DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. Developmental processes and proper biological functions are strongly dependent on hierarchical and regulated gene expression patterns. Numerous molecular processes control gene expression. DNA methylation is a physiological epigenetic process that leads to the long term-repression of gene expression. DNA methylation is a common epigenetic modification involving the methylation of 5'-cytosine residues and is often detected in the dinucleotides of CpG sequences. Methylation is often localized in promoter regions and occasionally in transcriptional regulatory regions in mammals, plants and even prokaryotes. Hypermethylation of the promoter region of genes is associated with gene silencing, whereas hypomethylation result in gene activation. Aberrant methylation in eukaryotic cells may lead to the silencing of important genes, such as tumour suppressor genes, affecting their related transcriptional pathways and ultimately leading to the development of disease such as cancer. Therefore, it is considered to be a hallmark of cancer, and it is detected in several types of cancer cells, including colon, breast, ovarian and cervical cancer cells and is associated with alterations in specific gene expression.
Hypermethylation of tumour suppressor gene promoters and the global disruption of many histone modifications are characteristic features of cancer. Deregulation of the epigenetic profile alters the transcription profile of many genes. In the case of tumour suppressors, DNA methylation reduces gene expression and subsequently removes regulatory proteins required for normal cell growth and development. Therefore, DNA methylation in cancer is predicted to influence multiple gene networks rather than single genes. Due to the heterogeneity of breast cancer at both histological and molecular levels, staging breast cancer fails to predict the prognosis or therapeutic response of the disease; therefore, DNA methylation targeted therapies have in recent years played an increasingly important role in the treatment of breast cancer. Two groups of agents targeting epigenetic modifications have previously been studied, i.e., histone deacetylase inhibitors and DNA methyltransferase inhibitors. The associations between DNA methylation mechanism and breast cancer classification and prognosis will be reviewed in detail in this chapter by describing the DNA methylation mechanism and gene expression in breast cancer, as well as functional genomics and genome-wide DNA methylation in breast cancer.
2. What is epigenetics?
Conard Waddington introduced the term 'epigenetic'in 1942 as a concept of environmental influence in inducing phenotype modification. His work on developmental plasticity states thatthe environmental influences during development could induce alternative phenotypes from one genotype, one of the clearest examples is polyphenisms in insects.Waddington showed that exposing the pupae of wild type Drosophila melanogaster to heat shock treatment resulted in altered wing vein patterns [1,2].Breeding individuals who have been exposed to these environmentally induced changes developed stable populations exhibiting the phenotype without the environmental stimulus. The concept of epigenetics was not clarified until the late 1990s, when Wolffe and Matzkeset's modern definition, i.e., "the study of heritable changes in gene expression that occur without a change in DNA sequence" . Birds come with a wider definition of epigenetic, that is, "the structural adaptation of chromosomal regions so as to register, signal or perpetuate activity states" . The term 'epigenome' has emerged to describe epigenetic modifications throughout the epigenome; thus, the epigenome controls the genome in both normal and abnormal cellular processes and events . Epigenetic mechanisms include DNA methylation, histone modification and non-coding RNAs, which work cooperatively to control gene expression.
3. DNA methylation
DNA methylation is a well-conserved process that occurs in eukaryotes and prokaryotes . DNA methylation refers to the covalent addition of a methyl group to carbon number five in the nitrogenous base cytosine at the DNA strand. Only cytosine residues adjacent to guanine are targets for methylation by the methyltransferase enzymes and the distribution of methylated and unmethylatedCpGs is tissue-specific, which leads to the cell-specific pattern of DNA methylation. The CpG may occur in multiple repeats which are known as CpG islands. CpG islands are often associated with the promoter regions of genes. Almost half of the genes in our genome have CpG-rich promoter regions. About 80% of the CpG dinucleotides not associated with CpG islands are heavily methylated within the entire gnome . In contrast, the CpG islands associated with gene promoters are usually unmethylated.
There are a number of factors that may maintain the undermethylated state of CpG islands, such as sequence feature, SP1 binding sites, specific acting enhancer elements, as well as specific histone methylation mark H3K4me3, which prevents the binding of
3.1. DNA Methyltransferases(DNMTs)
The methylation process is catalysed by the DNAmethyltransferases enzymes (DNMTs), which are known as DNMTs and include DNMT1,2, DNMT3A andDNMT3B . DNMT3A and DNMT3B are the de novomethyltransferases, while DNMT1 maintains methylation patterns during DNA replication (mitosis) . However, the actual function of DNMT2 remains unclear; several forms of DNMT1 have been detected, which differ in their translation start sites and prefer hemimethylatedDNA.Overexpression of DNMT1 has been reported in human tumours and may contribute to the global methylation abnormalities seen in cancer cells.However, increased expression of DNMTs is likely to be only partially responsible for observed methylation abnormalities, since not all tumours overexpress these enzymes .Cytosine (C5)-DNA methyltransferasescatalyse the transfer of a methyl group from S-adenosyl-methionine (SAM) onto cytosine residues in specific sequences of duplex DNA, alongside the production of 5-methyl cytosine and S-adenosyl-homocysteine (SAH) (Figure1). Cytosine (C5)-DNA methyltransferases have up to 10 conservative regions arranged in strictly defined sequences. The primary structures of cytosine (C5)-DNA methyltransferases reveals the association of their major functions with their conservative motifs, whereas the site-specific recognition belongs to a variable region of the target-recognizing domain (TRD) .The N-terminal domain of DNMT1 contains varied specific functional sequences, such as the nuclear localization signal (NLS), the cysteine-enriched zinc-binding motif and a special sequence directing the methylase into the area of DNA replication. In addition, DNMT1 interact with the proliferating cell nuclear antigen (PCNA), which is required for DNA replication and the DNMT1-PCNAinteraction allow for rapid remethylation of the newly synthesized daughter strands before being packed into chromatin .A null mutation of DNMT1 gene in mouse resulted in a considerable (up to 70%) decline in genome methylation and the death of developing embryos . The remaining30% level of DNA methylation and the ability of embryonic stem cells deprived of the DNMT1 methylase for de novo methylation of DNA suggest that these functions were performed by other DNA methylases. Such methylases were searched for in animals and new enzymes of the DNMT2 and DNMT3 families were found.Cell-cycle regulators p21 and retinoblastoma gene product Rb can bind to DNMT1 and inhibit its methyltransferase activity during DNA replication in the cell cycle. These observations indicate complex interaction between DNMT1 and the cellular proteins involved in gene regulation and epigenetic signalling during cell replication.
The DNMT3 family consists of two genes, DNMT3a and DNMT3b, which are highly expressed in undifferentiated ES cells but downregulated after differentiation and expressed at low levels in adult somatic tissues, and are overexpressed in tumour cells . Both DNMT3a and DNMT3b are required for genome-wide de novo methylation and are essential for mammalian development . Both DNMT3a and DNMT3b have been mapped by the UniGeneconsortium via polymorphisms in 3'-untranslated region sequences. DNMT3b was mapped to the region of chromosome 20q, which contains the trait for ICFNS (immunodeficiency centromeric instability, facial abnormalities) syndrome. This syndrome presents with variable combined immunodeficiency, mild facial anomalies and extravagant cytogenetic abnormalities which largely affect the pericentric region of chromosomes 1, 9 and 16. These regions contain a type of satellite DNA known as classical satellites,orsatellites 2 and 3.They are usually heavily methylated, but are almost entirely unmethylated in the DNA of ICF patients. It has been found that immunodeficiency centromeric instability (ICF) patients had mutations in the C-terminal DNA methyltransferase domain of DNMT3b. DNMT3b remains the only DNA methyltransferase shown to be mutated in a human disease . DNMT3b has been shown to play a crucial role in the hypermethylationof promoter CpG-rich regions of tumour suppressor genes and thus, its inactivation within human cancer cells .
3.2. How does methylation and demethylation occur?
A key question is how the enzymes know where to methylate. Two theories have been suggested. Firstly, it has been suggested that all genes are methylated by default, except for active genes .Actively transcribed genes have a preponderance of attached transcriptional factors, giving no physical access to methyltransferases for reaching their targets. On the other hand, inactive DNA is susceptible to methyltransferases and subsequently become methylated. This model was confirmed by the study of the transcription factor SP1. It has been shown that as long as SP1 is attached to its site, no methylation can occur in the adjacent CpG sites and removal of the SP1 leads to de novo methylation at this site . The second theory is that methylation is directed by sequence specific binding proteins, so the methyltransferases bind with certain proteins such as a histone deacetylases (HDACs) and other transcription repressors, and form a complex that binds to a specific sequence on the DNA .
Methylated genes may need to be activated in response to environmental signals and thus, demethylation is an important dynamic epigenetic mechanism, originally thought to only occur through passive demethylation(Figure 2). However, the rapid demethylation of paternal genomes upon fertilization and examples of the rapid demethylation of genes in post-mitotic neurons suggest that an active demethylase must exist[23, 25]. A number of enzymes have been suggested to have demethylase activity; these include MBD2b, MBD4, the DNA repair endonucleases XPG (Gadd45a) and a G/T mismatch repair DNA glycosylase, which is glycosidase dependent. In this mechanism, the methylated cytosine is recognized by glycosidase, which cleaves the bond between the DNA backbone and base.The base is subsequently removed and replaced with unmethylated cytosine by the DNA repair system.
Histones are five basic nuclear proteins that form the core of the nucleosome.The histone octamer, contains two molecules each of histones H2A, H2B, H3 and H4. Histone H1, the linker histone is located outside the core and is involved in the packing of DNA. Histone modifications play a major role in regulating gene expression and extend the information potential of the DNA, which explains the growing interest in the ‘Histone Code’ . Amino acids modifications on the N-terminal tails of histones protruding from the nucleosome core can induce both an open or closed chromatin structure and these affect the ability of transcription factors to access promoter regions in order to activate transcription. The covalent modification can be acetylation, methylation, phosphorylation and ubiquitination. Methylation of some residues is associated with both transcriptional repression, such as the methylation of histone 3 lysine 9 (H3 K9), andothers with transcriptional activation, such as methylation of histone 3 lysine 4 (H3 K4) [28, 29].
Histone methylation is performed by histone methyltransferase (HMTs), which can transfer up to three methyl groups to lysine residues within the tails of histones with different effects on gene activity. Acetylation occurs at lysine residue,whichis associated with transcriptional activation . This modification is performed by histone acetylases (HATs) and removed by the HDACs. The HDACs are critical in the regulation of the expression of genes that are important for cell survival, proliferation, differentiation and apoptosis .HDACs also act as members of a protein complex responsible for the recruitment of transcription factors to the promoter region of genes, including those of tumour suppressors, as well as the regulation of the acetylation status of specific cell cycle regulatory proteins . High HDAC expression and histone hypoacetylationhave been observed in cancer with the associated transcriptional repression of genes, providing a rationale for the investigation of HDAC inhibitors in cancer therapeutics .
Acetylation of histones has been extensively studied as one of the key regulatory mechanisms of gene expression . As early as the 1960s, histone acetylation was found to affect RNA transcription . The highly conserved lysine residue at the N-terminal of H3 at positions 9, 14, 18 and 23, and H4 lysine 5,8,12 and 16, are frequently targeted for modification . Acetylations of the lysine residues neutralize the positive charge of the histone tails and thereforedecrease their affinity for DNA, which results in open chromatin conformation and allowing the transcriptional machinery to reach its target . The acetyltransferases adds the acetyl groups from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues . There are eighteenHDAC enzymes in mammalian cells and these are classified into two families: (a) zinc metalloenzymes (catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs;(b) NAD-dependent Sir2 deacetylases (considered as class III HDACs)[40, 41].
Class I is a group of four enzymes known as HDAC1, 2, 3 and 8, and this class is associated with gene regulation. They are expressed ubiquitously and function exclusively in the nucleus . Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9, and class IIB, which includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus,and are mainly involvedin cell differentiation and are highly expressed in certain tissues . Class III includes the NAD-dependent deacetylases, which is a group of seven enzymes that are involved in maintaining chromatin stability. They can remove acetyl groups from histones, as well as other proteins . Class IV contains one member, HDAC11, which is closely related to class I; thus, some reviewers consider it a member of that class. The function of HDAC11 has not yet been characterized .
There is increasing evidence showingthat changes in chromatin structure will alter the DNA methylation process, and thatthe DNA methylation enzymes targeted to gene promoters are guided by chromatin modifying enzymes. The fact that chromatin configuration is dynamic and the chromatin modifying enzymes are activated by cellular signalling pathways. This provides a link between the extracellular environment and the state of DNA methylation . Evidence that this link between chromatin modelling and DNA methylation are involved in chromatin remodelling arises from mutations of the SWI-SNF proteins in humans and mice. These mutations result in defects in DNA methylation . A number of histone methyltransferases, such as G9a, SUV39H1 and EZH2, a member of the multi-proteinpolycomb complex PRC2, can regulate DNA methylation by either recruiting or regulating the stability of DNMTs. DNMTs in turn can recruit HDACs and MBPs to achieve chromatin condensation and gene silencing . This relationship between epigenetic devices renders the epigenetic mechanisms of genome expression a strictly regulated process.
5. DNA methylation and breast cancer
The study of epigenetic mechanisms in cancer during the last decade, including DNA methylation, histone modification, nucleosome positioningand miRNA expression, has provided extensive information about the mechanisms that contribute to the neoplastic phenotype through the regulation of the expression of genes critical to transformation pathways. Concerning DNA methylation, the low level of CpG methylation in tumours compared with that in their normal-tissue counterparts was one of the first epigenetic alterations to be found in human cancer.This indicates that cancer cells have a specific epigenome. Hypomethylation in cancer cells is associated with a number of adverse products, including chromosome instability, activation of transposable elementsand the loss of genomic imprinting .
Breast cancer has traditionally been staged by histopathologicalstandards that are based on size, level of invasiveness, lymph node infiltration and by immunochemical characterizationof cell surface receptors including theoestrogen receptor (ER), the progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Yetin many instances, staging breast cancer fails to predict prognosis or therapeutic response,due to the heterogeneity of the disease. Changes in gene expression that reset a cell programme from a normal to a diseased state involve multiple genetic circuitries, creating a characteristic signature of gene expression that defines the cell's unique identity and that classifies subtypes of breast cancers. Detailed knowledge of the DNA methylation status of all cytosines (methylome) is paramount for understanding the mechanisms and functions underlying DNA methylation, and will extend our ability to classify breast cancer and formulate outcome predictions. DNA methylation is a forceful biomarker and significantly more stable than proteins or RNA; it is therefore a promising target for the development of new approaches for the diagnosis and prognosis of breast cancer and other diseases. Since DNA methylation is critical in gene expression programming, modification in methylation from a normal to diseased state should be similarly reflected in a signature of DNA methylation that involves multiple gene pathways. Whole-genome approaches have been applied previouslyyielding different levels of achievementfor distinguishing breast-cancer-specific DNA methylation signatures and to test whether they can classify breast cancer, as well as whether they could be associated with specific clinical outcomes .
In addition to the use of immunohistochemistry and mRNA expression analysis in breast cancer diagnosis and prognosis, the application ofDNA methylation profiling became an important tool that canprovide additional classification value to the other methods currently used. For example, the Illumina 27 K arrays which is a whole-genome DNA methylation analysis suggests that DNA methylation profiling might expand current classifications of breast cancersubtypes[49, 50]. Whole-genome DNA methylation analysis of 248 breast cancer tumour samples, comprising a 'main set' of 123 samples (four normal and 119 infiltrating ductal carcinomas (IDCs)) and a 'validation set' of 125 samples (eight normal and 117 IDCs)revealed an immune 'signature' in a mixed tumour stromal population.. Methylome analysis performed on frozen primary tumour samplesled to the identification of six different methylation clusters . It was shown for the first time that DNA methylation profiles can reflect the cell-type composition of the tumour microenvironment, with T lymphocyte infiltration of these tumours in particular in HER2-enriched and basal-like tumours. High expression of certain immune-related genes were found to be associated with improved relapse-free survival, providing further insight into the importance of the immune system and tumour microenvironment in certain breast cancer subtypes.
Furthermore,altered DNA methylation patterns of the CpG islands in the promoter regions of tumour-suppressor genes are accepted as being a common mark of human cancer . Promoter hypermethylation affects genes from a wide range of cellular pathways such as DNA repair, toxic catabolism, cell adherence, cell cycle, apoptosis and angiogenesis, which may occur at various stages during the development of cancer [54, 55]. The CpG-island-containing gene promoters are usually unmethylated in normal cells to maintain euchromatic structure and transcriptionally active conformation, allowing gene expression. Yet during cancer development, many genes are hypermethylated at their CpG-island-containing promoters to inactivate their expression by changing open euchromatic structure to compact heterochromatic structure [56, 57]. These genes are selectively hypermethylated in tumourigenesis for inactivation of their functional involvement in various cellular pathways that prevent cancer formation. Some of the genes that have been identified in human cancers are classic tumour suppressor genes in which one mutationally inactivated allele is inherited. According to thetwo-hit model, complete inactivation of a tumour suppressor gene requires the loss-of-function of both gene copies . Epigenetic silencing of the remaining wild-type allele of the tumour suppressor gene by DNA methylationcan therefore be considered as the second hit in this model. For example, some well-known tumour suppressor genes, such as the cyclin-dependent kinase inhibitorp16INK4a, APC and BRCA1, are mutationally inactivated in the germline an occasionally lose function of the remaining functional allele in breast epithelial cells through DNA hypermethylation.These advanced knowledge in breast cancer methylome strongly indicate that the DNA hypermethylation mechanismplays a potential role in the initiation, promotion and maintenance of breast carcinogenesis, which in turn cooperatively interact with other genetic alterations to promote the development of breast cancer. In addition to cell-cycle regulatory genes, DNA methylation-mediated silencing of DNA repair genes such as BRCA1 and MGMT can result in further inactivation of tumour suppressor genes or in the activation of oncogenes, which further drive breast tumourigenesis. The genes that function as inhibitors of the WNT oncogenic pathway such as SFRP1 and WIF1have been found to be frequently hypermethylated in primary breast tumours .Accordingly, epigenetic gene silencing is another mechanism that fosters the malignant transformation of the mammary gland by aberrantly activating oncogenic signalling pathways, in addition to the genetic mutation-mediated mechanism.
In vitro experiments have shown that decreased BRCA1expression in cells led to increased levels of tumour growth, while increased expression of BRCA1 led to growth arrest and apoptosis. The magnitude of the decrease of functional BRCA1 protein correlates with disease prognosis . Phenotypically, BRCA1-methylated tumours are similar to tumours from carriers of germlineBRCA1 mutations. BRCA1 promoter hypermethylation was observed in one of two tumours among BRCA1 carriers . In a population-based ovarian tumours study, two of eight tumours with germlineBRCA1mutations lacked promoter methylation . Another study of 47 breast tumours among hereditary breast cancer families identified three BRCA1 carriers, two of which showedaltered BRCA1 promoter methylation in their tumours . .Tumours with BRCA1 mutations are usually more likely to be higher-grade, poorly differentiated, highly proliferative, ER negative and PR negative, as well as p53 mutations. BRCA1 mutated breast cancers are also associated with poor survival in some studies . BRCA1 promoter methylation was more frequent in invasive than in situ carcinoma and there were no correlation between BRCA1 promoter methylation and ER/PR status in a subset population .However, the findings that high prevalence ofBRCA1 promoter methylation in cases with at least one node involved and with a tumour size greater than 2cm,and high methylation levels, may correlate with a more advanced tumour stage at diagnosis. A 45% increase in the mortality of individuals with BRCA1 methylation positive tumours was also observed, compared to those who had unmethylated BRCA1 promoters . A familial breast cancer based study revealed no overall correlation regarding ER, PR or grade with hypermethylation of BRCA1 in the tumours from BRCA1 mutation negative families. However, seven individuals had promoter hypermethylation and the majority of these tumours had a basal-like phenotype, and were triple negative .
In addition, discriminating between tumour and normal or histologically non-malignant breast tissue has been applied widely by genome-wide DNA methylation. One of the first genome-wide DNA methylation studies in breast cancer developed methylation-specific digital karyotyping (MSDK) to assess epithelial, myoepithelial and stromal fibroblasts from normal breast and cancer tissues. Furthermore, genome-wide DNA methylation studies in breast cancer identified gene families that were commonly identified as differentially methylated between non-malignant and tumour groups and included FOX, KLF, PRDM, ZBTB, and ZNF transcription factors, as well as RAB and SLC gene families involved in the cell transportation of proteins or vesicles or involvement in cell adhesion [71-74]. The pathways and gene families do not appear to have a strong link to hormone metabolism or signalling; it is likely that these genes are not drivers of cancer but rather secondary events that occur as part of the tumourigenic process[75, 76].
Genome-wide DNA methylation studies have supported correlations between DNA methylation and gene expression, particularly the association between CpGislands,DNA hypermethylation and gene repression [49, 74, 77, 78].Using familial breast cancers and BRCA1/2-mutated tumours, combined with DNA methylation profiles, predicted BRCA status with gene expression, while copy number variation (CNV) found that reduced gene expression was more likely to be in genomic regions with loss of heterozygosity and/or high levels of DNA methylation. It has also been shown that the combination of gene dosage in breast cancer cell lines, allelic status and DNA methylation explains more gene expression changes than either genomic element on its own . Combining DNA methylation profiling with CNV and gene expression can be a promising tool for facilitating the identification of critical genes involved in tumourigenesis.In genome-wide methylation analysis, several platforms have recently been developed to allow genome-wide methylation analysis. The Golden Gate methylation array was the first platform that allowed methylation of 1536 CpG loci to be investigated. The InfiniumHumanMethylation27 method increased CpG investigation through the use of 27578 probes. Most recently, the Infinium HumanMethylation450K array was designed by Illumina. This array utilizes a florescence microarray hybridization technique, often associated with expression studies, to provide a methylation profile of 485764 CpG loci including CpG associated with CpG islands, shores, shelves and isolated loci in the open sea regions of the genome. Promoter regionshave also used the Infinium HumanMethylation27 BeadChip (Illumina) to analyse normal breast tissues from ten healthy individuals and compared this to 62 breast tumour samples (19 were inflammatory breast cancers).
Further studies have also compared tumours to non-malignant tissue; the number of genes identified that discriminates these tissues depends on the filtering or analyses utilized. Kim et al. (2012)used several filtering processes to identify six genes, whereas, Faryna et al. (2012)identified 214 CpG islands, but only one CpG island (TAC1) was methylated in all 10 cancer samples. The DNA methylation profiles characterized the samples into three groups based on high, intermediate and low DNA methylation levels, with the normal samples having low DNA methylation levels. When DNA methylation data were compared between normal and tumour samples, 1352 CpG loci (1134 genes) were differentially methylated . There was significantly greater methylation in tumours compared with normal tissue and 77% of these were CpG loci. Another study using the same technology found 6309 CpGs differentially methylated between 119 tumours and four normal breast tissue samples identified several hundred differentially methylated loci between 11 adjacent non-malignant breast tissues and 108 tumours[49, 74]. Kim et al. (2011)pooled DNA from 10 cancers and 10 non-malignant matched adjacent tissues and identified 1181 differentially methylated CpGs (corresponding to 1043 genes), with the vast majority (972) hypermethylated. Another study found 291 probes (264 genes) hypermethylated in breast cancer (n=39) compared to non-malignant breast tissue (n=4) following the removal of imprinted genes and X chromosome genes.
In addition, a number of studies have investigated whether genome-wide DNA methylation profiling can cluster breast cancers into hormone receptor status (ER/PR positive or negative) or subtypes (luminal A or B, basal or HER2). These investigations differentiate hormone receptor-positive breast cancers from hormone receptor-negative cases using DNA methylation profiles[49, 77, 83-85]. The majority of genome-wide DNA methylation studies have found that ER+PR+ tumours have higher levels of DNA methylation compared to ER−PR− tumours [77,82,85,86].Li et al. (2010)found 148 altered CpG sites (93 hypermethylated and 55 hypomethylated) in ER+PR+ breast cancers relative to ER−PR− tumours.Another studyrecognized 40 CpG probes that had an overall specificity of 89% and sensitivity of 90% for classifying ER+ from ER− tumours.
Moreover, Hill et al. (2011)used cluster analysis to show that ER+PR+ tumours had high methylation, whereas triple-negative breast cancers had low methylation status. Breast cancer cell lines have also shown clustering according to hormone receptor status based on DNA methylation levels . All these genome-wide DNA methylation studies therefore demonstrate that adequate results of appropriate clinical samples should identify methylation differences based on hormone receptor status. These studies may serve future studies as a basis for the development of an improved clinical test for identifying the hormone status of breast cancers.
In addition, in DNA methylation cluster analysis it was observed that one cluster was predominantly luminal A (22/30 samples), while the second cluster was highly correlated with luminal B (basal) (7/8 samples), while the third cluster contained a mixture of subtypes .In recent work, the Cancer Genome Atlas (TCGA)  and genome-wide profiling of DNA methylation has also been performed in primary breast tumours and have revealed that genes with hypermethylation was significantly correlated with deteriorate-free survival, including RECK, SFRP2 and ACADL. Tumour specificity of methylation was verifiedfor these genes by sequencing of an independent set of normal/breast tumour samples. Other investigations have observed that the reduction of RECK methylation is associated with worseprognoses in other tumours . Genomewide analysis has also been employed to characterize the DNA methylation profile of primary breast cancer with different metastatic potential. Identification of epigenetic profiles associated with low risk of metastases has been confirmed in theglobal breast CpG island methylation phenotype (B-CIMP). Comparable gene expression analyses identified genes with both significant hypermethylation and down-regulation in B-CIMP tumours, including those involved in epithelial-mesenchymal transition (EMT) such as LYN, MMP7, KLK10 and WNT6, and the genes in the B-CIMP repression signature showed genes with differential expression correlated with prognoses across several BC cohorts .
6. HDAC inhibitors and breast cancer
As previously noted, abnormal HDAC activity has been documented in a variety of tumour types and hasled to the development of HDAC inhibitors as anticancer therapeutics. Currently available HDAC inhibitors target a variety of HDAC isoenzymes with class 1 (HDAC 1, 2, 3 and 8), class 2 (HDAC 4-7 and 9-10) and class 4 (HDAC 11) activity. Modest clinical benefits were previously reported with relatively weak HDAC inhibitors such as valproic acid and phenylbutyrate in advanced solid tumours or hematologic malignancies . Laboratory research conducted to date supports the investigation of HDAC inhibitors for the treatment of breast cancer. Recently, vorinostat as HDAC inhibitor has shown to induce differentiation or arresting the growth of a wide variety of human carcinoma cells, including breast cancer cells .Vorinostat also reduced tumour incidence in NMU-induced rat mammary tumourigenesis by 40% . In vitro studies have demonstrated that vorinostat inhibits clonogenic growth of both ER-positive and ER-negative breast cancer cell lines by inducing G1 and G2/M cell cycle arrest and subsequent apoptosis .
The ability of the HDAC inhibitors to relieve transcriptional repression in preclinical breast cancer models has also been investigated. The accumulation of acetylated H3 and H4 histone tails in conjunction with the re-expression of a functional ER in ER-negative breast cancer cell lines has been observed using a novel HDAC inhibitor known as scriptaid .Treatment of ER-negative breast cancer cell lines with vorinostat is associated with reactivation of silenced ER, as well as downregulation of DNMT1 and EGFR protein expression . The significance of an epigenetically reactivated ER was demonstrated when tamoxifen sensitivity was restored in the ER-negative MDA-MB-231 breast cancer cells, following treatment with both HDAC (trichostatin A) and DNMT inhibitors (DAC) . Entinostat has been shown to induce not only re-expression of ERα, but also the androgen receptor and the aromatase enzyme (CYP19), both in vitro and in triple-negative breast cancer xenografts.In addition, the combination of letrozole and entinostat resulted in a significant and durable reduction in xenograft tumour volume when compared to treatment with either agent on its own. These experiments have provided a strong rationale for combining epigenetic modifiers with hormonal therapy in breast cancer clinical trials .Interestingly, many of these studies also indicate that a strategy combining HDAC and DNMT inhibitors is more effective than employing either agent on its own with respect to boththe re-expression of silenced genes and the restoration of response to tamoxifen and aromatase inhibitors [93,97].
Moreover, pretreatment of various tumour cell lines with HDAC inhibitors increases the cytotoxicity of chemotherapy. Administering the HDAC inhibitor after chemotherapy did not achieve the same results, suggesting that pretreatment with these agents may open the chromatin structure and thus facilitate an enhanced anti-cancer effect of chemotherapy drugs that targets DNA .In breast cancer cell lines with amplification and overexpression of HER2, the HDAC inhibitor use depleted HER2 by attenuation of its mRNA levels and the promotion of proteosomal degradation. HDAC inhibition has also been reported to enhanceapoptosis induction by trastuzumab, docetaxel, epothilone B and gemcitabine .HDAC inhibitors also significantly enhance trastuzumab-induced growth inhibition in trastuzumab-sensitive, HER2-overexpressing breast cancer cells, providing a strong rationale for clinical studies to use this combination in patients with HER2-positive disease .
Additionally,through in vitro and in vivo studies, HDAC inhibitors such as entinostat or valproic acid have been tested in breast cancer cells and efficiently restored both ERα expression and letrozole sensibility in ER- BC[101,102].The combination of treatment inducing the overexpression of TFAP2C with HDAC inhibitors or 5-azadeoxycytidine may improve ESR1 expression in ER- patients. CombiningHDAC inhibitors and 5-azadeoxycytidine treatment induces the most significant increase in ERα content. However, the addition of tamoxifen does not produce a tumourigenic response in ERBC cells, verifying a better response to tamoxifen in BC cells, whichcorrelates with a lower level of the RNA-stabilizing HuR protein.Tamoxifen treatment increased HuR content and contributed to its own resistance, while HDAC inhibitors/5-azadeoxycytidine decreased HuR. A first roundof treatment with HDAC inhibitors/5-azadeoxycytidine was given prior to tamoxifen delivery to obtain the best possible tamoxifen sensitivity. The precise functions of tamoxifen are complex; although it competes with 17β-estradiol to bind to ERα, ERα bound to tamoxifen is still able to target the TFF1 (also called pS2) promoter without the constitutive activation of gene transcription. Changes in the balance of co-activators/co-repressors and ERα-interacting partners were able to mediate the loss of transcriptional activity of the tamoxifen-ERα complex .
7. DNMTs inhibitorsand breast cancer
The human DNMTs 1, 3A and 3B coordinate mRNA expression in normal tissues and overexpression in tumours, and the expression levels of these DNMTs are reportedly elevated in breast cancer[105,106]. The levels of DNMT1, DNMT3a and DNMT3b overexpression have been shown to be similar among different tumour types. The DNMT3b gene has shown the highest range of expression (81.8 for DNMT3a compared to 16.6 and 14 for DNMT1 and DNMT3a, respectively). About 30% of patients revealed overexpression of DNMT3b in the tumour tissuecompared to normal breast tissue and the DNMT3b expression change was 82-fold . Interestingly, DNMT1 and DNMT3a were overexpressed in only five (3%) of breast carcinomas.These studies have shown thatDNMT3b plays a major role in breast tumourigenesis. This is consistent with a recent study in breast cancer cell lines that demonstrated a strong correlation between total DNMTs activity due to overexpression of DNMT3b, but not with the expression of DNMT3a or DNMT1 [107,108].
Cancer was the first group of diseases to be associated with DNA methylation and to be considered for DNA-methylation-targeted therapeutics, and serves as a prototype for determining the role of DNA methylation and DNA-methylation-targeted therapeutics in other diseases . As we noted previously, several types of aberration in DNA methylation and in the proteins involved in DNA methylation occur in cancer: hypermethylation of tumour suppressor genes, aberrant expression of DNMT1 and other DNMTs, and hypomethylation of unique genes and repetitive sequences[110,111]. Tumour suppressor gene silencing by DNA methylation offers influential molecular mechanisms by which DNA methylation can trigger cancerand also provides a rationale for therapeutics aimed at the inhibition of DNA methylation and the re-expression of silenced tumour suppressor genes.Multiple genes are hypermethylatedin breast cancer compared to non-cancerous tissue . These include genes involved in the evasion of apoptosis (RASSF1A, HOXA5, TWIST1), limitless replication potential (CCND2, p16, BRCA1, RARβ), growth (ERα, PGR)and tissue invasion and metastases (CDH1) . These genes are not only hypermethylated in tumour cells, but show increased epigenetic silencing in normal epithelium surrounding the tumour site.
Unlike geneticalterations that are almost impossible to revert, DNA methylation is a reversible event. Reactivation of hypermethylatedtumour-suppressor genes can be considered a possible therapeutic approach for developing pharmacological inhibitors of DNA methylation. Moreover, the use of DNMT inhibitors serve as usefultools for cancer treatment, because the restoration of the expression of tumour-suppressor genes can potentially restore the protective effect of these genes on tumour divisions. The nucleoside analogues 5-azacytidine (vidaza or AZA,) and 5-aza-2’-deoxycytidine (decitabine or DAC) are two DNMT inhibitors that serve as effective hypomethylating agentsand inhibit cell proliferation. For more than 30 years, these two drugs have been the two most prominent DNMT inhibitors under preclinical and clinical investigation .Moreover, these agents are pro-drugs that need to be incorporated into DNA in order to act as inhibitors of DNMTs. The nucleoside analogues are first phosphorylated to the triphosphate nucleotide and incorporated into DNA during DNA synthesis. Under normal conditions,DNMT1 transfers the methyl group from SAM to the fifth carbon position of the cytosine ring. This facilitates the release of the enzyme from its covalent bond with cytosine. Afterformation of an irreversible complex with DNMT1the5'-aza-cytosine ring is replaced with cytosine in the DNA, the methyl transfer does not take place and DNMT1degradation occurs (Figure 3). The replication fork progresses in the absence of DNMT1 which prevent methylation of daughter DNA in CpG islands during DNA replication, resulting in the passive loss of DNA methylation [116, 117]. In addition, AZA (but not DAC) is converted into a ribonucleoside moiety and is incorporated into RNA, interfering with protein translation . At low concentrations (e.g., 30nM DAC, 300nM AZA), these inhibitors exhibit potent DNA hypomethylation properties, whereas high concentrations (≈3 to10 μM) are cytotoxic .
The doses of AZA and DAC that were employed in many of the early clinical trials involving solid tumours were cytotoxic, reflecting maximum tolerated doses, which likely accounts for excessive toxicity and possibly also for the lack of overall efficacyobserved in these studies . A previous study indicated that DNMT inhibitors were associated with response rates as high as 18% in breast cancer . The doses of AZA that were employed in these studies, however, were far higher than doses used in clinical trials today and likely exerted cytotoxic activity as opposed to the relief of transcriptional repression as an anti-cancer strategy .
Current clinical studies including the administration of DNMT inhibitors at the presumed optimal epigenetic dose aim to elucidate the biological effects of these agents and to assess their clinical efficacy, alone or in combination with other anti-cancer agents. The ability of single agent AZA to induce the expression of ER and PR genes in patients with triple-negative breast cancer, and who are awaiting definitive breast cancer surgery, is under investigation using a 75 mg/m2/day dosing schedule . Based on the preclinical evidence previously described, which suggests that a combination of epigenetic modifiers may be more successful in the re-expression of silenced genes and the restoration of hormonal therapy responsiveness, patients with advanced triple-negative and hormone-resistant breast cancer are being enrolled in an ongoingmulti-centre phase 2 clinical trial and receive a combination of low dose AZA (40 mg/m2) on days 1 to 5 and 8 to 10, and entinostat 7 mg on day three and 10 of a 28 day cycle. Tumour biopsies prior to and after therapy are collected for assessing the modulation of candidate gene methylation and expression, such as the ER gene. Patients may transition to an optional continuation phase at the time of disease progression, in which the same epigenetic therapy is administered with the addition of hormonal therapy [122, 123].
One such putative agent is zebularine, that has been reported to prevent early tumour development and also inhibits the growth of mammary gland tumours and breast cancer celllines [124,125].Zebularine isknown as a novel DNMT inhibitor and is more stable with less toxicity .Zebularine, similar to AZA-CR and 5-AZA-CdR, incorporates into DNA and forms a covalent irreversible complex with DNMT,preventing the enzyme from methylating position 5 of cytosines clustered in regulatory CpG islands . In recent studies, zebularine has shown to be able to sustain the demethylation state of the 5'-region of the tumour suppressor gene CDKN2A/p16, as well as other methylated genes in T24, HCT15, CFPAC-1, SW48 and HT-29 cells. It was also reported that zebularine inhibits the growth of cancer cell lines, but not normal cells.
Zebularine acts as a cytidine analogue containing a 2-(1H)-pyrimidinone ring, which had originally been developed as a cytidinedeaminase inhibitor to prevent deamination of nucleoside analogues[129,130].Zebularine is also anadaptable starting material for the synthesis of complex nucleosides and is a mechanism based on DNA cytosine methyltransferase inhibitors . It acts primarily as a trap for DNMT protein by forming tight covalent complexes between DNMT protein and zebularine-substituted DNA . In contrast to other DNMT inhibitors, ithas low toxicity in most tested cell lines and is quite stable with a half-life of 510 h at pH 7.4 [131,133,134]. Because Zebularine has low toxicity, continuous administration of effective doses of zebularine on its own or in combination with other DNMT inhibitors is feasible and may result in the enhanced re-expression of epigenetically silenced genes in cancer cells .
Zebularine treatment leads to increased p21 protein expression coupled with decreased cyclinB and D protein expression in MCF-7 cells,and an increased percentage of cells in the S-phase, indicating a zebularine induced S-phase arrest ; this suggests errors in the chromatin assembly that contribute to genome instability . S-phase arrest can also be triggered by the repression of histone synthesis in human cells .The genomic instability induced by DNMT1 downregulation and the repression of histone synthesis triggers the activation of S-phase check point proteins like p21 (in MCF-7 cells) and/or downregulates cyclin-D to permit DNA repair prior to entering the G2 phase.
A zebularine-mediated decrease in the expression of global acetylated histones has been observed. Several preclinical studies have evaluated zebularine as a possible therapeutic in cancer cell lines. Zebularine incorporates into DNA, leading to cell growth inhibition and the increased expression of cell cycle regulatory genes in cancer cell lines, compared to normal fibroblasts.Additionally,to determine the ability of zebularine to prevent or treat breast cancer, Min et al.(2012) tested if daily oral treatment with zebularine affected mammary tumour growth in these MMTV-PyMT mice. They observed a significant delay in tumour growth and a reduction of total tumour burden in the zebularine-treated mice. They reported the depletion of DNMTs in tumours excised from zebularine-treated mice and identified upregulation of 12 genes previously characterized as silenced by DNA hypermethylation.Zebularine treatment was shown to be associated with a dose-dependent depletion of DNMT1, DNMT3a and DNMT3b proteins in breast cancer cell lines MCF-7 and MDA-MB-231 . Zebularine also depleted DNMT1 in T24 bladder carcinoma cells after 24 hours of treatment and partially depleted DNMT3b after three days of drug exposure. Recently,Chen et al. (2012) proved via an in vivo study that DNMT1 had been depleted and DNMT3b was significantly lowered (50% depletion) in the mammary tumours derived from zebularine-treated mice, compared with untreated mice. In addition to the mechanism of tumour growth inhibition, tumour cells eventually develop resistance to zebularine treatment and both zebularine and the HDAC inhibitor have shown a synergistic effect on the inhibition of breast cancer growth; therefore,combinatorial treatment with DNMT inhibitors, as well as combinatorial treatment with DNMT inhibitors and HDAC inhibitors may be warranted to overcome resistance to single-drug therapy.
Moreover, in most doses tested, zebularinehave been reported to deplete the expression of all three DNMT proteins post-transcriptionally in both breast cancer cell lines. It has been also reported that human cancer cells lacking DNMT1 or DNMT3b retain significant global methylation and gene silencing, but those lacking both DNMT1 and DNMT3b had >95% reduction in genomic DNA methylation and virtually absent DNMT activity. The zebularine treatment specifically targets DNMT1 and reduced DNMT 3a and 3b protein expression, implying that treated cells may still retain substantial methylation.Another study observed similar results in T24 bladder cancer cells continuously treated with zebularine for 40 days. Zebularine in these cells had no effect on the expression of DNMT1, 3a or 3b mRNA, but complete loss of DNMT1 and partial depletion of DNMT 3a and 3b protein were observed .
Previous findings have observed that ER can be epigenetically silenced in some human breast cancer cell lines and HDAC or DNMT inhibitors can re-express functional ER in ER negative breast cancer cells [140,141].Furthermore, investigations have demonstrated that the treatment of ER negative MDA-MB-231 breast cancer cells with zebularine results in functional ER reactivation, as manifested by the expression of ER mRNA and its target gene, PR. This has been reported with a dose as low as 50μM, far lower than doses that induced apoptosis. Chromatin immunoprecipitation analysis of the ER promoter in zebularine-treated cells showed characteristics of an active chromatin, as manifested by the accumulation of acetylated H3 and H4 and the release of DNMT1, 3a and 3b from the ER promoter region. Even though re-expression of ER with zebularine was not as robust as with 5-azaDc, the low toxicity can enable continuous administration for the sustained re-expression of ER cells .
However, several studies have shown that zebularinehas potential limitations such as beingless potent than the two FDA-approved DNMT inhibitors, azaC and 5-azaDc .It is hypothesized that the reduced inhibitor potency is due to sequestration of the drug by cytidinedeaminase, competitive inhibition of zebularine incorporation into DNA by increased cytidine and deoxycytidine, which accumulate as a consequence of its cytidinedeaminase properties and preferential incorporation of zebularine into RNA over DNA . Its efficacy, combined with a low toxicity profile,nonetheless renders it an attractive agent for combination or sequential therapy with other DNMT or HDAC inhibitors .
8. Combination of DNMT inhibitors
Based on the preclinical evidence previously described, which suggests that a combination of epigenetic modifiers may be more successful in the re-expression of silenced genes and the restoration of hormonal therapy responsiveness.We have noted previously that patients with advanced triple-negative and hormone-resistant breast cancer are being enrolled in an ongoingmulti-centre phase 2 clinical trial and are receiving the combination of a low dose of AZA andentinostat. Tumour biopsies prior to and after therapy are collected to assess the modulation of candidate gene methylation and expression, such as ER. Patients may transition to an optional continuation phase at the time of disease progression, in which the same epigenetic therapy is administered with the addition of hormonal therapy . Indeed, in a recently published trial exploring the combination of AZA and entinostat in advanced non-small cell lung cancer patients, investigators observed that the regimen was well tolerated and associated with a number of objective responses . These included a complete response, as well as a partial response in a patient without progression of the disease for two years after completing the clinical trial. Interestingly, a number of patients were found to have unexpected major objective responses to subsequent anti-cancer strategies, raising the question as to whether these agents may prime tumour cells to respond to subsequent therapies. A phase 1/2 Canadian trial investigating the combination of decitabine and vorinostat in patients with advanced solid tumours or hematologic malignancies also indicated clinical activity. Stabilization of disease for four or more cycles was observed in 29% of evaluable patients; two of these patients had metastatic breast cancer .
Moreover, cytidinedeaminase destabilizes DNMT inhibitors,similarly to 5-azaDc, leading to complete loss of their antineoplastic ability . Hence, administration of cytidinedeaminase inhibitors like zebularine should theoretically potentiate the therapeutic effects of 5-azaDc by slowing its degradation and stabilizing its activity. Indeed, the combination of 5-aza-Dc and zebularine produced better inhibition in cell proliferation and clonogenicity than either drug on its own in leukemic L1210 and HL-60 cell lines . Similarly, treatment of the AML-193 acute myeloid leukemic cell line, which has a densely methylated p15INK4B CpG island with zebularine, followed by the HDAC inhibitor trichostatin-A, synergistically enhanced p15INK4B expression . Consistent with these results, the combination of 50μMzebularine and 1μM 5-azaDc in breast cancer cells inhibited cell proliferation significantly compared with either drug on its own. In similar results, zebularine significantly inhibited cell proliferation and colony formation when combined with low doses of vorinostat.Cheishviliet al. (2014) investigated the combination of methylated DNA binding protein 2 (MBD2) depletion and DNMT inhibitor 5-azaCdR in breast cancer cells.This results in a combined effect in vitro and in vivo, enhancing tumour growth arrest on one hand,andinhibiting invasiveness which is triggered by 5-azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppressed and augmentedspecific gene networks that are induced only by DNMT inhibition. These data point to a possible new approach for targeting the DNA methylation machinery through the combination of MBD2 and DNMTinhibitors.
The combination of DNMT inhibitors with standard chemotherapy has not been extensively evaluated in the breast cancer setting. Based on strong preclinical evidence that the addition of AZA can possibly overcome platinum resistance through DNA hypomethylation, patients with both platinum resistant and refractory ovarian cancer received a combination of AZA and carboplatin after being enrolled into a phase 1b/2 study. An overall response rate of 22% was observed in the platinum-resistant patients (disease progression within six months of platinum, n=18), suggesting that further evaluation of the combination is warranted . Whether combining DNMT inhibitors with standard therapies or novel agents will result in clinical benefits for patients with breast cancer remains to be seen. In the meantime, robust preclinical data should support the development of new concepts in order to maximize the chance of success with these agents in the solid tumour arena.
Future studies need to include a more detailed investigation of the methylation differences between breast cancer subtypes to determine whether there is a methylation signature that can identify breast cancer subtypes. Laboratory studies have shown that AZA and DAC optimally inhibit DNA methylation when used at lower than cytotoxic doses and with prolonged exposure. The exact impact of using epigenetic modifiers at an optimally epigenetic dose instead of a cytotoxic dose remains unknown in the context of solid tumours, despite the supposition that anti-cancer activity will be enhanced. Ongoing clinical trials in breast cancer patients aim to elucidate this question.Optimizing the use of clinically available epigenetic modifiers is clearly important. An oral form of AZA is currently in development, which may be much more convenient for patients than the intravenous and subcutaneous routes employed at this time. A number of new agents are also in development that may circumvent some of the limitations of the currently available drugs such as their in vivo deamination by cytidinedeaminase and their tendency to be subject to drug resistance.
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