 µM including non-cancerous breast epithelial cells (M CF10A ) and NHDFs, therefore

Fig. 1

 1 Fig. 1 AHI analogues inhibit breast cancer cell proliferation. (a), AHI chemical structures. (b), Mean IC 50 values in breast cancer cell lines for DZA (open bars), DZNep (black bars) and Nep A (hatched bars). (c-e), Representative cell pro liferat ion assays are shown for (c) M CF7, (d ) MDA-MB-231 and (e) SKBr3 cells treated for 6 days with DZA (), DZNep () or Nep A (). (f), Representative cell p roliferat ion assay in MCF10A () and NHDF () cells following exposure to DZNep.

Fig. 2

 2 Fig. 2 AHIs induce G 2 /M cell cycle arrest in breast cancer cell lines. (a), Representative flo w cytometry histograms for PI stained MCF7 cells treated with 10 µM DZNep or DMSO solvent control. (b-d), bar charts representing percentages of cells in the indicated cell cycle phases, following treat ment with 10 μM DZNep (black bars) or DMSO solvent control (open bars) in (b) M CF7, (c) MDA -MB-231 or (d) SKBr3 cells. (e), bar chart representing percentages of cells in the sub-G 0 fract ion following treatment with 10 μM DZNep (black bars) or DMSO solvent control (open bars) in the indicated cell lines. (f), bar chart representing percentages of cells in the indicated cell cycle phases, following treat ment with 10 μM DZA (black bars) or DMSO solvent control (open bars) in MCF7 cells. (g), bar chart representing percentages of cells in the sub-G 0 fraction following treatment with 10 μM DZA (b lack bars) o r DM SO solvent control (open bars) in MCF7 cells. Bar charts show mean values fro m trip licate determinations in a representative experiment for each indicated cell line. * p<0.05, ** p<0.01 co mpared to DMSO control samples .

Fig. 3

 3 Fig. 3 AHIs induce apoptosis in breast cancer cell lines. (a), Representative flow cytometry raw data of FITCannexin V (FL1) and/or PI (FL2) stained MCF7 cells treated with 10 µM DZNep or DMSO solvent control for 144 hours. (b -d), Apoptosis analysis of (b) MCF7, (c) M DA-MB-231 or (d) SKBr3 breast cancer cells treated with 10 µM DZNep (b lack bars) or DMSO solvent control (open bars). Q1, annexin V negative/PI negative; Q2, annexin V positive/PI negative; Q3, annexin V positive/PI positive; Q4, annexin V negative/PI positive. * p<0.05, ** p<0.01 co mpared to DMSO control samples .

Fig. 4

 4 Fig. 4 DZNep induces cytoplasmic lipid droplet accu mu lation in breast cancer cell lines. (a), Representative brightfield images comparing mo rphology of the indicated breast cancer cell lines exposed for 72 hours to 10 μM DZNep or DMSO. (b), Representative fluorescence microscopy images for Nile red staining of cytoplasmic lip id droplets of cells exposed for 72 hours to 10 μM DZNep or DMSO. (c), Histogram of quantified Nile red staining. Black bars, DZNep; white bars, DMSO control. Data represent the mean number ± SEM of lipid droplets per field o f view. * p<0.05, ** p<0.01 co mpared to DMSO cont rol samp les.

Fig. 5

 5 Fig. 5 AdoHcy and HDAC inhib itors have synergistic effects in breast cancer cell lines. (a, c), h istograms representing percentage inhibition in cell proliferation assays in the indicated cell lines compared to DM SO exposed cells (open bars) following exposure to (a) 100 nM DZNep (black bars), 25 nM TSA (d iagonal hatched bars) or DZNep and TSA (horizontal hatched bars) or (c) 25 nM DZA (black bars), 25 nM TSA (diagonal hatched bars) or DZA and TSA (horizontal hatched bars). Data are mean ± SEM of t rip licate determinations fro m a representative experiment . Synergistic effects on cell pro liferat ion are indicated by CI values < 1.0. (b) and (d), Representative immunoblots to show expression of EZH2, acetylat ion of histone H4 at lysine 8 and trimethylation of h istone H3 at lysine 27. Cells were exposed for 48 hours to DZNep or DZA fo llo wed by the addition or not of TSA for 24 hours as indicated. All experiments were performed a minimu m of t wice.

Fig 6

 6 Fig 6 AdoHcy inhib ition and trastuzumab have synergistic effects in HER2 over-exp ressing breast cancer cells. (a), Histogram representing percentage inhibition in cell proliferation assays following indiv idual or comb ination exposure to 25 nM DZA and 1250 nM trastuzu mab in SKBr3 cells. Data represents the percentage inhibition of cell proliferation co mpared to cells exposed to DMSO solvent alone. Data are mean ± SEM of trip licate determinations fro m a representative experiment. Synergistic effects on cell p roliferat ion are indicated by a CI value < 1.0. (b), Representative immunoblot to demonstrate expression of HER2 at 48 hours following exposure to trastuzumab or DZA o r both. All experiments were performed a minimu m of t wice.

Acknowledgements

This work was supported by Cancer Research UK and Wessex Medical Research.

Supplementary Tables

Supplementary Table S1 Cell cycle analysis of breast cancer cell lines in response to 10µM