Cancer Letters xxx (2012) xxx xxx. Contents lists available at SciVerse ScienceDirect. Cancer Letters

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1 Cancer Letters xxx (2012) xxx xxx Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: Mini-review Epigenetic inactivation of DNA repair in breast cancer Somaira Nowsheen a, Khaled Aziz b, Phuoc T. Tran b, Vassilis G. Gorgoulis c, Eddy S. Yang a, Alexandros G. Georgakilas d, a Department of Radiation Oncology, Hazelrig-Salter Radiation Oncology Center, University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL 35294, USA b Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21231, USA c Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece d Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA article info abstract Article history: Available online xxxx Keywords: Epigenetics Biomarkers Cancer DNA damage Breast cancer Personalized treatment Carcinogenesis The study of epigenetic mechanisms in cancer, such as DNA methylation and histone modifications, has revealed a plethora of events that contribute to cancer through stable changes in the expression of genes critical to transformation pathways. In this mini review we look at the different epigenetic modifications prevalent in this neoplastic phenotype, focusing on breast cancer. Most encouragingly, research in epigenetics has led to improved survival of patients with certain forms of lymphoma and leukemia through the use of drugs that alter DNA methylation and histone acetylation. Thus, we look at the clinical utility of targeting epigenetic pathways. In addition, we explore numerous other clinical applications of epigenetics, in areas such as cancer screening and early detection, prevention, classification for epidemiology and prognostic purposes, and predicting outcomes after standard therapy. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Corresponding author. Address: Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858, USA. Tel.: ; fax: address: georgakilasa@ecu.edu (A.G. Georgakilas). The human genome controls cell fate in a very systematic, precise way. Genomic DNA is under constant assault which often results in alterations potentially transforming a normal cell to a precancerous lesion. Accumulation of these changes or mutations over time eventually leads to cancer with uncontrolled cell growth. Both genotoxic and non-genotoxic mechanisms have been implicated in malignant transformation. Genotoxic mechanisms involve changes in genomic DNA sequences leading to mutations while non-genotoxic mechanisms modulate gene expression directly [1]. Mutations can either be acquired or inherited and are caused by both endogenous and exogenous agents. On the other hand, epigenetics are heritable changes in phenotype or gene expression caused by mechanisms other than changes in DNA sequence. Both of these changes result in a differential expression and/or function of genes, such as the changes seen when cells differentiate or become malignant. Dysregulated cell growth and apoptosis in cancer are due to genetic and epigenetic changes such as point mutations, deletions, duplications, insertions, translocations, chromosome aberrations, viral infections (e.g. human papilloma virus, Epstein Barr virus, and Hepatitis virus), and epigenetic inactivation such as promoter hypermethylation and histone deacetylation. These mechanisms may affect the DNA sequence but ultimately change the function and regulation of the gene products or lead to gain/loss of function of critical genes. Several models of carcinogenesis have been proposed. One of the models put forth by Dr. Bert Vogelstein proposes a stepwise accumulation of genetic events including the loss of function of tumor suppressors such as p53 and the gain of function of oncogenes [2]. Loss of p53 function is observed in the majority of cancers and results in genomic instability, metabolic changes, insensitivity to apoptotic signals, invasiveness and motility [3,4]. Initially, an inactivating mutation in tumor suppressor gene leads to hyper-proliferation of epithelial cells and/or inactivation of DNA repair genes. The same mutation may inactivate several more tumor suppressor genes before resulting in cancer. An alternate theory accounting for both hereditary and nonhereditary cancer is Dr. Alfred Knudson s two-hit theory of cancer causation [5]. According to his proposal, people with a hereditary susceptibility to cancer take their first hit at conception and inherit a damaged gene on one of the two chromosomes. Others inherit two normal chromosomes but receive the first hit post-conception. A subsequent damage to the same gene on the second chromosome may lead to cancer. Therefore, people with a hereditary susceptibility to cancer just need one hit during their lifetime to transform a normal cell to cancer. This model fits perfectly for cancer such as retinoblastoma where inheritance of the first hit leads /$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.

2 2 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx to a far greater chance of developing a second cancer causing mutation [6]. It should be noted that most of the dominant models of cancer development are genetic. The two models of carcinogenesis outlined here are not competing; rather both can be considered as genetic models of carcinogenesis. The classical definition of epigenetic changes by Conrad Waddington described epigenetics as the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being. Today, an epigenetic trait is thought to be a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Epigenetic mechanisms contributing to malignant transformation cause us to deviate from the proposed models. Epigenetic mechanisms include, among others, DNA cytosine modification patterns, DNA methylation patterns, histone post-translational modifications, or deposition of certain histone variants along specific gene sequences (Fig. 1). These modifications modulate gene expression. The epigenetic pathway is considered to be a non-genotoxic mechanism capable of modulating gene expression and thus promoting malignant transformation [1]. The following sections will discuss in detail various epigenetic changes and their impact on carcinogenesis. 2. Epigenetic modifications The molecular basis of epigenetics is complex. It involves modifications of the activation of certain genes, but not the basic structure of DNA [7,8]. The chromatin proteins associated with DNA may be activated or silenced. This accounts for why the differentiated cells in a multicellular organism express only the genes that are necessary for their function. More importantly, epigenetic changes are preserved when cells divide and are passed on from one generation to the next. Genome-wide profiling has elucidated the chromatin structure, including the localization of histone post-translational modifications and histone variants, DNA methylation patterns and nucleosome occupancy [9]. Although some proteins that regulate chromatin structure are well defined, exactly how the histonemodifying enzymes, histone modifications and modification recognizing proteins are localized and restricted to specific loci is not fully understood. Chromatin is dynamically regulated to impact different cellular processes, often via post-translational modifications on histones. Histone modifications include acetylation, methylation, phosphorylation, sumoylation, poly(adp)-ribosylation, and ubiquitination (Table 1) [10]. Modifications can alter the chromatin structure and/or aid in the recruitment of other proteins involved in diverse cellular processes including gene transcription, DNA replication and DNA repair [11]. Epigenetic modifications can act as docking modules for different factors. Multiple histone modifications may occur simultaneously as well, the combination of which may change the behavior of the nucleosome. Thus, the cumulative effect of several dynamic modifications regulates gene transcription in a systematic and reproducible way. Histone modifications are proposed to affect chromosome function via distinct mechanisms. Modifications may alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA. The alterations may also act as binding sites for protein recognition modules, such as the bromodomains or chromodomains, which recognize acetylated lysines or methylated lysines, respectively. For each post-translational modification of histones, enzymes exist which either lay down the appropriate mark or remove it. Major factors in this regulation are the histone acetyltransferases, which acetylate the histone tails and induce chromatin decondensation; histone deacetylases (HDACs), which remove the acetyl groups and promote a tighter binding of histones to DNA; histone methyltransferases (HMTs), which promote or inhibit transcription depending on the target histone residue; and histone demethylases (HDMs), which counteract the HMTs [12,13]. The histone-modifying enzymes affect Fig. 1. Model of epigenetic modifications according to current knowledge. The different types of chemical modifications such as methylation, acetylation, etc. of promoter region and/or other regulatory DNA sequences outside the gene can have a severe impact on gene transcription and translation and a resulted high modulation of gene expression and product (protein) functionality.

3 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx 3 Table 1 Epigenetic modifications of histones. Modification Function Acetylation Histone acetylation enhances transcription Histone deacetylation represses transcription Histone acetylation is catalyzed by histone acetyltransferases Histone deacetylation is catalyzed by HDACs Histone acetylation is also involved in DNA repair Methylation Silences tumor suppressor genes Silences transcription of genes involved in DNA repair, chromatin remodeling, cell cycle regulation, metastasis, apoptosis, etc Changes chromatin structure DNA methyltransferases maintains DNA methylation patterns Phosphorylation Phosphorylation of histones is critical in DNA damage response Activates/deactivates proteins Sumoylation Affects DNA damage response, apoptosis, protein protein interactions, protein DNA interactions, protein locations, stability, and activity Poly(ADP)- Allows protein protein interaction ribosylation Non-covalent PARylation of histone protein increases the access of transcription factors and DNA binding proteins to the associated DNA Controls transcription, DNA repair, apoptosis Ubiquitination Involved in, among others, antigen processing, apoptosis, biogenesis of organelles, metastasis, cell cycle and division, DNA transcription and repair, differentiation and development, immune response and inflammation histones either locally, through targeted recruitment by sequence specific transcription factors, or globally throughout the genome in an untargeted manner affecting virtually all nucleosomes [14,15]. This has been translated into the histone code hypothesis which proposes that distinct combinations of covalent post-translational modifications of histones influence chromatin structure and lead to varied transcriptional results [16,17]. The concept was further generalized to the idea that various combinations of histone modifications are related to specific chromatin-related functions and processes [18]. For example, several histone modifications have been associated with apoptosis-induced chromatin changes. The histone N-terminal tails are crucial in the maintenance of chromatin stability and, as expected, are subject to numerous modifications. Most modifications play a role in transcriptional regulation and, thus, have the potential to be oncogenic if deregulated Methylation DNA methylation is a covalent chemical modification, resulting in the addition of a methyl (CH 3 ) group at the carbon 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. Even though most cytosine methylation occurs in the sequence context 5 0 CG3 0 (also called the CpG dinucleotide), some involves CpA and CpT dinucleotides. DNA is methylated by a group of enzymes known as the DNA methyltransferases (DNMT) [19]. The DNMTs known to date are DNMT1, DNMT1b, DNMT1o, DNMT1p, DNMT2, DNMT3A, DNMT3b with its isoforms, and DNMT3L [19]. Methylation can be de novo (when CpG dinucleotides on both DNA strands are unmethylated) or maintenance (when CpG dinucleotides on one strand are methylated). DNMT1 has de novo as well as maintenance methyltransferase activity, and DNMT3A and DNMT3b are powerful de novo methyltransferases since defects in these genes in mice have been shown to result in death either early in development or immediately after birth [20]. DNA methylation patterns are established early in embryogenesis and are delicately monitored during development. Other enzymes involved in methylation changes include demethylases such as 5-methylcytosine glycosylase and MBD2b. 5-methylcytosine glycosylase removes the methylated cytosine from DNA while MBD2b hydrolyzes 5-methylcytosine to cytosine and methanol [20]. One of the most common and stable mechanism of epigenetic gene inactivation is the methylation of the 5-carbon of the DNA base cytosine in the 5 0 -CpG-3 0 dinucleotide sequence context of CpG island or promoter regions which are executed by DNA cytosine methyltransferases [21]. In tumor tissues, tumor suppressor genes are often inactivated epigenetically by methylation when compared with normal tissue. These DNA methylation events are often preceded by changes in chromatin structure and histone modifications. Sequences that have undergone DNA methylation often harbor repressive histone modifications such as H3K9 trimethylation. Aberrant changes in DNA methylation such as global hypomethylation and CpG island hypermethylation were among the first events to be recognized in cancer. A link between DNA methylation and cancer was established when researchers demonstrated that the genomes of cancer cells are hypomethylated when compared to normal cells [22]. Global demethylation in the repetitive regions of the genome early during tumorigenesis predisposes cells to genomic instability [23]. Aberrant hypermethylation in cancer usually occurs at CpG islands which results in changes in chromatin structure and silencing of transcription [24]. Cancer cells frequently acquire aberrant methylation of multiple tumor-related genes that together confer a survival advantage [24]. Genes involved in cell-cycle regulation, tumor cell invasion, DNA repair, chromatin remodeling, cell signaling, transcription and apoptosis are aberrantly hypermethylated and silenced in tumor [7,9,11,15,17,18,23 26]. DNA methyltransferases are responsible for establishing and maintenance of DNA methylation patterns which results in stable long-term gene repression. However, there are significant crosstalk between DNA methylation and histone modification pathways that is mediated by interactions between HMTs and DNA methyltransferases [11]. Promoter CpG-island hypermethylation in cancer cells is associated to a particular pattern of histone marks. These include deacetylation of histones H3 and H4, loss of histone H3 lysine trimethylation, and gain of H3K9 methylation and H3K27 trimethylation [11]. Thus, epigenetic cross-talk and the interplay between DNA methylation and histone modification are involved in the process of gene transcription and aberrant gene silencing in tumors. All lysine methyltransferases that target histone N-terminal tails contain a SET domain. This domain possesses lysine methyltransferase activity and numerous SET domain-containing proteins are implicated in cancer. For example, mice devoid of the Suv39 family of enzymes that catalyze methylation of Histone H3 lysine 9 methylation (H3K9) are susceptible to cancer, especially B cell lymphomas [27]. Thus, H3K9 methylation has been proposed to provide a major switch for the functional organization of chromosomal subdomains.

4 4 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx Suv39H histone methyltransferases regulate H3K9 methylation at pericentric heterochromatin and induce a specialized histone methylation pattern that differs from the broad H3K9 methylation present at other chromosomal regions. Suv39H-deficient mice display severely impaired viability and chromosomal instabilities that are associated with an increased tumor risk and perturbed chromosome interactions during male meiosis. This points at a crucial role for pericentric H3-K9 methylation in protecting genome stability, and define the Suv39H HMTases as important epigenetic regulators for mammalian development [27]. DNA is more frequently hypomethylated than hypermethylated in cancer. Active transcription, active demethylation, replication timing, and local chromatin structure preventing access to the DNA methyltransferase may be to blame. A number of genes have been found to undergo hypermethylation in cancer: genes involved in cell cycle regulation (p16ink4a, p15ink4a, Rb, p14arf), DNA repair genes (BRCA1, MGMT), apoptotic genes (DAPK, TMS1), genes associated with drug resistance, detoxification, differentiation, angiogenesis, and metastasis [20]. Similarly, hypomethylation causes activation of oncogenes such as c-myc and H-RAS leading to chromosomal instability. O6-methylguanine DNA methyltransferase (MGMT) DNA-repair gene encodes a DNA-repair protein that removes alkyl groups from the O6 position of guanine, an important site of DNA alkylation. After removal it is marked for degradation by ubiquitination [28,29]. Interestingly, promoter hypermethylation of MGMT, which inactivates the MGMT gene transcript, has been shown in certain cancers, including glioblastoma, colon, head and neck squamous cell carcinoma, and lung carcinomas [28,29]. This epigenetic silencing of the MGMT gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma undergoing treatment with alkylating agents. Patients with glioblastoma containing a methylated MGMT promoter benefited from temozolomide, whereas those who did not have a methylated MGMT promoter did not have such a benefit [30,31]. Moreover, high levels of MGMT activity in cancer cells create a resistant phenotype by blunting the therapeutic effect of alkylating agents. Given these observations, MGMT promoter hypermethylation and its subsequent function is not only a predictor of response to therapy, but can also help clinicians decide to pursue more aggressive therapies for patients who have functional MGMT [28]. A number of factors can influence the DNA methylation levels of a cell without requiring a change in genomic DNA sequence. Aging causes DNA to become hypomethylated whereas certain CpG islands become hypermethylated [32]. Emerging research suggest that this age-dependent change in DNA methylation is linked to the increased cancer incidence in later life [33]. Diet is one of the more easily studied and therefore better understood environmental factors in epigenetic change. The nutrients enter metabolic pathways and are modified to usable forms. For example, the folate and methionine pathways are responsible for making methyl groups which are important epigenetic tags that silence genes. Since we cannot synthesize folate or methionine, a diet low in these molecules leads to alterations in DNA methylation and have been linked to cancer [34]. Nutrients like folic acid, B vitamins and S-Adenosyl methionine are key components of this methyl-generating pathway. Thus, diets high in these methyl-donating nutrients can rapidly alter gene expression. This is especially important during early development when the epigenome is first being established. Besides, agents present in the environment such as arsenic, nickel, chromium and cadmium can have substantial effects on DNA epigenetics [35]. Arsenic causes hypomethylation of the RAS gene whereas cadmium induces global hypomethylation by inactivating DNMT1 [36]. Furthermore, arsenic exposure has been shown to alter methylation levels of both global DNA and gene promoters including histone acetylation, methylation, and phosphorylation [36] Acetylation Besides changes in DNA methylation patterns, the chromatin or molecules organizing the genome have also been shown to regulate transcriptional activity. The addition of the COCH 3 functional group or acetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA [17]. This makes easier for RNA polymerase and transcription factors to access the promoter region. Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription. Acetyl groups are commonly transferred from acetyl-coa to coenzyme A which is an intermediate in the biological synthesis as well as in the breakdown of many organic molecules. Changes in histone acetylation and/or deacetylation are observed in a variety of cancers including breast, prostate, colon, testicular, renal and pancreatic cancer. Histone acetylation is catalyzed by histone acetyltransferases and histone deacetylation is catalyzed by HDACs. Several different forms of these enzymes have been identified. Among them, CBP/p300 is probably the most important, since it can interact with numerous transcription regulators including p53 [37]. PTEN, an important phosphatase involved in cell signaling via the AKT/PI3 kinase pathway, is subject to complex regulatory control via phosphorylation, ubiquitination, oxidation and acetylation. Acetylation of PTEN by the histone acetyltransferase p300/cbp-associated factor (PCAF) can repress its activity. On the other hand, deacetylation of PTEN by SIRT1 deacetylase and, by HDAC1, can stimulate its activity [38,39]. APE1/Ref-1 (APEX1) is a multifunctional protein possessing DNA repair activity on abasic and single-strand break sites as well as transcriptional regulatory activity associated with oxidative stress. APE1/Ref-1 is acetylated by PCAF; on the converse, it is stably associated with and deacetylated by Class I HDACs. The acetylation state of APE1/Ref-1 regulates its transcriptional activity [40]. Another example of a gene product that is acetylated is NFjB, a key transcription factor and effector molecule involved in responses to cell stress. It consists of a p50/p65 heterodimer. The p65 subunit is controlled by acetylation via PCAF and by deacetylation via HDAC3 and HDAC6 [41]. Acetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA. This makes RNA polymerase and transcription factors easier to access the promoter region. Thus, histone acetylation tends to open up chromatin structure. Accordingly, histone acetyltransferases (HATs) tend to be transcriptional activators whereas HDACs tend to be repressors. Many HAT genes are altered in some way in a variety of cancers. For instance, the p300 HAT gene is mutated in a number of gastrointestinal tumors. On the other hand, alteration of HDAC genes in cancer seems to be far less common. However, despite this low incidence of genetic mutation in cancer, HDAC inhibitors are performing well in the clinic as anti-cancer drugs. This is discussed in further detail in a subsequent section. Histone H2AZ (H2AZ) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression [42]. H2AZ is present at nucleosomes adjacent to the transcription start sites of both active and poised gene promoters. While H2AZ is ubiquitous across the entire promoter region of inactive genes in a deacetylated state, acetylated H2AZ is only localized at the transcription start sites of active genes. Interestingly in cancer cells, a gain of acetylated H2AZ at the transcription start sites occur with an overall decrease of H2AZ levels and results in oncogene activation. Furthermore, deacetylation of H2AZ at these sites is increased and results in silencing of tumor suppressor genes. This suggests that

5 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx 5 acetylation of H2AZ is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis [43]. As discussed above, DNA methylation can influence chromatin structure. Methylation is associated with repression of transcriptional initiation and is frequently found in transposable elements. Interestingly, DNA methylation can effect gene silencing by excluding H2AZ. This suggests that H2AZ protects genes from DNA methylation [42]. Histone acetylation is also involved in DNA repair. The acetyltransferase Tip60, a tumor suppressor, regulates transcription and is one of the proteins involved in the DNA damage response [44]. Moreover, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as MYC and p53 [45]. Tip60 is vital for both p53-mediated cell cycle arrest and apoptosis [45]. Tip60 also induces p53 acetylation at lysine 120 (K120) within the DNA-binding domain which is crucial for p53- dependent apoptosis. Interestingly, this acetylation is dispensable for p53-mediated cell cycle arrest [45]. Thus, Tip60-dependent acetylation of p53 at K120 modulates the decision between cell-cycle arrest and apoptosis. The human Tip60 locus is frequently mutated in breast cancer, head and neck squamous cell carcinoma and lymphomas. In response to DNA damage Tip60 also acetylates H2AX, a histone involved in repair of double strand breaks [46]. Acetylation of H2AX leads to its subsequent ubiquitylation and remodeling of chromatin near the break, facilitating DNA repair [46]. Tip60 is involved in the activation of the DNA damage repair pathway through the acetylation and activation of the ATM kinase [47]. Hyper-acetylation of histones leads to the activation of ATM. In addition to the specific role of acetylation at lysine 56 of histone H3 to facilitate chromatin assembly and serve as a marker for newly repaired chromatin, global acetylation levels must be kept at an intermediate, highly regulated level in order to maintain proper genome structure [48]. Besides its role in DNA replication and chromatin assembly, histone acetylation also plays a role in the timing of DNA replication and replication origin activity. In general, increases in histone acetylation of chromatin surrounding an origin of replication tend to cause the origin to initiate replication earlier, compared to when the origin is within hypoacetylated chromatin [49]. Thus, maintenance of a balance between acetylation and deacetylation is critical for proper DNA repair and cell survival Ubiquitination Ubiquitin is a small 8.5 kda peptide that can either target the conjugated protein to proteasomal degradation or serve as a modifier for protein function. Ubiquitination is an enzymatic, protein post-translational modification in which the carboxylic acid of the terminal glycine from the di-glycine motif in the activated ubiquitin forms an amide bond to the epsilon amine of the lysine in the modified protein. Modification of proteins by ubiquitin usually occurs via a three-step enzymatic reaction, involving ubiquitin-activating, -conjugating and -ligating (respectively E1, E2 and E3) enzymes that conjugate either one (monoubiquitination) or multiple (polyubiquitination) ubiquitin moieties on target polypeptides. The ubiquitination system functions in a wide variety of cellular processes, including antigen processing, apoptosis, biogenesis of organelles, cell cycle and division, DNA transcription and repair, differentiation and development, immune response and inflammation, neural and muscular degeneration, morphogenesis of neural networks, modulation of cell surface receptors, ion channels and the secretory pathway, response to stress and extracellular modulators, ribosome biogenesis, and viral infection [50]. Examples of ubiquitinylation include Lys119 of histone H2A and Lys120 of histone H2B [51]. These modifications regulate transcription and DNA repair [52,53]. Ubiquitinylation of H2A inhibits Lys4 H3 methylation and is strictly a repressive mark. Ubiquitinylation and deubiquitinylation of H2A and H2B occurs through the action of distinct enzymes, but in both cases it is common that the ubiquitinylation and deubiquitinylation machinery is associated with complexes having histone acetyltransferase, histone methylase and RNA polymerase activities [51]. These associations suggest that the histone ubiquitinylation is connected to a wide range of epigenetic and transcriptional mechanisms. The largest post-translational modification of histones is by conjugation of ubiquitin or ubiquitin-like moieties to lysine residues. Among others, H2A and H2B can be targeted for ubiquitination. H2A K119 ubiquitination is mediated by Bmi-1, an oncogene, and RING which acts as an E3 ubiquitin ligase. This is critical for gene silencing. While Bmi-1 is required for normal cell proliferation, increased Bmi-1 mediated H2AK119 ubiquitination has been linked to metastasis. During transcription, ubiquitination of H2A is generally associated with gene silencing whereas ubiquitination of H2B has been related to both gene activation and silencing [51,54,55]. UV irradiation not only causes an increase of H2A ubiquitination, but also induces a temporary H3 and H4 ubiquitination. This ubiquitination of H3 and H4 occurs early in the DNA damage response. Ubiquitinated H3 and H4 reduce nucleosomal stability and leads to a chromatin environment that facilitates the assembly of the nucleotide excision repair (NER) complex on damaged DNA [56]. DNA damage induced ubiquitination of H2A and H2AX is dependent on the ubiquitin-conjugating (E2) enzyme Ubc13 and the E3-ligase RING finger-containing protein RNF8 [52,53]. The ubiquitination depends on H2AX phosphorylation and the subsequent recruitment and activation of MDC1, to which RNF8 binds. Ubiquitinated targets (H2A and H2AX) are crucial for the formation of IRIF and assembly of downstream repair and checkpoint factors (RAP80, BRCA1 and 53BP1). The dynamic equilibrium of differential H2A ubiquitination are critical for genome stability as a mutant form of the H2A-specific deubiquitination enzyme USP3 causes delay of S phase progression and activation of checkpoints [57] Phosphorylation Phosphorylation is the addition of a phosphate (PO 3 4 )- group to a protein or other organic molecule. Phosphorylation events can activate or deactivate many genes and thus, reversible phosphorylation of proteins is an important regulatory mechanism. Kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in this process. Reversible phosphorylation results in a conformational change in the structure in enzymes and receptors, causing them to become activated or deactivated. Phosphorylation usually occurs on serine, threonine, tyrosine and histidine residues. The addition of a phosphate group to a polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of molecule. In this way it can introduce a conformational change in the structure of the protein via interaction with other hydrophobic and hydrophilic residues in the protein. This may facilitate the binding of other protein complexes and initiate a signaling cascade. Phosphorylation events can affect subcellular localization, protein interactions within complexes, enzymatic activity and chromatin association of proteins. For example, the tumor suppressor p53 is regulated by phosphorylation. Phosphorylation is also another important and long-appreciated histone modification that is often associated with chromosome/ chromatin condensation that includes mitosis, meiosis, apoptosis, and DNA damage. These processes are regulated by different histone kinases and are critical for gene regulation. Thus, defects in histone kinases can severely impact genomic stability and cell proliferation. H3S10 and H3S28 are phosphorylated at mitosis. JIL-1 mediated histone H3S10 phosphorylation induces a change in higher-order chromatin structure from a condensed heterochromatin-like state

6 6 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx to a more open euchromatic state [58]. Dysregulated phosphorylation has been associated with cancers of the breast and prostate. Moreover, Aurora kinases that perform this H3 phosphorylation have been implicated in cancer. Aurora kinases are important regulators of mitotic entry, centrosome function, mitotic spindle formation, chromosome segregation and cytokinesis, and their dysregulation leads to aneuploidy and tumorigenesis [59]. Aberrant expression of heterochromatin protein 1 (HP1) that detects H3K9ME3, can lead to aneuploidy. Aurora-kinase-B mediated phosphorylation of H3S10 might act as a switch resulting in dissociation of HP1 from heterochromatin while maintaining H3K9 methylation during mitosis. H3S10 phosphorylation is crucial for proper chromosome condensation and segregation during mitosis; overexpression of a non-phosphorylatable mutant of H3S10 results in retention of HP1 and defects in chromosome segregation [60]. Thus, dynamic phosphorylation of H3S10 by aurora kinase B during mitosis plays an integral part in maintaining chromosome stability. An early event following DNA damage is the phosphorylation of H2AX, a process that is required for efficient DNA repair. Upon introduction of a DNA double strand break, hundreds of H2AX molecules become phosphorylated within minutes in the chromatin flanking the break site, thus providing a rapid and highly amplified detection system and a focus for the accumulation of many other proteins involved in DNA repair and chromatin remodeling. This c-h2ax-containing region forms a focal point for the accumulation of DNA repair and chromatin remodeling factors. This local protein accumulation may facilitate the accurate repair of damaged DNA. We refer the readers to an excellent review by Bonner et al. on the role of H2AX in cancer [61] mirna mirnas are 22 nucleotides-long RNA molecules encoded in the genome that are transcribed by RNA polymerase II and regulates gene expression [62]. Thus they are involved in cellular differentiation, proliferation and apoptosis. mirnas bind to complementary mrnas and alter their stability by promoting their degradation or suppress their translation. Not surprisingly, mirnas can act as oncogenes or tumor suppressors and have been linked to cancer. For example, the proto-oncogene c-myc encodes a transcription factor that regulates cell proliferation, growth and apoptosis. c- MYC activates expression of a mirnas and expression of E2F1 is negatively regulated by two mirnas in this cluster, mir-17-5p and mir-20a [63]. mir-15a and mir-16-1 which target the antiapoptotic BCL2, are often down-regulated in chronic lymphocytic leukemia [64]. mir-15a and mir-16-1 expression is inversely correlated to BCL2 expression and both micrornas negatively regulate BCL2 at a posttranscriptional level [64]. The anti-apoptotic mir-21 is upregulated in glioblastomas and breast cancers [65,66]. Small interfering RNAs (sirnas), often considered to be closely related to mirnas, have been shown to be involved in both DNA methylation and histone modifications. For instance, DNA methylation enzymes DNMT1, 3a, and 3b are targets of mirnas [67]. Moreover, mirnas may regulate chromatin structure by regulating key histone modifiers [68]. Thus, mirnas can be considered important players in the epigenetic control of gene expression. Since epigenetic regulation of gene expression plays a critical role in the regulation of a variety of cellular processes including gene expression, DNA replication, DNA repair, stem cell maintenance and differentiation, and given the causal role of epigenetics in a variety of human diseases including carcinogenesis and aging, further knowledge in this interesting field will aid us in improving cancer care. We next discuss the potential application of epigenetics for diagnosis, prognosis, and treatment of breast cancer. 3. Epigenetic modifications in breast cancer Despite our efforts breast cancer continues to afflict thousands of women every year. The high histological and molecular heterogeneity of the disease poses a significant threat to therapy. There are several subtypes of breast cancers which display distinct clinical behaviors and responses to therapy: luminal or basal-like cancers, estrogen receptor (ER) and/or progesterone receptor (PR) and/ or the human epidermal growth factor receptor 2 (HER2) positive or negative breast cancer. Recently an additional classification described as a claudin-low phenotype has been described which correlate to poor prognosis in the clinic. The gene expression profiles of these stem cell derived cancer cells have low expression of luminal markers and high expression of mesenchymal markers [69 74]. Assessment of the epigenetic aspects of breast tumors could strongly improve our understanding of the biology and heterogeneity of breast cancers. Table 2 lists the major epigenetic changes in breast cancer. In an effort to repair damaged DNA and avoid passing the damaged DNA onto the progeny cells, the cell has evolved several repair pathways. These repair pathways include base excision repair (BER), NER, double strand break repair via homologous recombination (HR) or non-homologous end joining (NHEJ), and mismatch repair (MMR). The BRCA family of proteins is essential for HRmediated repair of DNA double strand breaks [75 78]. The tumor suppressor, BRCA1, is a nuclear cytoplasmic shuttling protein. It is not only critical for DNA damage repair but also plays a critical role in the induction of apoptosis. The function of BRCA1 is regulated by a variety of mechanisms including transcriptional control, phosphorylation, and protein protein interactions [79]. In addition to the critical DNA repair proteins such as ATM-, ATR-, and Chk2- dependent phosphorylation, cytoplasmic relocalization of BRCA1 protein is a mechanism whereby BRCA1 function is regulated in response to DNA damage [75 78]. Since as little as one unrepaired DNA double strand break is fatal to the cell, mutations in the BRCA gene lead to an increased risk for breast cancer as part of a hereditary breast-ovarian cancer syndrome [80,81]. Women with mutated BRCA1 or BRCA2 gene have up to a 60% risk of developing breast cancer [82,83]. Moreover, 55% increased risk of developing ovarian cancer is observed with BRCA1 mutations and about 25% for women with BRCA2 mutations [82]. DNA hypermethylation indicates which genes are turned off in breast tumors and a unique pattern is observed in breast tumors. Interestingly, 40% loss of methylated cytosine is observed in breast tumors. Genes important in familial breast cancer are also epigenetically silenced. In sporadic tumors, BRCA1 expression has been shown to be suppressed by a combination of gene deletion and epigenetic silencing via DNA hypermethylation. As mentioned above, one of the models of carcinogenesis is Dr. Alfred Knudson s two-hit theory of cancer causation. In familial BRCA1 tumors, hypermethylation occasionally serves as an alternate mechanism for the second hit. New epigenetic drugs targeting DNA methylation and histone deacetylation are in development for the treatment of breast cancer. Emerging research suggests hypermethylation of the BRCA1 promoter may be an inactivating mechanism for BRCA1 expression not only in breast and ovarian cancer but also lung and oral cancer [84,85]. Many women with breast cancer end up being over-treated or under-treated due to the lack of reliable biomarkers and thus, epigenetic modifications are a promising biomarker for this disease. Over the last decade, candidate gene approaches have highlighted several genes whose methylation in breast tumors varies according to the tumor s clinicopathological characteristics such

7 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx 7 Table 2 Epigenetic modifications in breast cancer. Epigenetic modification Function Genes Acetylation Deacetylation Metastasis Estrogen receptor (ER) Methylation Hypermethylation DNA repair BRCA1 O-6-methylguanine-DNA methyltransferase (MGMT) MutL homolog 1 (MLH1) RAD9 Metastasis Cadherin 13 (CDH13) Cystatin E/M (CST6) Spleen tyrosine kinase (SYK) Cell Cycle regulation Adenylate kinase 5 (AK5) Cyclin D2 (CCND2) Cyclin-dependent kinase inhibitors (CDKN1C and CDKN2A) ER FOX2A P16 INK4 Progesterone receptor (PR) Retinoic acid receptor (RAR) Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A) Runt-related transcription factor 3 (RUNX3) Stratifin (SFN) Secreted frizzled-related protein 1 (SFRP1) WNT inhibitory factor 1 (WIF1) Werner syndrome, RecQ helicase-like (WRN) Wilms tumor 1 (WT1) Apoptosis Adenomatous polyposis coli (APC) B-cell CLL/lymphoma 2 (BCL2) Death-associated protein kinase (DAPK) Deleted in colorectal carcinoma (DCC) Hypermethylated in cancer 1 (HIC1) Homeobox A5 (HOXA5) TMS1 TWIST Hypomethylation Metastasis Breast cancer-specific gene 1 protein (BCSG1) Caveolin 1 (CAV1) Cadherin (CDH1, CDH3) N-acetyltransferase 1 (NAT1) Plasminogen activator, urokinase (UPA) as the hormonal receptor status. For example, ER positivity has been linked to methylated RASSF1A, CCND2, GSTP1 and TWIST genes. On the other hand, methylated PGR, TFF1 and CDH13 are predominant in ER-negative tumors. Thus, distinct breast cancer epigenotypes may be present and DNA methylation may influence the tumor receptor status. Interestingly, Vesuna et al. recently reported that Twist1, a gene over-expressed in high-grade breast cancers, recruits HDAC1 to ER promoter leading to deacetylation and lower ER expression. Twist1 was also shown to recruit DNMT3B to the ER promoter leading to hypermethylation and reduced expression of ER. Reduced ER expression correlate with poor clinical prognosis and failure of hormonal therapy. Interestingly, ER expression was partially restored by using 5-azacytidine (demethylating agent) and valproic acid (HDAC inhibitor) in this study [86]. Other known cancer genes are also hypermethylated in breast cancers, such as those that affect cell cycle (p16 INK4a), and hormone receptors (ER, PR). New epigenomic approaches revealed the novel importance of potential tumor suppressor genes, such as the prolactin receptor and WRN, the Werner syndrome gene associated with premature aging and increased cancer risk, which is silenced in a subset of breast cancer. In addition to direct DNA hypermethylation, modification of histones is another epigenetic mechanism with implications in breast cancer. Overall, there is global loss of monomethylation and trimethylation of histone H4 in cancer. Recent epigenomic studies have revealed the existence of distinct breast cancer epigenotypes: alterations linked to tumor progression and the tumor s cell type of origin. This highlights the contribution of DNA methylation aberrations to breast cancer heterogeneity. Importantly, methylation signatures seem able to stratify breast cancer patients in terms of prognosis and will aid in better understanding of breast cancer biology. Genes that are critical for early embryonic development and subsequently silenced are sometimes re-expressed in cancer cells. The plasticity of epithelial cells is an important factor in breast cancer metastasis. The process of epithelial to mesenchymal transition (EMT) has been identified as a major player in the process of metastatic dissemination. Beginning with morphogenetic changes and concurrent loss of epithelial markers, cancer cells gain a mesenchymal phenotype. They can dissociate and invade the immediate extracellular matrix, intravasate in to vasculature, resist the susceptibility to undergo anoikis in the blood stream, migrate to a new location, extravasate from the blood stream (diapedesis) and colonize in hospitable soil. From an epigenetic standpoint, it has been shown that acetylation of histones H2A/H2B can prevent epithelial cells from undergoing epithelial to EMT [87]. Therefore it may be possible to independently target EMT in cells having a stemness phenotype. Interestingly, the EMT gene signature and loss of H2BK5Ac marker has been correlated with the clinically challenging claudin low phenotype discussed above [88,89], therefore suggesting acetylation of H2B to be crucial for maintenance of an epithelial state. 4. Role of epigenetics in cancer therapy The field of epigenetics and epigenetic molecular marker development has gained a lot of recent attention because of the ability to contribute to both cancer diagnosis and prognosis due to their high sensitivity and specificity. Furthermore, a number of epigenetic modifications have been detected in critical genes involved in

8 8 S. Nowsheen et al. / Cancer Letters xxx (2012) xxx xxx Table 3 Recent clinical trials targeting epigenetic changes for cancer therapy. Trial Cancer Details Adjuvant combined epigenetic therapy with 5- azacitidine and entinostat Treatment with hydralazine and magnesium valproate to overcome chemo resistance Epigenetic priming using azacitidine with neoadjuvant chemotherapy Treatment with hydralazine and valproate Re-expression of silenced estrogen receptor in triple negative breast cancer Chemoprevention of prostate Cancer, HDAC Inhibition and DNA methylation Oral HDAC inhibitor 4SC-202 in patients with advanced hematologic malignancies Lung This phase I/II trial combined epigenetic therapy with azacitidine and entinostat, inhibitors of DNA methylation and histone deacetylation, respectively, in extensively pretreated patients with recurrent metastatic non small cell lung cancer [91] Solid tumors, Valporate is a histone deacetylate inhibitor while hydralazine inhibits DNA methylation [92,93] breast Esophageal This trial evaluates the dose limiting toxicity of the DNA methyltransferase inhibitor [94] Ovarian and cervical Breast Prostate Leukemia, lymphoma, myeloma Valporate is a histone deacetylate inhibitor while hydralazine inhibits DNA methylation [92,93] Re-express epigenetically silenced ER using demethylating inhibitors (such as decitabine) and histone deacetylase inhibitors (such as LBH589) so that they respond to anti-er therapy such as Tamoxifen [95] The objective of the study is to identify mechanisms by which compounds found in cruciferous vegetables alter gene expression via epigenetic modifications. The role of sulforaphane that inhibits HDAC activity will be evaluated [96] Assessment of potential anti-cancer activity of 4SC-202 [97] various cancers that can potentially serve as clinical biomarkers. For example, epigenetic modifications such as promoter DNA hypomethylation has been observed for a number of genes including RAS in prostate and thyroid cancers and cancer testis antigen gene in prostate, laryngeal, lung and breast cancers [26]. On the other hand, promoter DNA hypermethylation modification has been associated with altered expression of critical genes associated with various cancers including BRCA1/BRCA2 in prostate, breast, pancreatic and ovarian cancers, Von Hippel Lindau tumor suppressor and p53 in breast cancer [26]. Since a critical role of epigenetic alterations in tumorigenesis has been established, epigenetic regulatory enzymes are important targets for cancer therapy. Recently, several drugs with an epigenetic activity have received approval for the treatment of cancer patients but are limited in their epigenetic specificity (Table 3). For instance, though histone deacetylase inhibitors are highly specific drugs, the enzymes have broad substrate specificity and deacetylate numerous proteins that are not associated with epigenetic regulation. Similarly, the induction of global DNA demethylation by nonspecific inhibition of DNA methyltransferases demonstrates pleiotropic effects on epigenetic regulation with no apparent tumor-specificity. So far, a histone deacetylase inhibitor and two DNA methyltransferase inhibitors (azanucleoside drugs) have been approved by the United States Food and Drug Administration to treat T cell cutaneous lymphoma and myelodysplastic syndrome, respectively. Additional drug candidates that inhibit histone deacetylases and DNA methyltransferases are in development as are histone methyltransferase inhibitors and DNA methylation inhibitors that do not require incorporation into DNA, like the azanucleoside drugs. The utility of combination therapies and development of more specific, targeted therapies remains areas of active research and interest. Table 3 lists some of the recent clinical trials targeting epigenetic changes for cancer therapy. Methylation of the promoter region of four genes (p16, CDH13, RASSF1A and APC) in patients with stage NSCLC was studied in 51 case samples in a retrospective study by Brock et al. Interestingly, methylation of both p16 and CDH13 in tumor and mediastinal lymph nodes was associated with a 5-year recurrence-free survival rate of 14.3% compared to 63.1% in the absence of methylation of these genes [90]. Analysis of the methylation status of these gene promoters may strengthen the ability to assess the risk of recurrence in these patients. Clinical inhibitors of DNA methylation are nucleoside analogs that are converted into deoxynucleotide triphosphates and become incorporated into DNA in place of cytosine during DNA replication. These inhibit DNA methyltransferases and target them for degradation. DNA methylation inhibitors do not affect cellular proliferation at low doses. These drugs, however, reactivate gene expression and have shown clinical activity as anti-cancer agents. Azacitidine or Vidaza was the first hypomethylating agent approved by the FDA for the treatment of myelodysplastic disorders and leukemia. Decitabine or Dacogen is also FDA approved. Over multiple cycles of therapy, both drugs have demonstrated clinical improvements in more than 30% of patients treated with few side effects. Demethylation, gene reactivation, and clonal elimination were observed in treated patients proving that epigenetic therapy may be a viable cancer treatment. Histone deacetylase inhibitors prevent histone deacetylation, thereby facilitating an open chromatin structure and leading to the activation of genes such as p21 which is involved in cell cycle regulation. A couple of these inhibitors have been approved by the FDA for treatment of cutaneous T cell lymphoma: vorinostat and romidepsin. Thus far, the FDA-approved epigenetic drugs have shown the greatest efficacy in hematopoietic malignancies and further research is warranted in deciphering why solid tumors fail to respond to this treatment strategy. In addition, because cancers are frequently associated with hypermethylated tumor suppressor genes and because tumor-derived DNA is present in various, easily accessible body fluids, methylated DNA has been proposed as a biomarker for detecting some cancers. Research into identification of predictors or markers of clinical responses has been actively pursued in the clinic. A recent study of elderly patients with AML who were treated with decitabine showed that higher pretreatment levels of microrna-29b (mir-29b), previously shown to target DNMTs, correlated with clinical responses; thus, if these results are validated, mir-29b levels could be used for stratification of patients. Unfortunately, second-generation azanucleoside drugs have integrated the knowledge about the cellular uptake and metabolic pathways, but do not show any increased specificity for cancer epigenotypes. Thus, the traditional rationale of epigenetic cancer therapy needs further analysis as we progress from the global inhibition of epigenetic modifications toward the identification and targeting of tumor-specific epigenetics. Epigenetic mechanisms that promote self-renewal and developmental plasticity in cancer cells warrant further investigation but bears promise as druggable targets. This will facilitate the development of epigenetic therapy approaches with improved tumor specificity. 5. Conclusion The epigenetic code is the result of histone modifications and DNA methylation on gene expression. While the genetic code in