The chromatin is a highly organized structure of DNA and protein. The organization of DNA in chromatin (euchromatin, active; heterochromatin, inactive) has many functions, such as packaging DNA into smaller volume, preventing DNA damage, and controlling DNA replication, transcription, and repair [100]. The fundamental unit of chromatin is the nucleosome, an octomeric structure containing two copies each of histones (H3, H4, H2A, and H2B) around which 147 base pairs of DNA are wrapped [101]. The states of chromatin are controlled by chemical modification of histone tail (N-terminus) via posttranscriptional including acetylation, methylation, phosphorylation, sumoylation, poly(ADP)-ribosylation, and ubiquitination and histone composition in conjunction with other nonhistone proteins [102, 103].

It was first proposed in 1964 that histone modifications may affect the regulation of gene expression, after demonstrating acetylation of the ε-amino group of lysine residues on histones [104]. After nearly half a century, it is has been elucidated that the posttranscriptional modifications of histone tails determine not only transcriptional activity but also all DNA-templated processes. The identification of the coexistence of histone modifications associated with activation or repression led to the proposal that the modification constitutes a code that could be recognized by transcription factors to determine the transcriptional state of a gene 10 years before [105]. However, these patterns appear to be not static, and a dynamically changing and complex landscape via the chromatin signaling pathway led to the new concept termed “histone cross talk.” This term represents the influence one or more coexisting histone modifications have on the deposition, interpretation, or erasure of other histone modifications [5, 106]. The recent investigations showed that histone cross talk mechanisms commonly seen and have a great importance for biological processes in organism [107, 108].

Histone modifications affect the chromosome function via several mechanisms. Generally it is believed that histone modifications cause structural changes in histone. This structural change may act as specific binding sites for protein domains (e.g., bromodomains, chromodomains, tudor domains) [109, 110]. Among the epigenetic mechanisms, histone modifications have further grown over the last decade with the discovery and characterization of a large number of histone-modifying molecules and protein complexes. The deregulation of these molecules or complexes may lead to deregulation of the control of chromatin-based processes by changing histone modifications and may have been associated with a large number of human malignancies. Genome-wide studies revealed that the histone modifications of malignant cells patterns disrupted when compared to healthy cells [111]. The posttranslational modification at amino acid tail of histone protein may result in changed transcription of important genes such as tumor suppressors. Changed patterns of histone modifications are a hallmark of cancer, and great amount of histone modifications have been linked to several cancer types to date [112]. The most well-known histone modifications types are acetylation/deacetylation and methylation/demethylation [113].

Scientists have long been aware of the existence of noncoding RNAs (ncRNAs). In spite of the great amount of knowledge about the function and types of ncRNAs, we are still far from fully knowing the role of large fractions of the transcriptome that do not encode for proteins [154]. Among ncRNAs, microRNAs are 18–25 nucleotides-long RNA molecules encoded in the genome that are transcribed by RNA polymerase II and important regulators of protein of gene expression that control both physiological and pathological processes, such as DNA methylation, development, differentiation, apoptosis, and proliferation [155, 156]. miRNAs are synthesized and processed in the nucleus, exported to the cytoplasm, and then bind to the target mRNA. The regulation of RNA transformation by miRNA is accomplished through RNA-induced silencing complex (RISC). miRNAs can inhibit mRNA translation or degrade mRNA [157, 158]. Major mechanisms of miRNA deregulation include genetic and epigenetic alterations as well as defects in the miRNA processing machinery. Each miRNA regulates multiple mRNAs and, conversely, each mRNA may be targeted by multiple RNAs (several hundreds). They can act as oncogenes or tumor suppressors and have been implicated in cancer initiation and progression, and the profiles of miRNA expression differ between normal and tumor tissues and between tumor types [159–161]. To date, several investigations relating to miRNA profiling has led to the identification of miRNAs’ changed expression level in human breast cancer [162, 163]. The expression level of these miRNAs was correlated with specific breast cancer biopathological features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation [164]. miRNAs act as tumor suppressors and are oncogenic in breast cancer like other cancer types. So, tumor formation may arise from the overexpression (or amplification) of oncogenic miRNA and/or reduction (or deletion) of a tumor-suppressor miRNA [165].

miRNA-21 is overexpressed in breast cancer like in other cancer types [164, 166]. p53 and programmed cell death 4 (PDCD4) are tumor-suppressor proteins, and the deregulation of them may lead to cancer development. miRNA have been linked to breast cancer by targeting these proteins in breast cancer cells [167].

Despite the extreme heterogeneity of breast cancer, global breast cancer survival rates have increased during the past decades due to advances in the central role of genetic alterations in the diagnosis, treatment, prevention of breast cancer, and prognosis [2, 168]. Survival rates should be further improved by finding epigenetic molecular markers associated with risk assessment and/or prognosis of breast cancer. The knowledge about epigenetic alterations profiles in detail might prove vital in many respects. First, it might help us to estimate breast cancer risk and take precautions before breast cancer develops. In addition, there are several subtypes of breast cancer and corresponding therapies currently used. Each subtype, even each individual, has unique molecular epigenetic characteristics. The elucidating of epigenetic characteristic might contribute to a better estimation of breast cancer prognosis and lead to the choice of the most useful therapy [169]. In this way, patients will not be exposed to ineffective toxins associated with expensive therapy. Several reports have proposed that hypermethylation or hypomethylation of specific genes and global methylation status might be useful epigenetic markers for breast cancer prognosis. The recent studies also included miRNAs’ expression profiles into putative epigenetic markers of breast cancer.

The major breast cancer subtype is ER-positive, and it has generally had a more favorable prognosis than ER-negative tumors. It is well established that ERα and E-cadherin are frequently involved in pathogenesis of breast cancer. The aberrant methylation of these genes is associated with malignant progression in human breast cancer [170]. ERα expression level is also regulated by miRNAs in the context of breast cancer. miRNA-206 [171] and miRNA-221/222 [77] target and regulate human ERα. miRNA-206 was upregulated in ERα-negative breast cancer. Another study found that miRNA-206 inhibits the expression of ESR1 mRNA through two binding sites in the ESR1 3′-untranslated region (3′-UTR). The researchers also found other miRNAs (miRNA-18a, miRNA-18b, miRNA-193b, and miRNA-302c) targeting to ESR1 mRNA in breast cancer cells [172]. Therefore, the aberrant methylation of the ESR1 gene and certain miRNAs altering the ESR1 gene expression might be putative epigenetic markers for human breast cancer prognosis.

BRCA1-associated breast cancer, hereditary or nonhereditary, occurs at early age due to involvement of the cellular DNA repair machinery. The inactivation of the BRCA1 by hypermethylation has been suggested to be the putative prognostic marker in breast cancer [173]. Besides the methylation, BRCA1 expression level could be regulated by miRNA-335. Overexpression of miR-335 resulted in an upregulation of BRCA1 mRNA expression, suggesting a functional dominance of ID4 signaling [174].

RASSF1A (Ras association domain family 1 isoform A) is a recently discovered tumor-suppressor gene. The protein encoded by RASSF1A interact is involved in the regulation of the cell cycle, apoptosis, and genetic instability. Thus, loss or altered expression level of the RASSF1A gene has been associated with several cancers. After illustrating the association between inactivation of the RASSF1A gene and the hypermethylation of its CpG-island promoter region, the RASSF1A gene has become the attractive biomarker for early cancer detection, diagnosis, and prognosis in many cancer types [175, 176]. The increased methylation level of the RASSF1A gene was observed in tumor size and lymph node status in breast cancer [177]. Similar results have been obtained by a meta-analysis of published data conducted with 1795 breast cancer patients. They concluded that RASSF1A promoter hypermethylation associates with worse survival in breast cancer patients [178]. These findings have indicated the great potential for the methylation of the RASSF1A gene in terms of the prognostic value of the breast cancer.

EZH2, histone-lysine N-methyltransferase acts as gene silencer by methylation and is related to several cancers. The overexpression of EZH2 is associated with aggressive breast cancer because of the enhanced cancer cell proliferation and a marker of poor prognosis in many solid tumor carcinomas including breast [179–181].

It has been investigated that several miRNAs are involved in breast cancer pathogenesis like cell regulation, and it has been proposed to be a prognostic factor for breast cancer. The miRNA-17-5p and miRNA-17/20 have been reported to be involved in breast cancer cell proliferation [182, 183]. miRNA-21 also could be a molecular prognostic marker for breast cancer and disease progression because of its association with advanced clinical stage, lymph node metastasis, and patient poor prognosis [184].

Another strategy to clarify the role of miRNA in breast cancer is the analysis of DNA methylation and expression miRNAs in combination. Alteration of methylation in the promoters of miRNAs has also been linked to transcriptional changes in cancers. Morita et al. found that DNA methylation in the proximal promoter of miRNAs is tightly linked to transcriptional silencing [185].

Cancer emerges not only because of the accumulation of genetic mutations, but also because of the reversible epigenetic changes. The dynamic alterations of the epigenetic mechanisms offer us a new field for developing novel cancer drugs that can react to epigenetically silenced tumor-suppressor genes [186]. So histone deacetylases and DNA methyltransferases have become the main targets for cancer therapy. In breast cancer, epigenetic silencing of tumor-suppressor genes due to alteration in both HATs and HDACs (histone modification) in combination with DNA hypermethylation is commonly observed [187]. The clarification of the epigenetic dysregulation mechanism in breast tumorigenesis has great importance in terms of the development of new therapies for breast cancer patients.

Aberrant HDAC activity has been investigated in several cancer types, especially in breast cancer. HDAC-1 expression and HDAC-3 protein expressions were analyzed immunohistochemically on a tissue microarray containing 600 core biopsies from 200 patients by Krusche et al. They found that moderate or strong nuclear immunoreactivity for HDAC-1 was observed in 39.8 % and for HDAC-3 in 43.9 % of breast carcinomas. HDAC-1 and HDAC-3 expressions correlated significantly with estrogen and progesterone receptor expression [188]. Another study concentrated on HDAC-6 expression levels in breast cancer has been done by Zhang et al. They also found that HDAC-6 mRNA expression is at significantly high levels in breast cancer patients with small tumors measuring less than 2 cm, with low histological grade, and in estrogen receptor α- and progesterone receptor-positive tumors. However, multivariate analysis concluded that the mRNA and protein of HDAC-6 were not independent prognostic factors for both overall survival and disease-free survival [189]. These studies led to the development of new therapies for breast cancer by finding suitable HDAC inhibitors. To date, a number of HDAC inhibitors have been designed and synthesized based on their chemical structure and are generally divided into four groups including hydroxamic acids, benzamides, cyclic peptide, and aliphatic acids (small chain fatty acids). The potential use of these inhibitors for breast cancer therapy has been investigated, as shown in Table 5.2.

Among them, some HDAC inhibitors like vorinostat (SAHA) and romidepsin (FK-228) have already been approved by the US Food and Drug Administration for clinical treatment of cutaneous T-cell lymphoma. Vorinostat is the first HDAC inhibitor and currently under evaluation in several phase II trials in breast cancer. It is already shown that vorinostat has profoundly antiproliferative activity and inhibits proliferation of both ER-positive and ER-negative breast cancer cell lines [190]. Entinostat (MS-275) and panobinostat (LBH-589) HDAC inhibitors are in phase I and II studies in combination with endocrine therapies, chemotherapeutic agents, or novel targeted therapy in women with breast cancer [12, 120]. A recent phase II study relating to the HDAC inhibitor vorinostat combined with tamoxifen for the treatment of patients with ER-positive metastatic breast cancer using 43 patients has been done. Even though the number of patients was small, they concluded that the combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. HDAC inhibitor with tamoxifen may restore hormone sensitivity by causing re-expression of a silenced ER gene [191].

In addition to phase trials, preclinical investigations have been widely done. The other idea for treatment of ER-negative breast cancer cells is using the synergistic effects of a combination treatment of HDAC inhibitors and DNMT inhibitors (demethylating agents). Fan et al. and Sharma et al. used 5-aza-2′-deoxycytidine (AZA) as a DNMT1 inhibitor and trichostatin A (TSA) as a HDAC inhibitor to investigate this synergistic effect. Both studies have shown the reactivate ERα and PR gene expression in ER-negative breast cancer cell lines, which are known to be aberrantly silenced in breast cancer [192, 193]. Other studies have shown that the HDAC inhibitors lead to reactive of ERα and PR expression by inhibition of the HDAC activity in breast cancer cells [194–196].

The other enzyme families to target for cancer therapy are HMTs and HDMs, previously implicated in cancer, inflammation, and diabetes [197]. The gene expressions level of the histone-modifying enzymes (HDMs and HTMs) are specific to cell types and highly correlated with target gene expression [198]. A recent study examined the expression profiles of 16 different histone-modifier genes including HATs, HDACs, and HDMs in breast cancer. They found that significantly different expression levels of histone-modifier genes exist between breast tumors and normal tissue, and their findings were significantly associated with conventional pathological parameters and clinical outcomes. So, it appears that histone-modifier enzymes offer utility as biomarkers and potential for targeted therapeutic strategies [199].

After these recent findings, miRNAs also have become the target for developing therapies for breast cancer. The miRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of drug-resistant breast cancers in the near future [200]. One of the initial studies has concluded that miRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways [201].