Targeted nutrigenomics as a means of breast cancer management: from DNA methylation to microRNAs

Breast Cancer (BC) is the most prevalent cancer affecting females and the leading cause of death around the world. The World Health Organization (WHO) has announced that the incidence of BC increases approximately 1.8-2 % annually. Hence, it is important that new prevention and treatment strategies involving epigenetics and nutrigenomics are explored. Epigenetics refers to potential changes in gene expression and chromatin structure without alteration of DNA sequence, but still modulates the expression of a particular phenotype. Nutrigenomics determines the effect of dietary habits on cancer risk and tumor behavior, both in progression and inhibition of cancer. The modulation of chromatin structure is an essential component in the regulation of transcriptional activation and repression. Therefore, identifying the regulators of gene expression changes during cancer progression is essential for early diagnosis and inhibition of this malignancy. The methylation of promoter genes, as well as the interplay between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes, is the primary component of epigenetics. Any defect in these processes is considered a crucial mechanism for gene and pathway dysregulation in all human cancers, including BC. Nutritional genomic and epigenetic mechanisms may play a pivotal role in prevention as well as early diagnosis of BC, especially for closely related female family members of BC patients. It seems cytosine methylation in Cytosine-phosphate-Guanine dinucleotide (CpG) Islands reflects changes in balance tissues rigidity. Hypo- and hypermethylation of CpG Islands (CGIs) play a crucial role in development of BC via up-regulation of oncogenes and down-regulation of tumor suppressor genes (TSGs). These could be the most effective mechanisms to distinguish BC from other types of cancer. beneficial effects of some nutritional components on the function and structure of non-coding RNA and DNA methylation. We also discuss the role of nutrigenomics as a non-invasive method to explore the epigenetic mechanisms involved in BC and also in the prevention, treatment, early diagnosis, and distinguishing a variety of cancers from each other. In addition, the importance of non-coding RNAs, including miRNAs in body fluids, also need to be further clarified.


Introduction
In 2018, with a 24.2% incidence rate and 15% mortality rate, breast cancer (BC) was the most commonly diagnosed cancer in women [1]. Epidemiological surveys have elucidated that individual risk increases with the number of first and second-degree female relatives diagnosed with BC, as well as the age of their disorder onset, compared with the general population [2,3].
Relatives of BC patients are 2 to 4 times more likely at risk than general population concerning BC [4,5]. The most understood forms of genetic alteration involve either mutation, amplification or deletion within the genome. Traditionally, it has become clear that the molecular mechanisms underlying the development and progression of cancers, including BC, involve gene modification and regulation beyond changes to the DNA sequence. More recently, it was brought forth that epigenetic mechanisms, in which no changes to the DNA sequence occurs but instead gene expression patterns are altered, dysregulate information inside the genome of a healthy cell, leading to malignancy such as cancer [6]. Moreover, hypo-and hyper-methylation of CGIs play a crucial role in development of BC via up-regulation of oncogenes and down-regulation of TSGs, respectively [7]. Even more recently, nutritional genomics is the latest method of investigation of gene modulation, and it appears that diet and the gut can cause significant malignancy [8,9]. Nutrigenomics can be used to understand the effect of nutrients on DNA/gene expression and the proteome and metabolome, as well as the effects of diet-gene interplay in the preservation of health conditions or development of diseases such as cancer [10,11].
It has been elucidated that many essential and non-essential nutrients such as micronutrients (e.g., vitamins and minerals), macronutrients (e.g., fatty acids and proteins) and some bio-reactive chemicals (e.g., flavonoids, carotenoids, phytosterols, eicosapentaenoic acid, and docosahexaenoic acid) can adjust gene expression during physiological and pathological processes via epigenetic mechanisms [12,13]. Nutritional genomics may also provide new information to develop treatments of diseases at the cellular level and preventative measures through the diet. Although it is challenging to determine the exact role of nutrients in epigenetic mechanisms because diet is often accompanied with dependent lifestyle factors such as poor diet, inappropriate body mass index (BMI), insufficient or lack of physical activity, and the use of drugs, alcohol or tobacco, there is accumulating evidence on the positive effects of food habits on reduction of cancer risk [14,15], and researchers have indicated that dietary and behavioral factors have a significant role in approximately one-third of cancerrelated deaths [16,17].
In the current study, we 1) discuss the role of DNA methylation and miRNAs in the occurrence of BC in relation to oncogene activation and tumor suppressor gene (TSG) inactivation, and 2) highlight the beneficial effects of some nutritional components on the function and structure of non-coding RNA and DNA methylation. We also discuss the role of nutrigenomics as a non-invasive method to explore the epigenetic mechanisms involved in BC and also in the prevention, treatment, early diagnosis, and distinguishing a variety of cancers from each other. In addition, the importance of non-coding RNAs, including miRNAs in body fluids, also need to be further clarified.

DNA methylation
DNA methylation is an epigenetic mechanism which almost modulating the function of the genes and impacting on gene expression, thereby it has a significant role in cellular differentiation, diversity of gene expression, as well as the development of nearly all types of cancer [18,19]. DNA methylation is a post-replicative process in which a methyl group is added at the 5-carbon of the cytosine ring, characterized as 5-methylcytosine (5-mC). These methyl groups project into the major groove of DNA and inhibit transcription. Most cytosine methylation occurs in the sequence context 5'CG3' and happens almost exclusively at cytosines that are followed immediately by a Guanine (CpG, Cytosine-phosphate-Guanine dinucleotide) [5]. The methyl transferase family catalyzes the methylation process, when a methyl group is transmitted from S-adenosyl methionine (SAM) to the cytosine. DNA methyl transferase 1 (DNMT1) is responsible for the maintenance of established patterns of DNA methylation, and DNMT3a and DNMT3b mediate establishment of de novo DNA methylation patterns (Figure 1) [20].
DNA methylation can occur in four types of regions including: 1) repetitive sequences, 2) CGI Promoters, 3) CGI Shore, and 4) the gene body (coding sequences) [21]. CGIs are located in approximately 60 percent of the repetitive DNA sequences and human gene promoters [22]. In constant regions, the CpG dinucleotides mostly tend to stay methylated, whilst in gene promoters, the CpGs are mostly unmethylated to prevent chromosome instability. In the first three cases, DNA methylation is typically related to quell of gene expression and transcription. However, DNA methylation can occur in the gene body rather than the promoters [21,23].

Mechanisms of DNA methylation in healthy cells and cancer cells
The expression of genes is facilitated by engagement of transcription factors (TF) to the gene promoter DNA [24]. However, the process of methylation leads to repression through changes in chromatin structure restricting access of TF to the gene promoter, preventing gene transcription and expression. Hyper-methylation of DNA causes the gene to be silenced ( Figure 1) [20].
DNA methylation patterns vary profoundly among healthy and cancerous cells ( Figure 2). For instance the total 5-methylcytosine can be 20-60% less in repetitive elements of cancerous cells compared to healthy cells, resulting in chromosomal instability [21,25]. Hypomethylation also occurs in specific regional DNA (promoters) of cancerous cells, resulting in the activation of potential oncogenes, as well as stimulating loss of imprinting at other DNA locations [25,26]. Conversely, hyper-methylation of CGI promoters of genes central to DNA repair and cell cycle control pathways correlates with very serious  (C) CpG Island Shore: The mechanism of DNA methylation templates is the same as in B, except that the CpG island shore is located up-stream. (D) Gene Bodies: CpG methylation in gene bodies prohibit gene expression in healthy cells, but is reversed in cancer cells, resulting in initiation of transcription at several incorrect loci tumor disease in humans. Moreover, tumor cells express atypical DNA methylation at CGI shores, and sometimes a specific enzyme that triggers the detachment of the methyl group from methylated DNA [27].

DNA methylation and BC
BC is a heterogeneous disease and has two main histological subtypes: ductal and lobular adenocarcinomas. These subtypes are distinct due to the clinical presentation and behavior, and morphological and molecular characteristics [6,28]. In recent years, DNA methylation patterns of target genes have been considered by investigators as a biomarker for BC prognosis (Table 1). Kim et al. examined the status of three genes; LHX2, WT1 and OTP, in primary tumors and adjacent and other healthy tissues, and found that the frequency of aberrant hyper-methylation in primary tumors (LHX2= 43.6%, WT1= 89.7% and OTP= 100%) was high. However, the methylation frequencies were intermediate and rare in adjacent healthy tissue and other healthy tissue [29]. DeRoo et al. assessed Line-1 methylation in blood samples, and found a dose dependent and negative correlation between undermethylation of Line-1 and risk of BC in non-hispanic caucasian women. They also found that three Single Nucleotide Polymorphism (SNP) genes; SLC19A1 (rs1051266), MTRR (rs10380) and MTHFR (rs1537514), related to Line-1 methylation, and MTHFR (rs1537514) is also linked to BC [30]. Chimonidou et al. indicated that in Cell-Free DNA (CF-DNA) of BC patients, CST6 promoters were hypermethylated compared to healthy individuals. For this reason, they suggested that CST6-promoter methylation, especially in CF-DNA of BC patients, could potentially be used as a novel plasma tumor biomarker for BC [31]. Rauscher   -Hyper-methylation of MAP9 gene in BC can be used as molecular biomarker for BC diagnosis. Ng et al., 2013 [39] -BC: n= 260 (clinically diagnosed), -Normal subjects: n=170 (pathologically verified not to have BC and no history of other cancers).
-Extraction total RNA from tissues: TRIzol reagent -Extraction total RNA containing small RNA from 500 ml of plasma: Trizol LS reagent and miRNeasy Mini Kit.  -Total RNA: extracted from a 300 µL serum sample using 3D Gene RNA extraction reagent from a liquid sample kit.
-Comprehensive miRNA expression analysis: done by using a 3D-Gene miRNA Labeling kit and a 3D-Gene Human miRNA Oligo Chip.
-miRNA to be present: The corresponding microarray signal was more than the (mean + 2 × standard deviation) signal of the negative controls.
-Quantitative RT-PCR assay: TaqMan  expression. It seems that, simultaneously with DNA, methylation and abrogation of SP1 binding to the proximal promoter region, suppression of progression and promotion of proliferation as a dual function of FOXF2 in related to SP1 will be influenced; therefore, the expression of FOXF2 will be silenced in BC cells [34]. Another study assessed DNA methylation profiles in breast tumors of women with a strong BC family history compared to adjacent non-tumor tissues, and selected 40 hypermethylated genes from The Cancer Genome Atlas (TCGA) based on breast tumor samples [35]. They demonstrated that only 32.5 percent of these genes including SEPW1, PCDHGA4, CCDC36, C1orf14, RPTOR, C1orf114, ZNF454, USP44, CSMD3, PRKAR1B, SLC7A14, SOX2OT and RYR2, were highly methylated in their breast tumor samples compared to the adjacent histologically healthy tissues. Accordingly, they suggested that identifying methylation markers as a biomarker for early diagnosis of BC cases in particularly high risk groups, like familial cases, can be examined [35]. Dehbid et al. suggested that the hypermethylation of MAP9 gene in BC patients causes the reduced gene expression consistently seen in these patients. Reduced gene expression of MAP9 in BC leads to instability of P53, which can inhibit apoptosis of the cancer cells. Therefore, Hyper-methylation of MAP9 gene and subsequent destabilization of P53 may be useful molecular biomarkers for BC diagnosis [36].
There are several atypical methylation patterns and subsequent changes in gene expression linked to BC that have proven useful for early detection, risk assessment and characterization of BC this last decade. Since DNA methylation is more stable and long-lasting than previously thought, and known to regulate gene expression triggering cells to become cancerous, it is now considered possible to use methylation patterns of genes involved in BC predisposition and development as biomarkers to diagnose patients most susceptible such as first relatives of patients [37][38][39][40][41][42][43][44][45][46].

Biosynthesis of miRNA in healthy tissue and its tumorigenesis mechanism in cancerous tissue
The process by which miRNAs adjust gene expression is complex, since one miRNA can target thousand numbers of mRNAs, and one mRNA can be modulated by various miRNAs. MiRNAs are small (18-24 nucleotides long) non-coding RNAs that regulate gene expression at the post-transcriptional level by attaching to the target (3´-UTR) of mRNA. Production of miRNA is completed in the nucleus and cytoplasm of cells [47,48]. In the nucleus, transcription of primary miRNAs (pri-miRNAs), 1-3 Kb long, are produced by RNA polymerase II from individual genes containing their promoter or intragenically from protein-coding genes [49]. The pri-miRNAs are then cleaved into stem-loop precursor miRNAs (pre-miRNA), 70-100 nucleotides long, by Drosha (RNA polymerase III) and DGCR8 cofactor (double-stranded RNA binding protein). Pre-miRNAs are transported to the cytoplasm by binding to Exportin-5 receptors (exporting-5-RanGTP), and then processed into a mature miRNA duplex by Dicer (RNase III enzyme), Trans Activating Response RNA-Binding Protein 2 (TARBP2) and AGO2 (DICER complex) [50]. The duplex strands are then separated and the complementary strand can be loaded in the RNA-induced silencing complex (RISC) or can also be degraded [51]. Recent findings show that the duplex strands have considerable biological functions, whereas the mature miRNAs (the guide strand) is incorporated into the RISC, including the GW182 and Argonaut (AGO) proteins. A special 6-8 nucleotide sequence at the 5´ end of the miRNA, called seed sequence, determines the role of miRNA in gene regulation. Thereby, the degree of homology between the seed sequences of miRNA and mRNA, triggered at 3´-UTR of the mRNA, delineates mRNA degradation, translational inhibition, or transcriptional activation. Seed matches can occur in any region of mRNA but the highest affinity occurs in the 3´ untranslated region (3´-UTR) of an mRNA (Figure 3) [51][52][53][54].
There is evidence of an intercellular communication function of miRNAs [55], and some miRNAs have been reported to intervene in gene cell-cycle regulatory processes and effect other proteins involved in cell proliferation [56,57]. Moreover, some miRNAs have a significant effect on tumor suppressor genes, decreasing proliferation and inducing apoptosis, and the levels of these miRNAs are low in cancer cells. In fact, in cancer cells the dysregulation of miRNA expression plays a significant role in the development of tumor progression, including invasion, tumor growth, angiogenesis, and metastatic capability (Figure 3) [58]. In addition, miRNAs are involved in tumor initiation by the regulation of cancer stem cell (CSC) properties, such as tumorigenicity, selfrenewal ability, and resistance to different therapies and drugs. Overall, dysregulated miRNAs can act as oncogenic miRNAs (oncomiRs) or TSGs. Moreover, both effects of miRNAs can be identified (Table 2). Chromosomal regions engaged in encoding oncogenic miRNAs may be amplified, resulting in up-regulation of oncogenic miRNAs and simultaneous silencing of TSGs. Alternatively, tumor-suppressive miRNAs are mostly placed in fragile sites along the chromosome, and omission or mutation of these regions leads to a reduction or loss of expression of tumor-suppressive miRNAs and subsequent upregulation of their target oncogenes [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77].

miRNAs and BC
There are several studies linking miRNA suppression to BC development, from cancer-initiation and BC metastasis, cell proliferation, angiogenesis, and invasion. Specifically, the expression of Drosha and Dicer enzymes, key to miRNA synthesis, is dysregulated, as is the DGCR8 protein complex [78]. The recently discovered absence of miRNAs in BC cells not only allows diagnosis, prognosis and determination of suitable therapeutic approaches, but may also provide novel targets for therapeutic replacement therapies in cancer diseases, in particular in BC [79]. Following synthesis, stable miRNAs are transferred outside the cell and are readily detectable in peripheral blood samples. These features can play a pivotal role in monitoring secreted miRNA levels from the expanding early-stage BC tissue, and could be a useful novel biomarker for early BC detection [  Initially, primary transcription of miRNAs (pri-miRNAs), 1-3 Kb long, are produced by RNA polymerase II. Next, the pri-miRNAs are cleaved into stem-loop pre-miRNA (70-100 nucleotides long) by Drosha (RNA polymerase III) and double-stranded RNA binding protein DGCR8. Pre-miRNAs are transported to the cytoplasm by binding to Exportin-5 receptors (exporting-5-RanGTP) and then processed into a mature miRNA duplex by Dicer (RNase III enzyme), Trans Activating Response RNA-Binding Protein 2 (TARBP2) and AGO2 (DICER complex). The duplex strands are then separated: the complementary strand can be loaded in the RISC or is degraded, and the mature miRNA (the guide strand) is incorporated into the RNA-induced silencing complex (RISC) which includes the GW182 and Argonaut (AGO) proteins. The degree of homology of the miRNA "seed" to the 3′ UTR target sequence of the mRNA determines the mRNA degradation, translational inhibition, or transcriptional activation. Nonetheless, dysregulation of miRNAs expression is able to have a major role in the development of tumor progression, including tumor growth, invasion, proliferation, cell death and angiogenesis. An oncogenic miRNA is intended to repress the translation of a tumor suppressor gene, provoking tumorigenesis, angiogenesis, invasion and leading to tumor formation. Conversely, a tumor suppressor miRNA can prohibit the expression of oncogene. Pri-miRNA: primary miRNA; pre-miRNA: precursor miRNA assessed miRNA profiles in tumor tissue, adjacent non-tumor tissue, corresponding plasma from the same BC patients, and plasma from matched healthy controls. In addition, they validated particular items by a case-control cohort and then blindly verified in an independent set of BC patients and healthy controls. Profiling of miRNAs showed that only three of them, including miRNA-16, miRNA-21 and miRNA-451, significantly increased in both plasma and tissue of BC patients and declined after surgery. Furthermore, miRNA-145 levels significantly decreased in BC patients. Interestingly, they found that the combination of plasma miRNA-451 and miRNA-145 levels was the best biomarker for BC prediction and prevention [39]. A study by Cuk et al. also emphasized the potential role of circulating miRNAs in plasma as early detection markers for BC. They found that four miRNAs; miRNA-148b, miRNA-376c, miRNA-409-3p and miRNA-801, were significantly up-regulated in the plasma of BC patients compared to healthy controls. The authors concluded that the identified miRNAs can be applied as multimarker blood-based tests for early detection of BC [40]. Si et al. introduced circulating miRNA-92a as a novel BC biomarker. The authors proposed that reduced levels of miRNA-92a and amplified levels of miRNA-21 in tissue and serum samples of BC were related to tumor size and a positive lymph node status compared to clinicopathologic data of the BC patients. In addition, miRNA-1, miRNA-92a, miRNA-133a, and miRNA-133b have been identified as the critical diagnostic biomarkers. These miRNAs were up-regulated and successfully validated in both serum and tumors of BC patients [41].
It seems that circulating miRNAs can be considered key biomarkers in the reduction of BC prevalence by enhancing the diagnosis, prognosis, prediction and the clinical management of patients and  their relatives. Accumulative evidence in recent years has highlighted the significant role of circulating nucleic acids in the blood, especially miRNAs, as early detection markers [81,82], and even for monitoring patient responses to various therapies for several unrelated diseases [83][84][85][86]. Moreover, assessment of miRNAs expression levels in other body fluids such as urine, is another non-invasive method for the detection of BC that works with miRNA markers [84].

Association between miRNA and DNA methylation
There is clear evidence of the prominent role of DNA methylation and miRNAs in cancer initiation, progression and even prevention [87]. It seems that in the epigenetic mechanisms, there is a bilateral relationship between DNA methylation pattern and miRNA expression profiles [88]. This mutual relationship can contribute to the diagnosis, prognosis and characterization of tumor tissue origination, metastatic capability and tumorigenesis potential in early stages of cancer [89]. In fact, by two major processes miRNAs can regulate DNA methylation in human cancers: 1) by modifying DNMTs and 2) by adjusting methylation of some crucial proteins, such as methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain proteins 2 and 4 (MBD2 and MBD4) [87]. In BC, miRNA-221 directly targets DNMT3b [88], miRNA-143 negatively correlate with DNMT3a [90], and miRNA-29b object DNMT3a and 3b [91].

Nutrigenomics and epigenetics in BC Essential and non-essential nutrients in BC
The role of some lifestyle factors, including BMI (overweight and obesity), habits, physical activity, smoking, and breastfeeding on BC diagnosis and prognosis have been assessed by various studies and the relationship between dietary patterns and nutrients with BC is inconsistent [96,97]. However, it has been suggested that one-third of all cancers in the USA are preventable by a change of diet and certain nutrients [98], and there are some studies showing that BC initiation and progression is at least in part, influenced by the diet. Essential nutrients, such as Folate, vitamin B 12 , vitamin D and non-essential nutrients like resveratrol, curcumin, tea phenols, genistein, sulforaphane, methionine, choline, and betaine can affect DNMT, DNA methylation, miRNAs oncogenic and tumor-suppressor miRNAs [99][100][101], highlighting a possible role of nutrigenomics in the epigenetic mechanisms of BC. The evidence shows that a healthy food plan specified by a high intake of vegetables, fruits, whole grains and legumes like beans, peas, lentils, and poultry, as well as low-fat and high-fiber dietary products, may impact on BC progression [101,102], whilst an unhealthy diet including a high intake of red or processed meat, high fat (saturated and trans fatty acid) dairy products and low intake of omega-3 fatty acids, refined grains and sugars, low intake of natural antioxidants, and fiber, can trigger the inflammation process, possibly enhancing the risk of BC [96,102].
Using nutritional genomics, we can elucidate if and how these different nutrients modify protein and gene expression leading to altered cellular function. Nutrition components can either act as carcinogenesis risk factors or show anti-cancer functions in cells and participate in initiation, progression and metastasis, or prevention and treatment of cancer cells [103]. The number of studies, which intend to clarify the effects of nutrients on the regulation of epigenetic mechanisms involved in BC prevention, is increasing and a few of them have been recapped in Table 3.

Bioactive components and epigenetics in BC
Roystone et al. explored the effects of Withaferin A (WA, found in the Indian winter cherry) and sulforaphane (SFN, found in cruciferous vegetables) acting as a DNMT inhibitor and histone deacetylase (HDAC) inhibitor, respectively. WA and SFN play a role in the regulation of cell cycle, and epigenetic-modifying enzymes in MCF-7 and MDA-MB-231BC cell lines, highlighting the capability of WA and SFN in enhancement of apoptosis, cell death and reduction of malignant expression of certain genes in BC cells [104][105][106][107][108][109][110][111][112]. -Although regulators of DNMT1, p21 and p53 over-expressed, there was no effect on DNMT1 suppression.  Administration of WA and SFN inhibited the cell cycle progression from S to G2 phase through down-regulation of cyclin D1 (CCND1), cyclin D kinase 4 (CDK4), phosphorylated retinoblastoma protein (pRB), and up-regulation of E2F mRNA. In addition, tumor suppressor p21 protein was increased in response to WA and SFN treatment, which may in part relate to the downregulation of pRB gene. In the same study, enhanced global methylation of MCF-7 and MDA-MB-231BC cell lines in BC cells was found [104]. Zhu et al. showed that trans-resveratrol can induce mammary promoter hyper-methylation in women at high risk for BC. The authors showed that two significant roles of trans-resveratrol include downregulation of DNMT [105] and cancer promoting prostaglandin (PG) E2 [113], as observed by in vitro investigations. It is also concluded that after increasing the Transisomer and the glucuronide metabolite predominate in the circulation throughout 12 week supplementation with trans-resveratrol, methylation of RASSF-1α which is known as a tumor suppressor of BC is decreased in addition to a reduction in PGE2 levels in breast cells [105]. Similarly, Qin et al. showed that resveratrol can reduce DNMT 1 and 3b expression in vitro and demethylate tumor suppressor RASSF-1α in individuals with a risk of BC development. In summary, they found that high doses of resveratrol (25 mg/kg/day) down-regulated DNMT 3b in tumor tissue compared to healthy tissues, while this effect was not observed for DNMT1. Additionally, tumor suppressive miRNAs: miRNA-21, miRNA-129, miRNA-204 and miRNA-489, were up-regulated more than two fold in tumor tissue and down-regulated two to 10 times in normal tissue, therefore negatively correlating with DNMT3b levels [106]. Pietruszewska et al. investigated the impact of sulforaphane (SFN) on the expression of DNA methylation-regulators in MCF-7 and MDA-MB-231 BC cells, including DNMT1, p53, and p21, as well as the methylation of PTEN and RARbeta2 tumor suppressor genes. They found that 22 µM or 10-46 µM of SFN best inhibited the growth of 50% of MCF-7 or MDA-MB-231 BC cells, and caused hypo-methylation of PTEN and RARbeta2 promoters, whilst their mRNA expression was up-regulated. Moreover, the expression of DNA methylation-regulators (p21 or p53) increased in both BC cell lines. Although the gene regulators of DNMT1, p21 and p53 were over-expressed in BC cell lines, no effect on DNMT1 suppression was observed [108]. However, in another study by this group observed an inverse relationship between p21 and DNMT1 expression because p21 competes with DNMT1 for the same binding site on a proliferating cell nuclear antigen (PCNA) during DNA replication [114]. Peng et al. suggest that 3,6-dihydroxyflavone (3,6-DHF) enhances miRNA-34a expression significantly in BC cells, and this component acts as a DNMT1 inhibitor in MDA-MB-231 cells [115]. More recently, 3,6-DHF has been found to up-regulate TSGs, such as miRNA-34a, through methylation of relevant promoters by suppressing DNMT1 during tumorigenesis. The level of ten-eleven translocation (TET) and 5-hydroxymethylcytosine (5hmc) are usually decreased in cancer cells, but administration of 3,6-DHF was found to increase the level of TET1 and 5hmc in MDA-MB-231 cells. Finally, because TET1 inhibition by siRNA was able to counteract the impact of 3,6-DHF on miRNA-34a promoter-demethylation and subsequent up-regulation, it has been proposed that an up-ward trend in TET1 expression through 3,6-DHF supplementation could be a nutritional measure to prevent BC [109]. A study by Kumar et al. investigated the effect of curcumin administration on the methylation status of glutathione S-transferase pi 1 (GSTP1) gene (a TSG) in MCF-7 BC cell line. While the ideal dose (IC50) dose of curcumin was 20µM, the non-toxic concentration of curcumin (10µM) reversed the hyper-methylation of the GSTP1 promoter and induce reexpression of GSTP1 protein [110]. Importantly, the authors noted that less than 3µM curcumin does not change the promoter methylation pattern of GSTP1 and greater than the IC50 and curcumin reinstates hyper-methylation of GSTP1 [110].

Essential components, BMI and epigenetics in BC
Adams et al. found a positive correlation between blood serum miRNA expression and the BMI of BC survivors, in the Hormones and Physical Exercise (HOPE) trial. In addition, the expression of 35 miRNAs were analyzed after a 6-month weight-loss trial (Lifestyle, Exercise, and Nutrition; LEAN). The HOPE trial reported that eight novel miRNAs, including miRNA-22-3p, miRNA-122-5p, miRNA-126-3p, miRNA-150-5p positively correlated with BMI, while miRNA-191-5p, miRNA-17-5p, miRNA-103a-3p, miRNA-93-5p negatively correlated. In the LEAN trial, miRNA-106b-5p, let-7b-5p, and miRNA-92a-3p were down-regulated and miRNA-27a-3p, miRNA-191-5p, miRNA-24-3p up-regulated after the 6 months of weight loss [107]. Pietruszewska et al. assessed the impact of 1, 4, and 8 mg/l folic acid on DNA methylation of PTEN, APC and RARbeta2 tumor suppressor genes in MCF-7 and MDA-MB-231 BC cells. They found that by increasing the concentration of folic acid, DNA methylation of these tumor suppressor gene promoters increased, transcriptional activities of these genes was impaired, and dose-dependent down-regulation of tumor suppressor genes occurred. These effects were more tangible in non-invasive MCF-7 cells as well as they indicated 30% up-regulation of DNMT1 expression at 8 mg/l dose [111].
The impact of nutrigenomics on cancer is still unclear and it needs further research to determine the exact mechanisms that food components use to influence cancer processes. Thus, investigating the processes utilized by dietary components in BC development is an important future direction for nutrigenomics. Evidence shows that the epigenetic impact of dietary compounds can intervene in various cell functions, such as proliferation and differentiation, cell cycle regulation, apoptosis, metastasis and angiogenesis by modifying miRNA expression and DNA methylation. Thus, it seems that nutrigenomics may provide new insight on treatment and prevention strategies to many diseases, including cancer.

Future direction
Hundreds of abnormal gene methylation patterns, either hypermethylation of TSGs or hypo-methylation of oncogenes, have been identified in BC. The involved genes can differ from one person to another according to many factors including age, ethnicity and family history. Epigenetic pathways, such as aberrant DNA methylation and improper expression of miRNAs, have particularly profound effects on initiation of BC and participate in BC metastasis, cell proliferation, division and angiogenesis. Nowadays, the most important question is whether it is possible to control and handle the epigenetic mechanisms by producing some nutraceutical or pharmaceutical substances. In other words, if the underlying cause of BC is dysregulation of tumor suppressor miRNAs or onco-miRNA secretion, is it possible to control BC by producing drugs based on miRNAs or natural products? Another avenue of research yet to be explored is the possible role of other small non-coding RNAs, including snRNA, snoRNA and siRNA in treatment and diagnosis of the onset of BC, especially in individuals with a high risk for BC development. This would lead on to testing the effects of food components in regulating the expression or function of these small non-coding RNAs. The most pressing area for nutrigenomic research in relation to BC, is in improving the diagnosis and treatment methodologies, especially in the early stage of the disease.

Concluding remarks
Assessment of circulating nucleic acids like miRNA, mRNA and CF-DNA in body fluids, such as saliva, bronchial aspirates, sera, plasma, urine, and even in stool could be a modern and noninvasive method for monitoring the occurrence or development of cancer, early detection and its recurrence. Hence, understanding the mechanisms of nutrigenomics in relation to epigenetics and disease has the potential to revolutionize BC diagnosis methods, prevention and treatment therapies. Particular focus on diagnosis and treatment at the onset of BC, and in patients most at risk, will be most effective in fighting the disease.