DNA methylation is the primary epigenetic mechanism extensively studied for its significant role in regulating gene expression. The process of DNA methylation entails the covalent addition of methyl group to the C-5 position of the cytosine in cytosine-guanine (CpG) dinucleotides by DNA methyltransferases (DNMTs). DNMT1 maintains the pre-existing methylation patterns during replication, while DNMT3A and DNMT3B govern de novo methylation [12]. Methylation of CpGs at the promoters of genes leads to suppression of gene expression, whereas methylation levels in the gene body positively correlate with expression. DNA methylation is critical in maintaining chromosome stability, X chromosome inactivation, transposable element suppression, aging, and genomic imprinting. Moreover, it influences several disease conditions, including cancer and autoimmune disorders [12].

Breast cancer cells often exhibit disruptions of normal methylation patterns. The hypermethylation of promoter region leads to inactivation of tumor suppressor genes, such as BRCA1, APC, CDH1, CCND2, and CTNNB1. On the other hand, breast cancer exhibits global DNA hypomethylation, with up to 50% of instances exhibiting lower 5-methylcytosine content than their counterparts in normal tissue [13]. Hypomethylation in repetitive sequences and pericentromeric satellite DNA is prominent in cancer cells, and it generally remains highly methylated in normal breast cells. For example, retrotransposons, including long interspersed nuclear elements (LINEs), are hypomethylated in breast cancer [14]. In addition, Sat2 and Sat$\alpha$ repeats are frequently hypomethylated in a variety of malignancies, including breast cancer. Hypomethylation of these transposable elements leads to their transcriptional activation, further contributing to genomic instability [14, 15]. Moreover, several lines of evidence demonstrated gene-specific hypomethylation in breast cancer, such as hypomethylation of Urokinase-type plasminogen activator (uPA), synuclein-$\gamma$ gene (SNCG), and multidrug resistance 1 gene (MDR1) [16, 17, 18].

Another key epigenetic mechanism is the function of non-coding RNAs (ncRNAs), which constitute approximately 62–75% of the genome [19, 20]. To date, numerous ncRNAs have been found, including circular RNA (circRNA), short hairpin RNA (shRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), small-interfering RNA (siRNA), and long non-coding RNA (lncRNA). These ncRNAs have emerged as an essential source of biomarkers and targeted therapies due to a growing understanding of their role in various diseases [21].

The dysregulation of miRNAs, one of the crucial ncRNAs, has come to light in the emergence of multifactorial disorders, including breast cancer. miRNAs are short (18–22 nucleotides) endogenous single-stranded RNA molecules that attach to the target mRNA to limit translation. Aberrant miRNA activities are associated with breast cancer onset and progression [22]. For instance, metastatic breast cancer has been associated with overexpression of miR-21 and miR-155 [23]. Similarly, patients with higher expression levels of miR-1307-3p, miR-940, and miR-340-3p were observed to experience decreased overall survival rates [24]. Furthermore, miR-497 regulates cell growth and invasion of breast cancer cells through inhibiting the expression of cyclin E1 [25].

LncRNAs, another important class of non-coding RNAs ranging from 200 nucleotides to 100 kilobases in length, can regulate gene expression at various levels by interacting with DNA, RNA, and proteins [26]. Dysregulation in lncRNA regulatory activity facilitates breast cancer development and dissemination. For example, growth arrest-specific 5 (GAS5) remains highly downregulated in breast cancer and regulates the expression of several tumor-associated genes. Employing various pathways, such as mitochondrial signaling pathways and cell death receptors, GAS5 can induce apoptosis in breast cancer. Moreover, it plays a critical role in regulating PI3K/AKT/mTOR, NF-$\kappa$B, and Wnt/$\beta$-catenin pathway [27]. Another crucial lncRNA, metastasis associated lung adenocarcinoma transcript 1 (MALAT1), is aberrantly expressed in several kinds of breast cancer types, potentiates metastasis and a poor prognosis [28].

Histones serve as essential DNA packaging proteins, thereby maintaining chromatin structure. Histone proteins (H2A, H2B, H3, and H4) assist in generating nucleosomes by wrapping octamers around 147 bp of DNA. Post-translational modifications (PTMs) of histone cause changes in the state of chromatin, subsequently leading to gene expression regulation [29]. Specific residues of the amino and carboxy ends of the histone tails undergo several modifications, including acetylation, phosphorylation, methylation, sumoylation, ubiquitination, ADP-ribosylation, glycosylation, and carbonylation. Different enzymes, such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and histone demethylases (HDMs), catalyze these modifications. Alterations in the expression of these enzymes also implicate the onset and advancement of cancer [29]. Numerous alterations have been identified in breast cancer, including the upregulation of p300, HBO1, HDAC1, HDAC2, HDAC3, and HDAC6 [30].

Histone methyltransferases, including lysine methyltransferase 2 (KMT2), were found to be linked with the proliferation and metastasis of breast cancer cells. KMT2 activates oncogenes and pro-metastatic genes by methylating H3K4 at both enhancer and promoter regions [31]. Amplification and overexpression of a crucial histone methyltransferase enhancer of zeste homolog 2 (EZH2) are prominent in breast cancer. By catalyzing H3K27 methylation, EZH2 facilitates transcriptional silencing of several genes and persuades epithelial to mesenchymal transition (EMT) and metastasis in breast cancer [32, 33]. Another prominent HMT, disruptor silencing 1 like (DOT1L), is also known to potentiate metastatic behavior of breast cancer cells [34].

Several histone demethylases, including Lysine-specific demethylase 4A (KDM4A), Lysine-specific demethylase 4B (KDM4B), and Lysine-specific demethylase 4C (KDM4C) are also associated with breast cancer. ER$\alpha$ positive subtype cells exhibit overexpression of KDM4A and KDM4B, whereas in the case of TNBC, enhanced levels of KDM4C are observed [35]. KDM4A triggers a Notch1-NICD-dependent signaling pathway to promote breast cancer growth and metastasis [36]. KDM4B regulates estrogen signaling, and downregulation in KDM4B expression limits breast cancer growth [37]. KDM4C acts as a co-activator of HIF-1$\alpha$/VEGF signaling, thereby promoting breast cancer progression [38].

Estrogens, the predominant sex hormones in women, play a crucial role in reproductive maturity, bone growth, and energy homeostasis. The five primary subtypes of estrogen are estrone (E1), 17-$\beta$ estradiol (E2), estriol (E3), estetrol (E4), and estrone-sulfate (E1s) [39]. Among them, E1 and E2 constitute the majority of estrogens in the body. Besides, E1 primarily functions as a reservoir for estrogen undergoes a reversible conversion to E2, which is the more physiologically active form. Whereas, E3 and E4 are found only during pregnancy, with E3 being the most prevalent. Actions of estrogens are facilitated through two primary types of intracellular estrogen receptors (ERs), namely ER$\alpha$ and ER$\beta$, encoded by the genes ESR1 and ESR2, respectively [40]. ER$\alpha$ positive subtype, which constitutes around 75% of breast tumors, exhibits a better prognosis due to their sensitivity towards hormonal therapy. Conversely, poor prognosis and aggressive tumor growth are observed in the case of ER$\alpha$ negative subtypes [41]. Epigenetic silencing via abnormal methylation of the ER$\alpha$ promoter is one mechanism implicated in the suppression of ER$\alpha$ expression (Fig. 2). Recruitment of Zinc Finger E-box Binding Homeobox 1 (ZEB1)/DNA Methyltransferase 3 $\beta$ (DNMT 3$\beta$)/Histone Deacetylase 1 (HDAC 1) complex on the ESR1 promoter contributes to hypermethylation of ESR1 (Fig. 2). Likewise, enhancement in histone deacetylation also suppresses ESR1 transcription by inducing condensation of the nucleosome structure [42].

Fig. 2.

Epigenetic mechanisms regulating the expression of ER$\alpha$ and ESR1 in breast cancer. (A) DNA methylation mediated suppression of ER$\alpha$ expression, (B) suppression of ESR1 expression by ZEB1/DNMT 3B/HDAC1 complex. DNMT, DNA Methyltransferase; Me, Methylated; ER$\alpha$ , Estrogen Receptor $\alpha$; ESR1 , Estrogen Receptor 1; ZEB1, Zinc Finger E-box Binding Homeobox 1; DNMT3B, DNA Methyltransferase 3 Beta; HDAC1 , Histone Deacetylase 1. Created with ChemDraw Professional (version: 16.0, PerkinElmer Inc., Waltham, MA, USA).

Upon binding of E2, dimerization of ER$\alpha$ monomers takes place, which further translocates to the nucleus and serves as a transcription factor regulating the expression of various genes [43]. E2-ER$\alpha$ dimers recruit ATP-dependent chromatin remodeling complexes (SWI/SNF) and HATs, including p300, p160, CREB-binding protein (CBP), and p300/CBP-associated factor (pCAF), to estrogen-responsive promoters. Eventually, it facilitates breast cancer cell proliferation and promotes tumor growth [44].

Several studies demonstrated a significant role of miRNAs in suppressing ER expression [45, 46]. For instance, overexpression of miR-221 and miR-222 results in post-transcriptional suppression of ER$\alpha$. Therefore, miR-221 and miR-222 facilitate downregulation in the expression of several tumor suppressors, including Phosphatase and Tensin Homolog (PTEN), Cyclin-Dependent Kinase Inhibitor 1B (CDKN1B), Cyclin-Dependent Kinase Inhibitor 1C (CDKN1C), BCL2 Interacting Mediator of Cell Death (BIM), Tissue Inhibitor of Metalloproteinases 3 (TIMP3), and Forkhead Box O3 (FOXO3), as well as lead to estrogen-independent uncontrolled proliferation [45, 46].

EMT is a cellular process wherein cells transform, losing their epithelial traits and adopting mesenchymal characteristics. EMT plays a pivotal role in wound healing, development, and progression of cancer. It makes cancer cells more migratory and endows drug resistance, stemness, and recurrence. Different growth factors (Transforming Growth Factor $\beta$ (TGF-$\beta$), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Insulin-like Growth Factor (IGF), and Platelet-Derived Growth Factor (PDGF)) trigger signaling pathways to instigate EMT transcription factors (Snail Family Transcriptional Repressor (SNAI), Zinc Finger E-box Binding Homeobox (ZEB), and Twist Family BHLH Transcription Factor (TWIST)) and eventually orchestrate EMT. These Transcription factors (TFs) have been demonstrated to form regulatory complexes with various proteins involved in transcriptional regulation, including epigenetic regulators [47]. In different cancer conditions, including breast cancer, the collaboration between the transcription factors of EMT and epigenetic regulators is prominent, which leads to EMT induction by regulating the expression of genes involved in EMT [48].

For example, in breast cancer, DOT1L collaborates with c-Myc and p300 to promote the methylation and acetylation of H3K79 in the promoter areas of EMT transcription factors, which leads to increased expression of these genes (Fig. 3). Consequently, this process promotes EMT-associated cancer stemness characteristics [47]. Furthermore, invasive breast cancer cells exhibit aberrantly high levels of methylation in CDH1, which is associated with downregulation in the expression of E-cadherin [48]. E-cadherin is a tumor suppressor that facilitates cell-to-cell adhesion between neighboring cells [49]. G9a silences transcription at the E-cadherin promoter in breast cancer by interacting with Snail (Fig. 3) [50].

Fig. 3.

Role of epigenetic mechanisms in epithelial to mesenchymal transition (EMT) dynamics of breast cancer. CDH, Cadherin; DOT1L, Disrupter of telomere silencing protein 1; c-MYC, Cellular Myelocytomatosis Oncogene; EMT-TF, Epithelial to Mesenchymal Transition Transcription Factor; G9a/EHMT2, Euchromatic Histone Lysine Methyltransferase 2; SNAIL: Snail Family Transcriptional Repressor. Created with ChemDraw Professional (version: 16.0, PerkinElmer Inc., Waltham, MA, USA).

Histone deacetylase inhibitor Trichostatin A (TSA) reverses EMT in breast cancer by inhibiting Slug expression [51]. Additionally, inhibiting Bromodomain protein 4 (BRD4) leads to suppression of Gli1, which is necessary for the transcriptional activation of Snail. This suggests that BRD4 regulates the aggressiveness of breast cancer cells through both transcriptional regulation of Snail and post-translational mechanisms [52]. Thus, understanding the interplay between EMT and epigenetics offers new avenues for cancer treatment.

Emerging evidence suggests that a tiny population of cancer cells promotes the recurrence and dissemination of the disease known as cancer stem cells (CSC). Like other CSCs, breast cancer stem cells (BCSCs) comprise a subset of diverse breast cancer cells with substantial potential for proliferation and self-renewal. Several studies demonstrated that BCSCs are the main contributory factor of drug resistance, recurrence, metastasis, and the onset of the disease [53]. During development, epigenetic machinery is crucial in reprogramming stem cells to facilitate differentiation into specific cellular and tissue lineages. In several malignancies, including breast cancer, aberrant epigenetic alterations often potentiate the generation of CSCs, which lack differentiation capacity [54].

Epigenetic mechanisms are pivotal in regulating the expression of numerous genes involved in signaling pathways associated with cancer stemness, including Wnt, Notch, Hedgehog, and Hippo/yes-associated protein 1 (YAP) [55]. In breast cancer cells, the promoters of genes involved in Wnt/$\beta$-catenin signaling pathways are often found to be hypermethylated [56]. Furthermore, the prominent downregulation of DNA methylation leads to decreased expression of Wnt inhibitors, such as Wnt inhibitory factor 1 (WIF-1), several frizzled-related proteins (SFRP1-5), and Dickkopf-Related Protein 1 (DKK1), in cancer cells (Fig. 4). Consequently, this enhances Wnt/$\beta$-catenin signaling activity in breast cancer stem cells compared to non-CSCs [55, 56]. Another study unveiled an abundance of H3K27me3 and decreased H3K4me2 levels in triple-negative breast cancer stem cells (TNBCSCs) [57]. These epigenetic alterations significantly impact various stemness-associated signaling pathways, including Wnt and human gonadotropin-releasing hormone (GNRH), consequently fostering aggressiveness and drug resistance in TNBCSCs [55]. Epigenetic alterations can also regulate Hedgehog pathway machinery and instigate the onset and advancement of several tumors. In breast and gastric cancer, hypomethylation of the promoter region of the SHH gene results in increased expression of the ligand (Fig. 4). Overexpression of SHH leads to the expression of hedgehog pathway downstream genes, thus facilitating cancer progression and self-renewal capacity [58, 59]. Hyperactivation of Notch signaling, particularly Notch 1 and 4, enhances metastatic and self-renewal traits of BCSCs [55]. In BCSCs, EZH2 serves as a Notch 1 activator, correspondingly regulating Notch signaling to potentiate the growth of CSC populations and cancer development [60]. Histone acetylases and deacetylases are recruited by Hairy and enhancer of split-1 (HES) (Fig. 4), a Notch effector and transcriptional repressor, one of the epigenetic processes controlling Notch signaling [61]. Furthermore, in breast cancer and pancreatic adenocarcinomas, downregulation of miR-200 members leads to activation of the Notch pathway and consequences cell survival, CSC maintenance, and therapeutic resistance [62].

Fig. 4.

Regulation of signaling pathways associated with cancer stemness, including Wnt, Notch Hedgehog, and Hippo pathways by epigenetic mechanisms. LRP, Low-Density Lipoprotein Receptor-Related Protein; WNT, Wingless-Type Integration Site; DVL, Dishevelled Segment Polarity Protein; GSK, Glycogen Synthase Kinase; APC, Adenomatous Polyposis Coli; WIF1, WNT Inhibitory Factor 1; SFRP1, Secreted Frizzled-Related Protein 1; DKK1, Dickkopf-Related Protein 1; NICD, Notch Intracellular Domain; MAML, Mastermind-Like Protein; CSL, Suppressor of Hairless; LIFR, Leukemia Inhibitory Factor Receptor; LATS1/2, Large Tumor Suppressor Kinase 1/2; YAP, Yes-Associated Protein; TAZ, Transcriptional Coactivator with PDZ-Binding Motif; TEAD, TEA domain transcription factor; SHH, Sonic Hedgehog Signaling Molecule. Created with ChemDraw Professional (version: 16.0, PerkinElmer Inc., Waltham, MA, USA).

Dysregulation in Hippo signaling has been linked to increased stem cell proliferative and self-renewal capacities of cancer cells. It has been reported that by altering Leukemia inhibitory factor receptor (LIFR) expression, miRNA-125a indirectly regulates the activity of Transcriptional coactivator with PDZ-binding motif (TAZ), an effector molecule in the Hippo pathway [63]. Furthermore, another crucial miRNA, miR-520b, activates Hippo/YAP signaling by targeting Large tumor suppressor kinase 2 (LATS2), thereby promoting stemness in breast cancer patients (Fig. 4) [64].

Innate and acquired resistance towards conventional chemotherapeutic drugs facilitates cancer recurrence and poor prognosis. The occurrence of cancer stem cells (CSCs) and the EMT process are thought to be the leading causes of cancer therapeutic resistance. According to accumulating evidence, drug resistance in several cancers, including breast cancer, is also influenced by aberrant epigenetic regulation [65]. Tamoxifen resistance in breast tumors is caused by EZH2-mediated suppression of Growth Regulating Estrogen Receptor Binding 1 (GREB1) expression, an ER$\alpha$ cofactor [66]. In breast cancer cells, loss of ten-eleven translocation 2 (TET2) resulted in decreased expression of ER$\alpha$, leading to endocrine resistance [67]. Through reducing p53 stability and enhancing the expression of Amplified in breast cancer 1 (AIB1), Histone acetyltransferase Lysine Acetyltransferase 2A (LAT2A) promotes tamoxifen resistance in breast cancer [68]. Another study reported that inhibition of CBP/p300 activity suppresses ER$\alpha$ function and eventually restricts breast cancer cell growth [69]. Histone demethylase LSD1 is involved in the regulation of self-renewal of breast CSCs, which contributes to the development of chemoresistance in breast cancer [70]. Additionally, in breast cancer, lysine-specific demethylase 1A (LSD1) regulates EMT by interacting with Protein kinase C $\theta$ (PKC-$\theta$) and induces drug resistance [71]. According to Metzger et al. [72], KDM4A governs the growth and self-renewal of breast CSCs, which facilitates therapeutic resistance. Moreover, insulin like growth factor 1 receptor (IGF1R) and erythroblastic oncogene B (ErbB) signaling can also be triggered by KDM5A, which then activates the PI3K/AKT/mTOR pathway and tamoxifen resistance [73]. Zhou et al. [74] showed that the overexpression of peptidyl arginine deiminase 4 (PAD4) induced apoptosis and elevated the levels of GSK3$\beta$ and p53, making cells more susceptible to Adriamycin. Overall, epigenetic machinery is implicated in therapeutic failure, and targeting epigenetic regulators shows significant potential for overcoming breast cancer drug resistance.

DNA methylation entails the transfer of a methyl group to the fifth carbon of cytosine, orchestrated by a set of enzymes known as DNA methyltransferases (DNMTs), comprising DNMT1, DNMT3A, DNMT3B, and DNMT3L. DNA methylation leads to the silencing of gene expression by impeding the binding of transcription factors to the DNA [12]. The DNMT inhibitors work by incorporating themselves between the DNA base pairs and preventing the methylation of the promoter sequence (CpG islands). Decitabine and Azacitidine are well-known first-generation DNMT inhibitors that follow this mechanism [78].

DNMT inhibitors are of three types, namely non-nucleosidic, nucleosidic, and natural compounds. Natural compounds such as Epigallocatechin-3-gallate (EGCG), catechin, and quercetin demonstrated the ability to inhibit the activity of DNMT1. This inhibition leads to the demethylation of DNA, reactivation of tumor suppressor genes, and reduction of breast cancer cell proliferation [79]. A non-nucleosidic compound called hydralazine is conventionally used as an antihypertensive drug but serves to reduce the expression of DNMTs in mammals [80]. Hydralazine, combined with valproic acid, a mild demethylating drug, decreases cancer cell survival dose-dependently [81]. Zebularine, a potent nucleoside DNMT inhibitor, upregulates p21 expression, downregulates cyclin-D expression, induces apoptosis, and arrests MCF-7 and MDA-MB-231 cells in the S-phase [82]. Procainamide, an antiarrhythmic drug, is a non-nucleoside DNMT analog that sensitizes the ER$\alpha$ positive breast cancer cells to tamoxifen therapy by upregulating ER$\beta$ expression [83]. An anti-inflammatory compound used to treat ulcerative colitis, called Olsalazine, is found to escalate the expression of the CDH1 gene, which encodes for E-cadherin; thereby, it abridges EMT in MDA-MB-231 cells [84]. Liraglutide, a type II diabetes drug, has been found to function as a DNMT inhibitor by curtailing the methylation of ESR1, CDH1, and ADAM33 promoter regions in MCF-7, MDA-MB-231, and MDA-MB-436 cell lines [85]. In addition, five potential candidates, namely C-7756, C-5769, C-1723, C-2129, and C-2140, were reported as potential DNMT1 inhibitors as per the in-silico studies [86].

Histone acetyltransferases (HATs) and Histone Deacetylases (HDACs) are essential enzymes involved in the regulation of gene expression and chromatin remodeling [87]. HATs mediate the addition of an acetyl group from acetyl-CoA to the lysine residues, resulting in the neutralization of the charge of lysine. This process results in the transcriptional activation of the underlying DNA sequence due to the slackened chromatin structure [87]. On the contrary, HDACs catalyze the acetyl group removal from the histones, leading to a more stable and condensed chromatin structure contributing to transcriptional repression [88]. Any dysregulation in the dynamic interplay between these enzymes may result in life-threatening diseases like cancer [75].

Extensive in silico studies, including expression and survival studies, have recognized p300 acetyltransferase as the critical tumorigenic driver. Screening from 1293 FDA-approved drugs using Molecular Docking (MD) and molecular dynamic simulation (MDS) have identified Netarsudil and Imatinib as potential repurposed drugs against the p300 HAT domain. Further, these drugs exhibited significant anti-proliferative activity against breast cancer cell lines [89]. In addition, several HAT inhibitors from natural sources such as Carnosol, Garcinol, Anacardic acid, and Curcumin are investigated in breast cancer. Carnosol, a polyphenol abundant in rosemary, oregano, and sage plants, serves as a potential inhibitor of p300. It blocks the acetyltransferase activity of p300 by impeding the acetyl CoA binding site, which impels histone hypoacetylation in MDA-MB-231 cells [90]. Another natural compound, Garcinol, is reported to be an effective inhibitor of CBP/p300 mediated acetylation of p53 in Michigan Cancer Foundation-7 (MCF-7) cells. Furthermore, a significant decrease in H3K18 methylation was observed, thereby hindering the MCF-7 cell growth by arresting the cells at the G1 phase. Curcumin also inhibits the activity of p300/CBP, which blocks the MCF-7 cells at the G2/M phase [91, 92]. Anacardic acid is predominantly found in Anacardiaceae members like Amphipterygium adstringens, cashew, ginko, etc. It is a non-selective HAT inhibitor with HSP90 ATPase inhibitory potential to suppress Heat shock protein 90 (Hsp90) oncoproteins overexpressed in TNBC cells [93]. Pentamidine is a chemical compound used to treat protozoal infection. It has been shown to repress DNA and protein synthesis by targeting Tip60, a HAT from the MYST family (MOZ, Ybf2/ Sas3, Sas2 and Tip60 family), thereby dwindling the H2A acetylation [94]. In addition, bisubstrate inhibitor A is more capable of restraining the HAT activity by resembling the HAT substrate possessing a lysine peptide and an acetyl-CoA group [95]. Some other notable HAT inhibitors include NU9056, MG-149, TH1834, and Lys-CoA [94].

The HDAC inhibitors can be subdivided into three groups, namely, Hydroxyamates (Vorinostat, Belinostat, Dacinostat, Panobinostat, CUDC-10, Quisinostat, and Tefinostat), Benzamides (Tacedinaline, Entinostat, Mocetinostat, and Chidamide) and Carboxylic acid HDAC inhibitors (Butyric acid, Pivanex, Phenylbutyric acid, and Valproic acid). Electrophilic ketones and cyclic peptides are recently explored for their HDAC inhibitory activity [96]. Vorinostat, commonly known as suberoylanilide hydroxamic acid (SAHA), is one of the most prevalent first-generation HDAC inhibitors to be discovered first. It functions by inhibiting the class I and II family of HDAC and requires a Zn²⁺ ion for the catalytic binding. Vorinostat is known to deplete the ER$\alpha$ expression in breast cancer cells by channeling it to the ubiquitin-proteasome degradation pathway, which mitigates cell proliferation, migration, and invasion [97]. Quisinostat, a second-generation class I and II HDAC inhibitor, disrupts the self-renewability of cancer stem cells by inducing the expression of histone H1, which acts as a tumor suppressor without affecting the normal stem cells [98]. Tacedinaline, a class I and II HDAC inhibitor, yielded promising outcomes by suppressing the BIRC5 gene, which is typically overexpressed in TNBC and inhibits the expression of apoptotic proteins [99]. Mocetinostat, a class I and IV HDAC inhibitor, was found to trigger apoptotic cell death in 4T1 breast cancer cells in combination with capecitabine [100]. Chidamide, an inhibitor of HDAC 1, 2, 3, and 10, belongs to the benzamide class of HDAC inhibitors exhibited promising efficacy in the treatment of human epidermal growth factor receptor (HER)+ breast cancer patients along with exemestane [101]. Following the failure of cyclin-dependent kinases 4/6 (CDK4/6) inhibitor administration in Hormone receptor (HR)+ and HER2-negative advanced breast cancer patients, Tucidinostat combined with endocrine therapy was an emphatic strategy for such patients [102].

Histone methyltransferases are essential for adding methyl groups to specific lysine or arginine residues on histone proteins. This process is catalyzed by lysine methyltransferases (KMTs) or arginine methyltransferases (RMTs), which derive the methyl groups from S-adenosyl-l-methionine (SAM). KMTs append 1, 2, or 3 methyl groups to the lysine residues, while RMTs add 1 or 2 methyl groups to the arginine residues. It is further classified based on the presence or absence of Su(var)3-9, Enhancer-of-zeste, and Trithorax domain (SET domain) as SET domain lysine methyltransferases and Disruptor of telomeric silencing 1 (DOT1) domain lysine N-methyltransferase [75]. A few histone methyltransferases include EZH2, G9a, DOT1L, and Protein arginine methyl transferase (PRMT). Chaetocin, derived from Chaetomium fungi, explicitly targets the H3K9 tri-methylation mediated by SUV39H1. In MDA-MB-231 cells, it inhibited cell proliferation and induced apoptosis by activating caspase 3 and Poly (ADP-ribose) polymerase (PARP) proteins [103]. A PRMT5 inhibitor, GSK3326595, is in a phase II clinical trial for treating early-stage HR+ breast cancer. JNJ-64619178 is one of the potent inhibitors of PRMT5, and it is currently under clinical trial for advanced tumors [104]. Pemrametostat is an oral small molecule inhibitor under investigation, which inhibits PRMT5 and reduces the arginine methylation levels of histones H2A, H3, and H4, thus downregulating the cancer cell proliferation. Sinefungin is a natural inhibitor of EHMT1/2 derived from Streptomyces incamatus and S. Griseolus [105]. EZH2 specific inhibitors that inhibit its activity by competing with S-Adenosyl methionine (SAM) are 3-deazaneplanocin A hydrochloride (DZNep), GSK2816126, EPZ005687, GSK343, GSK926, Tazemetostat, CPI-1205, etc. Some inhibitors broadly curb the activity of both EZH1/2, like UNC1999, ORS1/ORS2, and DS-3201b, while the others target the EZH2 degradation, such as GNA022, ANCR, FBW7, ZRANB1 [106].

Histone demethylases are divided into two classes based on their specificity as Flavin adenine dinucleotide (FAD) dependant lysine-specific demethylases (LSDs) and 2-oxoglutarate- ferrous iron and oxygen-dependent demethylases, which encompasses the Jumonji C domain-containing family. The former can demethylate mono and dimethylated substrates, while the latter can demethylate mono, di, and trimethylated substrates [107]. Phenelzine, a monoamine oxidase inhibitor, inhibits the activity of LSD1 irreversibly by downregulating the mesenchymal markers alongside nab-paclitaxel in metastatic breast cancer patients. It was also found to inhibit the cancer stem cells and reduce the number of circulating tumor cells that give rise to metastasis [108]. Pargyline is a monoamine oxidase inhibitor that blocks the activity of LSD1, exhibits a dose-dependent decrease in cell proliferation, and elevated expression of cleaved PARP proteins that consequences apoptosis [109]. The MAO-A gene is primarily associated with depression but is often found to be overexpressed in breast cancer cells, contributing to poor prognosis. Clorgyline, an MAO-A inhibitor, facilitates the mesenchymal to epithelial transition (MET) in metastatic MDA-MB-231 cells by promoting the expression of epithelial markers such as E-cadherin while concurrently altering the expression of mesenchymal markers like vimentin. In addition, it suppresses cell proliferation, anchorage-independent growth, and invasiveness [110]. ORY-1001 (Iadademstat) is another LSD1 inhibitor that suppresses androgen receptor expression in TNBC cells like MDA-MB-231 and BT549. It also abridges the multiplication of cells and provokes apoptosis [111]. 4SC-202, also referred to as Domatinostat is a potent inhibitor targeting both HDAC class I and LSD1 [112]. It demonstrates cytostatic and cytotoxic effects, making it a promising candidate for cancer therapy. Treatment of MDA-MB-231 and 4T1 TNBC cells with 4SC-202 led to cytotoxic effects, prohibited migration, invasion, and mitigated lung metastasis [112].

Combining epidrugs with chemotherapy or immunotherapy offers potential benefits such as reduced drug resistance and improved overall treatment outcomes [113, 114]. This integrated strategy holds immense potential to fight against breast cancer, paving the way for personalized and effective therapeutic interventions. Several such combinations are under clinical trial to optimize the dosage and evaluate long-term safety and efficacy (Table 2, Ref. [75, 82, 85, 97, 99, 100, 101, 108, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126]). Combining HDAC inhibitors has proven to be more efficient than using them individually [113, 127]. The co-administration of Estrogen Related Receptor $\alpha$ (ERR$\alpha$) inhibitor C29 with DNMT inhibitor like 5-aza-2${}^{\prime }$-deoxycytidine (decitabine) inhibits the crosstalk between DNMT1 and ERR$\alpha$, which contributes to the stability of ERR$\alpha$ that in turn contributes to DNA methylation. The conjunction of these drugs helps to reactivate the tumor suppressor gene Interferon Regulatory Factor-4 (IRF4), which mitigates the proliferation of breast cancer cells [115]. Panobinostat, an HDAC inhibitor in combination with Trastuzumab, is under clinical trials for treating HER+ metastatic breast cancer [128]. Vorinostat and Valproic acid intensified Trastuzumab mediated antibody dependent cell-mediated phagocytosis and cytotoxicity. As well as it decreased the expression of anti-apoptotic protein myeloid leukemia cell differentiation 1 (MCL1) in HER+ SKBR3 cells [116]. Entinostat, along with Lapatinib, was found to reduce the colony-forming potential by inhibiting phosphorylated AKT and FOXO3 mediated Bim expression that triggers apoptosis [117]. In the case of Vorinostat and Olaparib combined treatment, Vorinostat inhibits HDAC while Olaparib blocks polyadenosine 5${}^{\prime }$ diphosphoribose polymerase (PARP). Together, it affects the ability of cancer cells to repair DNA damage, which results in tumor cell death [97]. Valproic acid, a popular HDAC inhibitor, has shown to fuel up the anti-cancer activity of Capecitabine by upregulating the expression of thymidine phosphorylase (TP). TP is a key enzyme involved in the conversion of 5${}^{\prime }$-deoxy-5-fluorouridine (5${}^{\prime }$-DFUR) to 5-FU, which is further metabolized to 5-fluoro-2-deoxyuridine monophosphate (FdUMP). Further, FdUMP curbs the thymidine synthesis by interfering with DNA replication by inhibiting thymidylate synthase (TS). These two drugs synergistically induced apoptosis, RNA, and DNA damage in the breast cancer cell lines, including MCF-7, MDA-MB-231, MDA-MB-468, and SKBR3 [118]. Azacitidine and Entinostat combination, under phase 2 clinical trial, was synergistically found to inhibit DNA synthesis and class I HDAC activity [119]. Another HDAC inhibitor, Panobinostat, was proven to enhance the efficacy of Trastuzumab by evoking Natural killer (NK)-cell mediated immune response in a C-X-C Motif Chemokine Receptor 3 (CXCR3) and Interferon$\gamma$ (IFN$\gamma$) dependant manner in HER+ breast cancer patients [120]. Combining Entinostat and Atezolizumab mutually targeted the HDAC activity and protein-programmed cell death ligand 1 (PD-L1) in TNBC cells [121].

Moreover, Letrozole, a widely recognized aromatase inhibitor, when paired with Panobinostat, successfully reinstated ER$\alpha$ expression and enhanced cell sensitivity to endocrine therapy. This effect was characterized by elevated acetylation of H3 and H4 histones and resulted in the inhibition of tumor progression in ER-negative breast cancer cells [122]. Vorinostat has been shown to augment the anti-apoptotic effect of paclitaxel through acetylation of histone and $\alpha$-tubulin. It also deterred the interaction of Hsp90 with AKT, c-RAF, and HER2 through proteasomal degradation of Hsp90 and exhibited antiangiogenic activity by altering the Vascular endothelial growth factor (VEGF) signaling pathway [123]. Hydralazine, a DNA demethylating agent, in combination with an HDAC inhibitor like magnesium valproate, synergistically induced gene reactivation and reduction in global C5 methylation in locally advanced breast cancer patients along with Doxorubicin and Cyclophosphamide [124]. A combination therapy using Romidepsin, an HDAC inhibitor, Gemcitabine, a DNA synthesis inhibitor, and DNA damaging agent, Cisplatin, was found to induce cell death in TNBC cells like MDA-MB-231, MDA-MB-468, and MCF-7 by the generation of reactive oxygen species (ROS) and proteolytic cleavage of PARP [125]. DNMT inhibitor Decitabine successfully abridged the cancer cell proliferation, DNA methylation, and DNMT1 activity along with Doxorubicin [6].