Histone Methylation (HM) also occurs at lysine-(K) and arginine-(R) residues without changing the chromatic architecture, but is active as binding sites for the alternative proteins that may be involved in chromatin condensation (Nielsen et al. 2001). Editing of methyl marks (MM)is performed by S-adenosylmethionine (SAM)- dependent methyltransferases(13- Tsukada et al. 2006) and erasing of MM by either the Jumonji family of demethylases or lysine-specific histone demethylases 1 (LSD1) and 2 (LSD2) (Shi et al. 2004). Arginine methylation of histone proteins is known to antagonize the alternative histone marks, followed by raising the histone code complexity (Guccione et al. 2007). A brief insight of HM-process is provided (Fig. 11.2).

Epigenetic modification play a key role in cancer development. In this platform, genomic hypomethylation is considered as a global event, and CPG methylation is responsible for silencing of tumor suppressor genes (Esteller 2008; Dobrovic and Simpfendorfer 1997; Deng et al. 1999; Hatzimichael et al. 2009; Hatzimichael et al. 2012; Esteller 2000, 2006) (Fig. 11.3). In addition, there are complementary events such as distraction of the histone modification territory, and histone modifiers include mutation of deacetylases, and amplification of methyltransferases and demethylases (Fraga et al. 2005; Ropero et al. 2006; Cloos et al. 2006). At a glance there are three major and interactive platforms in cancer and epigenetic modifications; (1) genomic hypomethylation, (2) CPG island, as a contrast territory against platform 1, and (3) Distraction of Histone modification territory (Fig. 11.3).

Furthermore, epigenetic reservoir may be altered through protein activation or inactivation.

More specifically, the epigenomic stabilization and instabilization relies on enzymatic territory and could be predisposed to mutation in different malignancies (Hatzimichael et al. 2013; Yan et al. 2011; Simo-Riudalbas et al. 2011; Ley et al. 2010; Thol et al. 2011; Delhommeau et al. 2009; Weissmann et al. 2012) (Fig. 11.4). Besides, the metabolic genomic mutations are reported for isocitrate dehydrogenase (IDH) 1 and IDH2 genes in patients with myelodysplastic syndromes (MDS) and acute myelocytic leukaemia (AML) (Ward et al. 2010). In another study in AML, IDH1 and IDH2 mutations revealed to have common epigenetic defects with TET2 mutations. However, specific hypermethylation signature has been found in AML patients with mutations of IDH1/2 (Figueroa et al. 2010). Interestingly, influential mutation on the Polycomb repressive complex (PRC), per se EZH2, are capable to modify histone elements. Moreover, EZH2 overexpression are found in the epithelial neoplasms and leukemia (Varambally et al. 2012; Benetatos et al. 2013; Simon and Lange 2008). Diversiy in the type of mutations include activating mutations of EZH2 in B-cell lymphomas, missense, nonsense, and frameshift mutations in myeloid malignancies (Sneeringe et al. 2010; Ernst et al. 2010; Nikolosk et al. 2010). Furthermore, silencing of tumor suppressor genes (TSGs) by promoter CpG methylation in breast tumorigenesis has been reported. They have highlighted that the TSG methylation in BCcould be used as a potential marker for cancer management. In a review article the epigenetic alterations in BC, biological and clinical insinuation are provided (Xian et al. 2013).

By considering the multistep carcinogenesis, divese capability of tumor cells through initiation and progression does not, solely, rely on the genetic alterations. But, epigenetic changes assist and enable the neoplastic cells to behave as the cancer cells in malignant phase with the possible reversible capacity (Fig. 11.5). However, still there are some unmasked facts in cancer epigenetics which require the harmonic and complementary approaches by considering the clinical follow-up data.

Details about epigenetic diversity in cancer is, previously, reviewd (Mehdipour et al. 2012) and the summary of crutial facts is provided (Fig. 11.6). In addition, epigenetic alterations play an essential role in malignant transformation.

Retinoids with capability of antiproliferative and proapoptotic impacts, are involved in the decisive biological processes and also act as the suppresser of carcinogenic development. Retinoic acid (RA), as a derivative of vitamin A or retinol, has a remarkable epigenetic regulatory role in gene transcription (Chambon 1996). It has also a crucial impact on the morphogenic progress and development of postpuberty mammary gland (Montesano et al. 2002; Wang et al. 2005). Retinoids and their products induce differentiation in various cell types as well.

Retinoids and all-trans retinoic acid, 13-cis retinoic acid, bexarotene as the Retinoids’ natural metabolized and synthetic products have the key role in cell differentiation. Retinoids apply their actions by binding to the nuclear retinoic acid receptors (α, β, γ), in those the regulatory impact is due to transcriptional and homeostatic characteristics and their role in neoplastic transformation. By considering the retinoids’ efficacy in cancer prevention and treatment, an achievement is the treatment of of leukemic subtypes characterized with chromosomal translocations. So far, the therapeutic limitation in the prevention and treatment of solid tumors may be due to the epigenetic silencing of retinoic acid receptor beta (RARβ) which could be developed by including assessment of RARβ and downstream genes in the solid tumor (Bistulfi et al. 2006; Connolly et al. 2013). Moreover, the “dualistic role” of the retinoic acid signaling pathway in cancer is reviewed (Coyle et al. 2013). They have emphasized on the quadro-radial factors including gene transcription, interactions with other transcription targets, apoptotic pathways, and the immune machinery. It was also referred to the therapeutic impact of retinoid on innovation of an appropriate cancer therapy. Then it could be stated that retinoic acid could be considered as a hero in cancer.

Retinoids are the promising targets in cancer prevention and treatment, especially in breast cancer. Partly, the therapeutic success in the prevention and treatment of solid tumors may may be due to the epigenetic silencing of RARβ (Connolly et al. 2013).

Retinoic acid receptors (RARs) belong to the nuclear hormone receptor superfamily, and are characterized with the heterodimeric partners, known as the retinoid X receptors (RXRs).

They are capable to regulate genes having a direct repeat five (DR5) retinoic acid response element (RARE) within the promoter regions. Regarding the pharmacologic levels of retinoid-derived ligands, all-trans retinoic acid (AT-RA) and 9-cis-retinoic acid, for RARs and RXRs are respectively aimed to transactive the heterodimeric partners. In addition, the reduction of RARβ2 or RARβ4 mRNA is reported in breast cancer cell lines (Hoffmann et al. 1990; Swisshelm et al. 1994). Besides, Reduction or lack of the RARβ2 mRNA expression in primary breast cancers have been also reported (Widschwendter et al. 1995; Zhang et al. 1994).

The biological effects of RAR are summarized (Evans and Kaye 1999; Chambon 1996; Altucci and Gronemeyer 2001; Balmer and Blomhoff 2002):

Epigenetic silencing could repress the transcriptional function which lead to the reduction of RARβ2 expression (Xu et al. 1994; Houle et al. 1993; Liu et al. 1996). It is also reported that the RARβ-prime as a truncated, oncogenic RARβ protein is entirely expressed in breast cancer cell lines. Its worth to highlight that this isoform could block the functions of tumor suppressor of RARβ2 and RARβ4 protein isoforms as well (Lee et al. 1995; Sabichi et al. 1998). RARβ2 as a two edged sward, is capable to activate either tumor suppressor or antimetastatic programming. However, “the RARβ2 could be defined as a reflective mirror with multi-potential territory which facilitates diverted interactions with variety of targets at biological and molecular levels” (Mehdipour et al. 2012).

Interstingly, an incorporated process is essential through the RA receptor α (RARα), RA signal elicit chromatin modifications in RA signal leads to the transcription of the RA receptor β2 which is located at chromosome 3p24 (Chambon 1996; Dilworth and Chambon 2001). Furthermore, RARβ2 maintain an independent transcription accompanied by only few downstream RA-responsive genes (Brand et al. 1988; Sucov et al. 1990; Husmann et al. 1991). Retinoids have also multi-influential capacities, however its informative characteristics is summarized (Fig. 11.7) (Mehdipour et al. 2012).

In RA-sensitive BC cell line, an inactive mode of transcription was found for RARβ2 in which notable value of repressive chromatin modifications including DNA hypermethylation could be characterized. Besides, simultaneously, with the conversion of RARβ2 alleles from a permissive transcriptional status into a nonpermissive mode which is characterized by aberrant DNA hypermethylation, cells are converted to RA resistance (Ren et al. 2005). In addition, numerous proteins involved in retinol or RA metabolism found as unbalanced or down-regulated in breast cancer cells (Andreola et al. 2000; Guo et al. 2000; Mira-y-Lopez et al. 2000; Rexer et al. 2001), including retinol binding protein 1 (CRBP1) which is involved in the morphological differentiation of human breast epithelial cells (Farias et al. 2005). The CRBP1 is encoded by the RBP1 gene, was characterized as a downstream RAR-regulated gene, as one of the RAR targets (Smith et al. 1991; Husmann et al. 1992). RARβ2 is also involved in transcription of RA-responsive gene and retinoid signaling (Pozzi et al. 2006; Kawakami et al. 2005; Vermot et al. 2005a, b). RARβ has four isoforms with different biological functions of those RARβ2 play an influential capacities in carcinogenesis and therapy and could be listed as cancer preventive and therapeutic agents in BC. The characteristics of RARβ2 is summarized in Fig. 11.8.

Ectopic RARβ2 expression regulate downstream factors which are presentive of differential expression of transcription factors, signalling molecules and metabolic enzymes (Feinberg et al. 2006). RARβ2 is also capable to change the gene expression pattern of AT-RA in cell lines (Kurbel 2013). In addition, the ectopic RARβ2 expression, the type and origin of cell may have role in characterizing the expression profile. Besides, an interactive and regulatory manner may be involved between mode of RARβ2 expression and multi- pathways. So a global genetic programming may be involved for managing the status of gene regulation. In this regard, three upregulated genes by RARβ2 on chromosome Xq28 have been reported at cell line level including ARHGAP4 as a GTPase-activating prote, RPL10 as a 60S ribosomal proteand SSR4 as signal sequence receptor delta 4 (Tribioli et al. 1996; Oh et al. 2002; Mangelsdorf et al. 1994; OMIM: 300090). However, the importance of X chromosome may be due to the involvement of interactive targets in this chromosome with beta retinoic acid. Furthermore, the highlighted points of a review on RARβ2 gene in breast cancer is presented in Tables 11.1, 11.2, 11.3 and 11.4 (Mehdipour et al. 2012).

Cancer prevention is an optimal and end point hope in cancer world, in this regard the list of some environmental factors, their characteristics, and key mechanisms which are involved in the altered epigenetic and genetic make up is provided (Table 11.2). For instance, the dietary methionine, as a vital amino acid, plays a key role in epigenetic which could provide a normal firm for genome-wide DNA methylation patterns through cytosine methylation. In addition, the CpG methylation patterns is important during the early stages of embryon. Therefore, the nutritional behaviors could be translated to the gestations through ancestral line by influencing the epigenetic and genetic composition. So, the fate of individual health, not only, depends on each individual, but on the previous generations as well. Furthermore, research was conducted to investigate an association between folate intake, vitamins B(2), B(6), B(12) and methionine with methylation of promoter region in E-cadherin, p16, and RAR-β(2) genes in the primary BC tumors (Tao et al. 2011). They have stated that intake of these nutrient may be capable to alter promoter methylation in normal or dysplastic breast tissue. In recent publication the multidisciplinary role of epigenetics in cancer prevention as “Nutri- epigenetic” has been, complementary, reviewed (Gerhauser 2013). They have highlighted the influential impact of natural chemopreventive agents on the expression or manner of action of histone modifying enzymes and DNA methyltransferases. The major deregulated events during carcinogenic process by epigenetic alterations include drug metabolism, cell cycle regulation, potential to repair DNA damage/ to induce apoptosis, response to inflammatory stimuli, cell signaling, cell growth control and differentiation. The provided scheme illustrates a complementary view of these machinery (Fig. 11.9). It was also emphasized that epigenetics has influential impact on gene regulation during developmental stages (Gerhauser 2013). It was also highlighted that epigenetic changes occur at early phase of cancer development. In addition they have stated that an interventions with chemopreventive agents, probably, starts early after birth, but I believe that the programming of epigenetic machinery has its root in two complementary territories including heritage and the life style of embryon, i.e. maternal and also ancestral lines including paternal and maternal sides. In addition, a comprehensive insight has been, recently, provided in a book review (Gray SG 2014) in which the basic aspects of epigenetics have been explored.

Cancer stem cells (CSCs) as a cooperative network interact with many molecular and biological event. The characteristics of CSCs are previously highlighted (Clarke et al. 2006). CSC is a cell within a tumor property which instinctly acquires the capability to self-renew and to establish the heterogeneous lineages of cancer cells. CSC self renew capacity include two manners, (1) “Symmetrical selfrenewing cell division” in which the identical CSCs having self-renewal ability; and (2) “Asymmetrical self-renewing cell division” in which one stem cell and one more differentiated progenitor cell are characterized. CSCs are characterized by their capability to reiterate the generation of an incessantly growing neoplasm. The putative CSCs are also named as ‘‘tumorinitiating cell’’ and ‘‘tumorigenic cell’’. However, symmetrical division of stem cells may form two progenitor cells, leading to depletion of CSCs population or leading to cancer celll death which may be considered as an medication for cancer therapy.

The CSCs have been, initially, isolated in human breast carcinoma (Al-Hajj et al. 2003). The key related characteristics of ductal invasive carcinomas include; (1) Heterogenic intratumoral differentiation of this breast disease, (2) The epithelial to mesenchymal transition (EMT) status, (3) Expression of TWIST1, as a repressor of an EMT-inducing transcriptional factor in invasive lobular breast cancer, (4) Disuniting of invasive tumor cells in EMT-like condition, (5) different pathways are involved in formation of cancer stem cells, and (6) Distribution of associated tumor cell, are all correlated with the malignant progression leading to a poor clinical prognosis (Yang et al. 2004; Fujita et al. 2003; Xue et al. 2003; Blanco et al. 2002).

Due to these characteristics breast cancer is a focal territory in which two paradigms including cancer stem cells and EMT of cancer stem cell migration is remarkably highlighted.

Moreover, the intestinal gastric cancer, pancreatic cancer, and squamous cell carcinomas are also the examples for this category in which the intra-tumor heterogeneity and EMT could be detected (Nakajima et al. 2004; Rosivatz et al. 2002; McAlhany et al. 2004).

Three main fscts about stem cells in solid tumors are highlighted as followings:

Regarding the genetic and epigenetic signatures of ‘‘Stemness’’ Clarke and his colleagues (Clarke et al. 2006) have stated very important considerations as:

However, as a complementary information the essential renewal assay in vivo is not adequate to be translated in human and more specific definition is required for evaluating the quantitative values of stemness in human cancers. Such attempts require the follow-up strategy during different stages of cancer patients. Although microarray and genomewide technologies are applied to unmask tendency in genetic and epigenetic for CSCs, but specific Cell Based Strategy (CBS) would, appropriately, solve the heterogeneity insight of diverse tumors.

Labelling a cell as stem cell and cancer stem cell is a rather difficult aim. There are some key questions; (1) Where is the stem cell originated from? Is it derived from other stem cell populations? Or (2) is it derived from an initially normal cell or (3) Or derived from cancer cell?

Now lets to characterize the capability of in vitro assay:

Reliability of an in vitro assay to define a cell as “stemness” and cancer stem cell:

As the matter of fact and by considering the epithelial to mesenchymal transition/mesenchymal to epithelial transition (EMT/MET) processes, there are a triangle in cancer initiation including programming/reprogramming/cancer progression. Furthermore, stem cells are associated with embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs), immortalized mammary epithelial cells that have undergone epithelial to EMT transition which is the reverse manner of the MET process (Mani et al. 2008; Morel et al. 2008). There is also a cooperation between two capacities including EMT and stemness through cancer progression, and separation of cells from the primry tumor that facilitate migration and metastasis. Interestingly, such evocative process of EMT is similar to the embryonic tissue development which is the state of art and fascinating journey of cells from far away departure station to diverse destinations (Thiery et al. 2009). Also, CSCs are capable to kernel new neoplasms to self-renew and lead to generate non-stem differentiated cells. CSCs is not apparently pluripotent which differentiate them from iPSCs; due to this point, cancer cells do not produce any cell type; instead may be considered as relapse of the original primary tumor cells occurs through metastatic process (Gupta et al. 2009; Polyak et al. 2009). However, the machinery of stem cell and invasion is rather a core item in cancer metastasis. In this regard, it worths to refer to the recent work which was focused on the “mouse haematopoietic stem cell regulator Latexin (LXN)” (Oldridge et al. 2013). This target was considerd as a unique “homologue of the retinoic acid receptor responder 1 (RARRES1) gene”. The co-expression of either RARRES1 or LXN was suppressed by DNA methylation in prostate cancer (PC) cell lines and furthermore inhibition of RARRES1 and LXN led to increase the invasive capability of primary PC cell line.

Additionally, it is recently reported that phosphorylation of the RARγ2 play a key role for the neuronal differentiation embryonic stem cells in mouse (Al Tanoury et al. 2014).

One of the serious concern in breast cancer is the possible developmental capacity of malignancy in premalignant breast neoplasm. Such process by considering the status of DNA methylation including Methylated-IN-Tumor (MINT)17, MINT31, RARβ2 and RASSF1A genomic markers have been studied through benign, premalignant and malignant status of breast cancer (van Hoesel et al. 2013). They have found DNA hypermethylation at early stage of BC development with diverse degree of tumor during the cancer progression.