Advances in the epigenetic regulation of breast cancer

Abstract

The clonal evolution of breast cancer involves a complex dialogue between tumor cells and their environment. In this process, epigenetic mechanisms play a crucial role in regulating the cellular transcriptome without altering the underlying DNA sequence. Here, we provide an updated summary of three main epigenetic mechanisms: histone modifications, long non-coding RNAs (lncRNAs), and higher-order chromatin structures. Post-translational modifications of DNA or histones influence gene expression by altering chromatin accessibility and/or recruiting regulatory protein complexes. This process is dynamically regulated by enzymes that add or remove these marks, as well as by reader proteins that recognize them. Dysregulated expression or malfunction of these regulators creates an aberrant epigenetic landscape and gene expression profile, contributing to breast cancer initiation, metastasis, and drug resistance. Notably, the donor molecules for chromatin modifications are largely derived from intermediate metabolites shaped by environmental cues, highlighting the intricate crosstalk between epigenetic regulation and both cellular and systemic metabolic states. DNA and histone modifications are further interrelated with lncRNAs and higher-order chromatin architectures, which have been actively investigated in breast carcinogenesis. We also briefly introduce the role of epigenetics in other chromatin-associated events such as replication initiation. Aberrant replication initiation can drive gene duplication and genomic alterations resembling those observed in clinical breast cancer, endowing tumor cells with growth advantages and therapeutic resistance. Finally, we summarize emerging therapeutic strategies that target epigenetic vulnerabilities in breast cancer and discuss their current limitations and future directions.

Introduction

Breast cancer is the most common malignancy among women worldwide.^[1] According to a recent report from the National Cancer Center of China, 357,200 new breast cancer cases and 75,000 breast cancer-related deaths were recorded nationwide in 2022.^[2] The incidence has risen sharply over the past four decades, placing an increasing burden on the public health system.^[3] Breast cancer is a highly heterogeneous disease. The classic stratification is based on immunohistochemical detection of hormone receptor (HR) expression—estrogen receptor (ER) and progesterone receptor (PR)—as well as human epidermal growth factor receptor 2 (HER2). This categorization defines the following subtypes: (1) ER⁺ and/or PR⁺ tumors, which account for approximately 70–80% of cases.^[4] Within this group, tumors with low Ki-67 and negative HER2 expression are classified as luminal A, which tend to grow slowly and have a better prognosis. By contrast, luminal B tumors show high Ki-67 expression, HER2^-/+, and are more proliferative; (2) HER2⁺ tumors, which represent 15–20% of cases, are defined by overexpression or amplification of the HER2 gene.^[4] These tumors are generally more aggressive but often respond well to targeted therapies such as trastuzumab (Herceptin); and (3) triple-negative breast cancer (TNBC), a heterogeneous subtype, is defined by the absence of HR expression and HER2 overexpression. TNBC is more common in younger women, tends to be aggressive, and has fewer targeted treatment options.^[4,5]

Breast cancer can also be classified according to more complex molecular signatures, which have advanced rapidly with the development of multi-omic sequencing technologies at both bulk-tissue and single-cell levels. For example, combined whole-genome and transcriptome sequencing in a meta-cohort of 1828 breast tumors—spanning pre-invasive, primary invasive, and metastatic disease—showed that complex focal amplifications, including cyclic extrachromosomal DNA amplification and the apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B) editing, characterize both the ER⁺ high-risk integrative subgroup and the HER2⁺ subgroup.^[6] TNBC tumors, by contrast, display genome-wide instability and tandem duplications and are enriched for signatures of homologous recombination repair deficiency. In ER⁺ typical-risk tumors, the genomes remain largely stable. These findings suggest that complex structural alterations persist throughout the evolution of breast cancer subtypes, reflecting distinct therapeutic vulnerabilities.^[6]

Among all breast cancers, only 10–15% of cases are related to genetic predisposition or family history.^[7] The most commonly mutated germline genes in patients with breast cancer are BRCA1 and BRCA2.^[7,8] Genetic testing for BRCA mutations is routinely conducted in high-risk populations and is recognized as a companion diagnostic for patients who may benefit from poly (ADP-ribose) polymerase inhibitors.^[9,10] Mutations in other genes—such as ATM, PALB2, CHEK2, BARD1, RAD51C, RAD51D, and TP53, most of which are involved in the DNA damage repair pathway—have also been associated with a significantly increased risk of breast cancer.^[7,8] By contrast, the etiology of most breast cancers cannot be fully explained by genetic background. Epigenetic mechanisms, which regulate how cells interpret genetic information without altering the DNA sequence itself, may instead play a more central role in disease progression.

The primary aspects of epigenetic regulation include post-translational modifications of DNA, RNA, and histones, as well as various non-coding RNAs and chromatin structure remodeling. These mechanisms reshape chromosome structure into either an “open” or “closed” state, creating a local environment that promotes gene activation or repression. Alternatively, modified DNA or histones can act as platforms for “reader” proteins, which recognize these signals and recruit additional regulatory factors. Recently, we and others have shown that epigenetic mechanisms also play an important role in the initiation of DNA replication. Dysregulated replication initiation may influence clonal evolution during breast cancer progression, providing tumor cells with growth advantages and drug resistance. In this review, we summarize recent advances in the study of epigenetic mechanisms in breast cancer, with particular emphasis on histone modifications, long non-coding RNAs (lncRNAs), and higher-order chromatin structures. We also discuss the potential of epigenetic therapies in breast cancer.

Histone Modifications in Breast Cancer

The link between histone modifications and eukaryotic gene expression was first proposed in the early 1960s, following Vincent Allfrey’s discovery of histone acetylation. However, the rapid expansion of epigenetics did not begin until the mid-1990s, when Charles David Allis and Stuart Lee Schreiber identified the first histone acetyltransferases and histone deacetylases, which are associated with transcriptional activation and repression, respectively.^[11,12] Post-translational histone modifications influence gene expression either by altering histone charges to disrupt nucleosome–DNA interactions, by recruiting reader-associated regulatory protein complexes, or through both mechanisms. Several reviews have examined the role of histone modifications and histone-modifying enzymes in breast carcinogenesis from different perspectives.^[13–15]

Using immunohistochemical staining, researchers collected 880 breast cancer samples and systematically examined multiple histone modifications, including acetylation of H3K9, H3K18, H4K12, and H4K16; dimethylation of H3K4; trimethylation of H4K20; and dimethylation of H4R3. The results showed low or absent H4K16ac levels in 78.9% of samples. Clusters of histone modification patterns were also linked to breast cancer subtypes, prognostic factors, and patient outcomes.^[16] However, only limited follow-up mechanistic studies have addressed the functional significance of these widespread increases or decreases in specific histone modifications. Instead, accumulating evidence suggests that breast carcinogenesis is more closely related to dysregulation of histone-modifying enzymes and/or aberrant distribution of histone marks on chromatin, which can lead to oncogene upregulation or tumor suppressor gene silencing. For example, the H3K9 trimethyl demethylase JMJD2B has been shown to coordinate with the H3K4 methyltransferase mixed-lineage leukemia protein 2 to enhance ERα-regulated transcription, thereby promoting hormonally responsive breast carcinogenesis.^[17] Similarly, SET8, a SET domain–containing methyltransferase that catalyzes H4K20 monomethylation, drives epithelial mesenchymal transition (EMT) and metastasis in breast cancer through its dual role as an epigenetic modifier: Repressing E-cadherin while activating N-cadherin.^[18] Notably, the advent of new genetic tools capable of spatiotemporally manipulating histone modifications at high resolution has prompted a reevaluation of long-standing views on certain histone marks in transcriptional regulation. For instance, Polycomb complexes, traditionally thought to mediate transcriptional repression, have recently been shown to promote transcriptional activation. In particular, the non-canonical PRC1.1 can deposit H2AK119ub1 at active promoters, thereby licensing gene expression and enabling regulatory T-cell plasticity during immune adaptation.^[19] This highlights the functional complexity of histone modifications in fine-tuning oncogenic transcriptional programs in a context-dependent manner. Recent mechanistic studies have also increasingly emphasized translational potential, especially the development of novel drugs for subgroups of patients with breast cancer who have a poor prognosis under conventional therapies. One example is CTx-648 (PF-9363), a highly potent and selective orally bioavailable inhibitor of KAT6A, a histone H3K23 acetyltransferase frequently amplified or overexpressed in multiple tumors. This compound has demonstrated robust antitumor activity in KAT6A-overexpressing ER⁺ breast cancers, including those refractory to endocrine therapy.^[20]

Driven by advances in modern mass spectrometry technology, the catalog of histone modifications has rapidly expanded beyond the classic acetylation, methylation, phosphorylation, and ubiquitination. In particular, histone acylation refers to a group of chemical modifications structurally similar to acetylation but differing in carbon chain length, hydrophobicity, and charge. Like acetylation, acylation occurs on lysine residues. To date, identified acylations include propionylation, butyrylation, crotonylation, succinylation, malonylation, lactylation, β-hydroxybutyrylation, myristoylation, and palmitoylation. The donor molecules for histone acylation are acyl-CoAs, which are primarily intermediate metabolites. Consequently, histone acylation levels can be influenced by intracellular metabolic states and, in turn, regulate gene expression profiles during different stages of breast cancer. For example, cancer cells often display increased lactate production through aerobic glycolysis, a phenomenon known as the Warburg effect. It was recently discovered that chromatin histones are frequently modified by lactate-derived lactylation, an epigenetic modification that directly stimulates gene transcription.^[21] In breast cancer, studies have shown that KCNK1, a member of the potassium-selective leak channels, promotes cancer cell proliferation and invasion by binding to and activating lactate dehydrogenase A (LDHA). This activation stimulates histone lysine lactylation, inducing the expression of a panel of genes—including LDHA itself—thus establishing a vicious positive feedback loop that reduces tumor cell stiffness and adhesion while promoting metastasis.^[22] Another study demonstrated that aerobic glycolysis–driven lactylation of H3K18 (H3K18la) activates c-Myc expression, which in turn upregulates serine/arginine splicing factor 10. This cascade drives alternative splicing of MDM4 and Bcl-x, thereby promoting breast tumorigenesis.^[23] Notably, upregulated histone lactylation is not always associated with transcriptional activation. A recent study showed that lactate exposure–induced histone H4K12la led to transcriptional repression of SLFN5, a member of the Schlafen family that functions as a tumor suppressor in breast cancer. This repression impeded tumor cell apoptosis and promoted TNBC progression.^[24]

The biological behavior of breast cancer cells is strongly influenced by the tumor microenvironment (TME), which contains a wide range of metabolites, including lactate, fatty acids, and ketone bodies. Metabolites secreted by different cellular sources within the TME can reshape the epigenetic landscape of tumor cells. For example, octanoate, a medium-chain fatty acid, has been shown to activate the serine, one-carbon, glycine pathway via 3-phosphoglycerate dehydrogenase. This activation promotes the synthesis of S-adenosylmethionine (SAM) and the accumulation of 2-hydroxyglutarate, thereby contributing to epigenomic reprogramming and phenotypic plasticity in ER⁻ breast cancer.^[25] Systemic metabolic changes can also influence breast cancer progression. Hyperinsulinemia, for instance, has been reported to remodel the epigenetic landscape by increasing histone acetylation through the phosphoinositide 3-kinase (PI3K)/protein Kinase B (AKT)/mammalian target of rapamycin signaling pathway. This insulin-driven chromatin remodeling has been linked to heightened genomic instability and more aggressive tumor behavior in TNBC.^[26] Because breast tissue is inherently rich in adipocytes, it is especially vulnerable to systemic metabolic disturbances associated with obesity. Evidence indicates that obesity is correlated with widespread DNA methylation changes, particularly in genes involved in immune regulation, cell proliferation, and genome maintenance in ER⁺ breast cancer.^[27] Together, these findings highlight the complex interplay between epigenetic regulation in breast cancer and both local and systemic metabolic states.

To date, the function of a particular histone modification is often studied by overexpressing or depleting the corresponding enzymes that catalyze its addition or removal. However, it is important to note that these enzymes can also act on non-histone substrates. As a result, the role of histone modifications and the enzymes that catalyze them in gene regulation is not always equivalent, adding further complexity to epigenetic mechanisms in breast cancer. For example, PRMT5, a histone arginine methyltransferase, has been shown to maintain breast cancer stemness by catalyzing dimethylation of H3R2.^[28] Beyond histones, PRMT5 also modifies non-histone proteins such as KEAP1 at R596, preventing its ubiquitination and degradation. Because KEAP1 serves as a substrate-recognition component of the E3 complex that targets NRF2 for degradation, this mechanism may contribute to lowering intracellular NRF2 levels, thereby protecting TNBC cells from ferroptosis during immunotherapy.^[29] In addition, the activity of histone-modifying enzymes can itself be regulated by post-translational modifications, adding another layer of complexity to the epigenetic network in breast cancer. For instance, one study demonstrated that the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) is stabilized by SMYD2-mediated methylation at K307, a modification that can subsequently be removed by LSD1. Together, SMYD2 and EZH2 act to repress gene transcription, thereby promoting breast cancer tumorigenesis and metastasis.^[30] A summary of recent mechanistic studies on DNA and histone modifications in breast cancer is presented in Table 1.

Table 1.

Modifications of DNA and histones in breast cancer.

Modification	Modification type	Reaction type	Enzymes	Tumor features	Mechanisms	References
DNA methylation	5mC	Methylation	DNMT1	–	DNMT1 enhances breast cancer stemness and tumor formation by downregulating FOXO3a via promoter methylation, modulating the FOXO3a/FOXM1/SOX2 signaling pathway.	[31]
			DNMT1 DNMT3A	–	DNMT1 and DNMT3A mediate hypermethylation of RGMA promoter and downregulation of RGMA, activating the FAK/Src/PI3K/AKT signaling pathway and stimulating breast cancer growth.	[32]
			DNMT3A	–	DNMT3A-mediated promoter hypermethylation leads to the downregulation of DPT, reversing DPT-mediated YAP cytoplasmic retention and promoting breast cancer growth.	[33]
	5hmC	Demethylation	TET1	TNBC	TET1-mediated hypomethylation activates oncogenic pathways, including PI3K, EGFR, and PDGF, promoting TNBC progression.	[34]
			TET1	Basal-like	TET1-mediated promoter demethylation maintains high expression of POU4F1, a key transcription factor in basal-like breast cancer, thus promoting tumor growth and metastasis.	[35]
			TET1, TET3	TNBC	TET1 and TET3 enhance UCHL1 transcription by promoter demethylation, while UCHL1 deubiquitinates and stabilizes KLF5 in addition to ERa. This axis contributes to the endocrine resistance in TNBC.	[36]
			TET1	Lymph node metastatic luminal A and luminal B	Genome-wide 5-hmC profiling has identified MAP7D1 in promoting lymph node metastasis of breast cancer. TET1-dependent 5hmC leads to high expression of MAP7D1.	[37]
Histone methylation	H3K4me3	Methylation	SETD1A/COMPASS	–	The SETD1A/COMPASS complex facilitates the transcription factor ZNF408 in promoting H3K4me3 of STING1. Loss of ZNF408 attenuates STING-mediated immune surveillance of breast cancer.	[38]
			KMT2/MLL	TNBC	FOXQ1 recruits the KMT2/MLL complex to activate transcription of EMT genes, thereby promoting TNBC metastasis.	[39]
			KDM5A	TNBC	KDM5A demethylates H3K4me3 to downregulate p16 expression, thereby inhibiting DNA damage and promoting metastasis. Fbxo22 has been identified as an E3 ligase of KDM5A, which inhibits this process.	[40]
		Demethylation	UTX/KDM6A	–	GATA3 recruits UTX to activate the transcription of genes involved in EMT, thereby suppressing breast cancer metastasis.	[41]
	H3K9me3	Demethylation	KDM4A	–	KDM4A promotes breast cancer progression by demethylation of H3K9me3, leading to activation of Notch1-NICD–dependent signaling.	[42]
	H3R2me2a	Methylation	PRMT6	–	PRMT6 represses p21 transcription by directly binding to the p21 promoter and catalyzing H3R2me2a, thereby acting as an oncogene that promotes growth and prevents senescence in breast cancer cells.	[43]
	H3R2me2s	Methylation	PRMT5	ER+	PRMT5 catalyzes H3R2me2s on the FOXP1 promoter and upregulates gene expression, thereby enhancing breast cancer stem cell function.	[28]
Histone acetylation	H3K9ac	Acetylation	MYST3 (KAT6A)	MYST3 amplification, ER+/HER2+	MYST3 promotes H3K9ac on the ESR1 promoter, thereby activating ERα expression and promoting breast cancer cell growth.	[44]
	H3K9ac	Deacetylation	HDAC4	–	Inhibition of HDAC4 enhances H3K9 acetylation on the NEDD9 promoter, activating the FAK/NF-κB signaling pathway and promoting breast cancer cell metastasis.	[45]
	H3K23ac	Acetylation	KAT6A/B	KAT6A amplification, ER+	KAT6A/B acetyltransferases promote H3K23 acetylation and drive breast cancer growth, a process that can be blocked by a highly potent, selective, orally bioavailable inhibitor.	[20]
	H3K27ac	Acetylation	P300/CBP, BET family readers	TNBC	Targeting P300/CBP but not BET could curtail the epigenetic remodeling in IFNγ-driven transcription.	[46]
	H3K27ac	Acetylation	P300/CBP	TNBC	The ZNF451-SLUG complex in TNBC cells recruits P300/CBP to the CCL5 promoter and activates gene transcription. This leads to recruitment and activation of macrophages, promoting immune evasion.	[47]
	H3K27ac	Deacetylation	HDAC5	–	HDAC5 represses a subset of cell cycle-related genes. Loss of HDAC5 impairs RB-mediated repression of pro-oncogenic genes and confers CDK4/6 inhibitor resistance in breast cancer.	[48]
Histone ubiquitination	H2AK119ub1	Ubiquitination	Ring1b	–	Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically inhibit E-cadherin expression and promote breast cancer metastasis.	[49]
	H2Bub1		RNF40	ER+	The proteasome inhibitor bortezomib causes a global decrease in H2Bub1, leading to transcriptional elongation defects in estrogen target genes. Silencing RNF40 expression decreases H2Bub1 and inhibits ERα-induced gene transcription.	[50]
Histone phosphorylation	H2A.X Y39ph	Phosphorylation	JMJD6	TNBC	The tyrosine kinase activity of JMJD6 enhances histone H2A.X phosphorylation, leading to gene upregulation and autophagy in TNBC.	[51]
Histone lactylation	H3K18la	Lactylation	LDHA	–	KCNK1 binds and activates LDHA, which promotes H3K18la of targeted genes and LDHA itself. This results in proliferation, invasion, and metastasis of breast cancer.	[22]

AKT: AKT Serine/threonine kinase; CCL5: C-C motif chemokine ligand 5; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase 3A; DPT: Dermatopontin; EMT: Epithelial-mesenchymal transition; ER: Estrogen receptor; FAK: Focal adhesion kinase; FOXM1: Forkhead box M1; FOXO3: Forkhead box O3; H2A: Histone H2A; H2AK119ub1: Histone H2A lysine 119 ubiquitination; H2Bub1: Histone H2B ubiquitination; H3K9ac: Histone H3 lysine 9 acetylation; H3K18la: Histone H3 lysine 18 lactylation; H3K23ac: Histone H3 lysine 23 acetylation; H3K27ac: Histone H3 lysine 27 acetylation; H3K4me3: Histone H3 lysine 4 trimethylation; H3K9me3: Histone H3 lysine 9 trimethylation; H3R2me2a: Histone H3 arginine 2 asymmetric dimethylation; H3R2me2s: Histone H3 arginine 2 symmetric dimethylation; HER2: Human epidermal growth factor receptor 2; IFN-γ: Interferon-gamma; KLF5: Krüppel-like factor 5; KMT2: Lysine methyltransferase 2; MAP7D1: Microtubule-associated protein 7 domain-containing 1; MLL: Mixed-lineage leukemia; MYST3: Lysine acetyltransferase 6A (KAT6A); NF-κB: Nuclear factor kappa-B; PI3K: Phosphoinositide 3-kinase; POU4F1: POU class 4 homeobox 1; p21: Cyclin-dependent kinase inhibitor 1A; RGMA: Repulsive guidance molecule A; SETD1A: SET domain containing 1A; SOX2: SRY-box transcription factor 2; SRC: Proto-oncogene tyrosine-protein kinase Src; TET1: Ten-eleven translocation methylcytosine dioxygenase 1; TNBC: Triple-negative breast cancer; UCHL1: Ubiquitin carboxyl-terminal hydrolase L1; X Y39ph: Histone H2A X Y39 phosphorylation; YAP: Yes-associated protein; ZNF408: Zinc finger protein 408; 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; –: Not available.

LncRNAs in Breast Cancer

Protein-coding genes account for only approximately 2% of the human genome. With advances in modern molecular biology, deciphering the information embedded in the non-coding genome has received increasing attention. In particular, the epigenetic landscape of breast cancer can be regulated by non-coding RNAs, among which lncRNAs play a critical role in both tumor cells and tumor-associated cells within the microenvironment. LncRNAs are transcribed by RNA polymerase II, carry a 5′ cap and a poly(A) tail, and typically exceed 200 nucleotides in length. Most are localized in the nucleus. In general, lncRNAs regulate transcription either in cis (affecting neighboring genes) or in trans (modulating the activity of distant transcriptional activators or repressors). An early pivotal study demonstrated that the lncRNA HOX transcript antisense RNA (HOTAIR) recruits the PRC2 complex, which catalyzes histone H3 lysine 27 methylation. Elevated HOTAIR expression in primary breast tumors induces genome-wide retargeting of PRC2 to a chromatin state resembling that of embryonic fibroblasts, leading to widespread gene expression changes and increased cancer invasiveness and metastasis.^[52] In this case, dysregulated HOTAIR expression redirects its associated histone-modifying enzymes, reshaping the epigenomic landscape and producing a more aggressive tumor transcriptome. LncRNAs can also function as scaffold factors, either facilitating or inhibiting the assembly of transcriptional regulatory complexes, thereby shaping the gene expression profile of breast cancer cells. For example, estrogen-inducible lncRNA (ERINA) promotes breast cancer proliferation and cell cycle progression by binding the transcription factor E2F1 and preventing its interaction with the tumor suppressor RB transcriptional corepressor 1.^[53] Mammary tumor-associated RNA 25 (MaTAR25), a chromatin-associated lncRNA, interacts with the transcription factor PURB to transactivate tensin 1, a focal adhesion component linking the extracellular matrix to the actin cytoskeleton. Elevated expression of the human orthologue of MaTAR25 correlates with poor prognosis and metastasis in patients with breast cancer.^[54]

Some lncRNAs regulate gene expression post-transcriptionally by influencing splicing, editing, transport, translation, or degradation of the corresponding mRNA transcripts.^[55] For instance, lncRNA-BC069792 suppresses breast cancer progression by acting as a molecular sponge for the microRNAs hsa-miR-658 and hsa-miR-4739. By sequestering these miRNAs, it upregulates KCNQ4 expression and inhibits activation of the PI3K/AKT signaling pathway.^[56] LINC02568, a highly expressed lncRNA in ER⁺ breast cancer, plays a dual role in breast carcinogenesis and endocrine resistance. In the cytoplasm, it regulates estrogen/ERα-induced transcriptional activation in trans by stabilizing ESR1 mRNA through sponging miR-1233-5p. In the nucleus, LINC02568 acts in cis to maintain tumor-specific pH homeostasis by inhibiting carbonic anhydrase CA12.^[57] Notably, the term “long non-coding RNA” may not fully capture the functions of some lncRNAs. Recent studies have shown that particular lncRNAs can also encode proteins. For example, lncRNA LY6E divergent transcript (LY6E-DT) functions as a scaffold RNA to promote nuclear entry of YBX1, which activates transcription of ZEB1, thereby enhancing metastasis. At the same time, LY6E-DT encodes a conserved 153-amino-acid protein called metastatic-related protein, which stabilizes epidermal growth factor receptor (EGFR) mRNA and enhances EGFR protein expression, leading to PI3K/AKT pathway activation and promotion of breast cancer progression.^[58]

Throughout disease progression, breast cancer cells engage in continuous dialogue with the surrounding TME, which consists of fibroblasts, immune cells, stromal cells, blood vessels, the extracellular matrix, and extracellular vesicles. Recent studies have shown that many lncRNAs play critical roles in regulating the function of cells within the TME. For example, HIF-1α-stabilizing lncRNA (HISLA), a myeloid-specific lncRNA, can be transported by extracellular vesicles into breast cancer cells. There, it blocks the interaction between PHD2 and HIF-1α, preventing hydroxylation and degradation of HIF-1α, which in turn enhances aerobic glycolysis and anti-apoptotic activity in tumor cells. Lactate released from glycolytic breast cancer cells further upregulates HISLA in macrophages, creating a feed-forward loop between tumor cells and their microenvironment.^[59] Another example is interferon-responsive nuclear factor-κB activator (IRENA), an interferon (IFN)-induced cytoplasmic lncRNA expressed in post-chemotherapy macrophages. IRENA triggers activation of Nuclear factor kappa-B–mediated pro-tumor inflammatory cytokines in breast cancer. In macrophage-conditional IRENA-knockout mice, targeting IRENA in IFN-activated macrophages abolished their pro-tumor effects while preserving their ability to enhance antitumor immunity.^[60] We summarize the latest studies on the biological functions, molecular mechanisms, and therapeutic potential of lncRNAs in breast cancer in Table 2.

Table 2.

LncRNAs in breast cancer: Expression features, functions, and mechanisms.

LncRNA	Tumor features	Expression status		Mechanisms	References
		Tumor cells	Tumor-infiltrated cells
ERINA	ER+	High	–	ERINA facilitates cancer cell cycle progression by preventing E2F1 binding to RB1.	[53]
MaTAR25	HER2+, luminal, and TNBC	High	–	MaTAR25 promotes breast cancer proliferation, migration, and invasion by interacting with PURB to transactivate the expression of Tns1, a key component of focal adhesions that links the extracellular matrix and the actin cytoskeleton.	[54]
LINC02568	ER+	High	–	LINC02568 promotes tumor development and drug resistance by stabilizing ESR1 mRNA in a trans manner and regulating CA12-mediated pH homeostasis in a cis manner.	[57]
HOTTIP	BCSCs	High	–	HOTTIP maintains the stemness of BCSCs by sponging miR-148a-3p to upregulate WNT1.	[61]
LINC00115	Chemoresistant breast cancer stem-like cells	High	–	LINC00115 induces chemoresistance and metastasis of breast cancer by linking SETDB1 to PLK3, promoting PLK3 methylation and HIF1α stabilization.	[62]
LINC00173	TNBC	High	–	LINC00173 promotes TNBC proliferation and invasion by antagonizing miR-490-3p.	[63]
LINC00839	Chemoresistant cells	High	–	LIN00839 promotes breast cancer proliferation and chemoresistance via the Myc/LINC00839/LINC28B feedback loop.	[64]
LINC02273	TNBC	High in metastatic lesions	–	hnRNPL stabilizes LINC02273, which promotes TNBC metastasis by epigenetically increasing AGR2 transcription.	[65]
LINK-A	TNBC	High	–	LINK-A interacts with the EGFR-GPNMB heterodimer, recruiting and activating BRK and LRRK2 and stabilizing HIF1α. This process promotes glycolysis reprogramming and tumorigenesis in TNBC.	[66]
lncSNHG5	–	–	High	High expression of lncSNHG5 in cancer-associated fibroblasts stabilizes ZNF281 mRNA and activates the CCL2/CCL5 signaling pathway, promoting angiogenesis and vascular permeability in breast cancer.	[67]
MALAT1	–	High	–	MALAT1 promotes progression and doxorubicin resistance of breast cancer cells by sponging miR-5703p.	[68]
MCM3AP-AS1	–	High	–	MCM3AP-AS1 promotes breast cancer proliferation, migration, and invasion by modulating the miR-28-5p/CENPF axis.	[69]
NEAT1	p53 wild-type	High	–	NEAT1 repression by MED12 enhances chemosensitivity in p53 wild-type breast cancer cells.	[70]
RP11-19E11	Basal breast cancer	High	–	RP11-19E11 is an E2F1 target required for the proliferation and survival of cancer cells.	[71]
SNHG6	–	High	–	SNHG6 promotes the proliferation, migration, and EMT of cancer cells by sponging miR-543, thereby alleviating the inhibition of LAMC1/PI3K/AKT signaling.	[72]
TINCR	–	High	–	TINCR impairs the efficacy of immunotherapy by acting as a molecular sponge for miR-199a-5p and upregulating the stability of USP20 mRNA, thereby promoting PD-L1 expression by decreasing its ubiquitination level.	[73]
TROJAN	TNBC	High	–	TROJAN promotes TNBC proliferation and invasion by blocking ZMYND8-ZNF592 interaction, leading to ZMYND8 degradation.	[74]
Xist	–	High	–	Knockdown of Xist in tumor-associated macrophages suppresses the expression of C/EBPα and KLF6, inducing M1-to-M2 conversion and promoting breast cancer proliferation and migration.	[75]
BC069792	–	Low	–	LncRNA BC069792 inhibits breast cancer proliferation, invasion, and metastasis by absorbing microRNAs hsa-miR-658 and hsa-miR-4739, thereby up-regulating KCNQ4 and inhibiting the PI3K/AKT signaling.	[56]
SEMA3B-AS1	TNBC	Low	–	LncRNA SEMA3B-AS1 inhibits breast cancer progression by sponging miR-3940-3p, preventing the degradation of its target gene KLLN, which acts as a tumor suppressor in TNBC.	[76]

AKT: AKT Serine/threonine kinase; AGR2: Anterior gradient 2; BCSCs: Breast cancer stem cells; CCL2: C-C motif chemokine ligand 2; CCL5: C-C motif chemokine ligand 5; CENPF: Centromere protein F; EGFR-GPNMB: Epidermal growth factor receptor-glycoprotein NMB; ER: Estrogen receptor; ERINA: Estrogen inducible lncRNA; GPNMB: Glycoprotein nonmetastatic melanoma protein B; HIF1α: Hypoxia-inducible factor 1 subunit alpha; HOTTIP: HOXA distal transcript antisense RNA; KCNQ4: Potassium voltage-gated channel subfamily Q member 4; KLF6: Krüppel-like factor 6; LAMC1: Laminin subunit gamma-1; LINK-A: long intergenic non-coding RNA for kinase activation; LncRNA: Long non-coding RNA; lncSNHG: LncRNA small nucleolar RNA host gene 5; MALAT1: Metastasis-associated lung adenocarcinoma transcript 1; MaTAR25: Mammary tumor-associated RNA 25; MED12: Mediator complex subunit 12; mRNA: Messenger RNA; NEAT1: Nuclear paraspeckle assembly transcript 1; PI3K: Phosphoinositide 3-Kinase; SNHG6: Small nucleolar RNA host gene 6; TNBC: Triple-negative breast cancer; TINCR: Tissue differentiation inducing noncoding RNA; XIST: X-inactive specific transcript; miR-570-3p: MicroRNA-570-3p; -: Indicates the specific tumor feature was absent or not evaluated; –: Not available.

Higher-Order Chromatin Structures in Breast Cancer

Histone modifications and lncRNAs act not on linear chromatin but within a three-dimensional (3D) chromatin architecture inside the nucleus. Thus, understanding higher-order chromatin structures and their relationship to pathological changes in breast cancer has become increasingly important [Figure 1]. The hierarchical chromatin structure is organized into four levels: chromosome territories, chromatin compartments, topologically associating domains (TADs), and chromatin loops. Chromosome territories refer to distinct regions within the nucleus, typically with the nucleolus surrounded by clusters of telomeres, heterochromatin, and centromeres, while euchromatin is more freely dispersed. Genome-wide analyses of epigenetic marks using methods such as chromatin immunoprecipitation sequencing (ChIP-seq), cleavage under targets and release using nuclease (CUT&RUN), assay for transposase-accessible chromatin sequencing (ATAC-seq), and DNase I hypersensitive sites sequencing (DNase-seq) have shown that euchromatin and heterochromatin regions alternate along the length of chromosomes. Chromosome conformation capture methods—including Hi-C, Split-Pool Recognition of Interactions by Tag Extension (SPRITE), and tyramide signal amplification sequencing (TSA-seq)—further demonstrate that these domains are spatially segregated. Generally, the A compartment corresponds to euchromatin, which is gene-rich, marked by active epigenetic modifications, and transcriptionally active. By contrast, the B compartment corresponds to heterochromatin, which has higher transposon density and repressive epigenetic features. TADs function as relatively independent local units, often arranged in a nested, hierarchical manner, ranging in size from tens of kilobases to several megabases. Interactions within a TAD are significantly stronger than those between different TADs. At an even finer scale, chromatin loops bring regulatory elements such as enhancers into physical proximity with their target genes [Figure 2].^[77,78] Dysregulation of higher-order chromatin structures—caused by mechanisms such as aberrant expression of chromatin remodeling complexes or global redistribution of histone modifications—can profoundly influence breast cancer initiation, metastasis, and drug resistance.

Figure 1.

Interplay between histone modifications, lncRNAs, and higher-order chromatin structures in regulating gene expression. Histone modifications, lncRNAs, and higher-order chromatin structures orchestrate gene expression through reciprocal and multilayered interactions. Histone modifications regulate lncRNA expression by altering local chromatin accessibility and/or recruiting transcriptional regulatory complexes. In turn, lncRNAs can reshape histone modification landscapes by acting as scaffolds that guide chromatin-modifying enzymes to specific genomic regions or as decoys that sequester epigenetic regulators. Both histone modifications and lncRNAs also influence the organization of the three-dimensional genome, which in turn modulates gene expression and other chromatin-associated processes. LncRNA: Long non-coding RNA.

Figure 2.

Higher-order chromatin structures in eukaryotic cells. The hierarchical chromatin structure in eukaryotic cells is organized into four levels: chromosome territories, chromatin compartments, TADs, and chromatin loops. Chromosome territories represent distinct regions within the nucleus, typically with the nucleolus surrounded by clusters of telomeres, heterochromatin, and centromeres, while euchromatin is more freely dispersed. The A compartment (shaded in red) corresponds to euchromatin, characterized by high gene density, active epigenetic modifications, and transcriptional activity. By contrast, the B compartment (shaded in blue) corresponds to heterochromatin, enriched for transposons and repressive epigenetic features. TADs are relatively independent local units, often nested in a hierarchical manner, ranging in size from tens of kilobases to several megabases. They are demarcated by boundary elements such as CTCF–cohesin complexes. At a finer scale, promoter–enhancer loops bring regulatory elements into proximity to control transcription. Nucleosomes decorated with distinct histone modifications are illustrated at this level. Local chromatin assembly can also be remodeled by ATP-dependent complexes such as SWI/SNF, ISWI, INO80, and CHD, which reposition, evict, or restructure nucleosomes. Aberrant expression of histone modification regulators and chromatin remodelers drives global epigenetic changes, leading to dysregulated transcription and oncogenic progression. CHD: Chromodomain helicase DNA-binding; CTCF: CCCTC-binding factor; ISWI: Imitation switch; SWI/SNF: Switch/sucrose non-fermentable; TADs: Topologically associating domains.

Dysregulated enhancer activity is a frequent feature of breast cancer. Analysis of the epigenetic landscape in CDK4/6 inhibitor–treated breast cancer cells revealed widespread enhancer activation, including super-enhancers that drive luminal differentiation and apoptotic evasion, as well as enhancers overlapping with endogenous retroviral elements near IFN-driven genes. These findings help explain some of the effects of CDK4/6 inhibitors beyond cell cycle arrest.^[79] Several breast cancer risk loci identified in large-scale genome-wide association studies have later been shown to function as enhancers, regulating gene expression through chromatin loops with their target gene promoters. For example, we previously reported that the region surrounding the breast cancer risk SNP rs4971059 functions as an active enhancer of TRIM46, which encodes an E3 ligase of HDAC1, a classic histone deacetylase. Genome-wide profiling revealed that the TRIM46–HDAC1 axis regulates multiple genes involved in DNA replication and repair, thereby conferring oncogenic potential and chemoresistance in breast cancer.^[80] This illustrates how genetic variation that predisposes individuals to breast cancer can operate through a pathway involving an epigenetic enzyme, underscoring the close relationship between genetics and epigenetics as the molecular basis of cancer origin. In another study, the architectural protein CCCTC-binding factor (CTCF), which mediates both interchromosomal and intrachromosomal interactions, was shown to promote chromatin accessibility at ERα-enriched enhancers and facilitate enhancer–promoter looping. This process is aided by BAP18, a reader protein that recognizes H3K4 trimethylation. Depletion of BAP18 sensitized MCF-7 cells to both anti-estrogen and anti-enhancer treatment.^[81] Here, a histone modification regulator (e.g., BAP18) and a chromatin organizer (e.g., CTCF) act in coordination to regulate enhancer activity, illustrating the crosstalk among different epigenetic mechanisms. At a broader chromatin-wide scale, subtype-restricted gene expression can be controlled by multi-way interactions among promoters and enhancers within spatial hubs, orchestrated by oncogenic transcription factors such as MYC and SOX9 in TNBC cells.^[82] These findings highlight the critical role of chromatin loops in shaping cancer-associated transcriptome alterations.

Beyond enhancer–promoter loops, higher-order chromatin structures are generally thought to remain relatively stable within a given cell type. Thus, their role in breast cancer—across different subtypes and disease stages—remains to be fully clarified. A recent study showed that 3D chromatin interactions within and between TADs change frequently in ER⁺ endocrine-resistant breast cancer cells. These differential interactions are enriched for resistance-associated genetic variants at CTCF-defined anchors. Ectopic chromatin interactions were observed at active enhancers and promoters and were linked to altered expression of ER target genes. Furthermore, changes in active A compartments and inactive B compartments were associated with reduced ER binding and atypical gene expression,^[83] suggesting that chromatin structure dynamics at multiple levels may contribute to breast cancer progression.

In addition to shaping transcriptomes, epigenetic landscapes and 3D chromatin structures also influence other chromatin-related processes, such as DNA replication initiation. Dysregulation of this process can profoundly impact tumor clonal selection and evolution.^[84] For example, NFIB, a member of the nuclear factor I (NFI) family, was initially identified in host cells as a factor that promotes adenoviral DNA replication, but it has since been studied primarily in the context of transcription regulation. Our recent work demonstrated that NFIB physically associates with the pre-replication complex and functions as a “replication pioneer factor” in mammalian cells. Mechanistically, NFIB binds directly to nucleosomes, facilitating the eviction of parental histones and thereby increasing chromatin accessibility. This enhanced accessibility, especially at euchromatic regions marked by H3K4me3, promotes pre-replication complex assembly at replication origins and licenses replication initiation. NFIB deficiency alters chromosome contacts and compartments during both the G1 and S phases, impairing the firing of a subset of origins at early-replication domains. Importantly, cancer-associated NFIB overexpression drives gene duplication and genomic alterations that recapitulate aberrations observed in clinical breast cancer, endowing tumor cells with growth advantages and drug resistance.^[85] Moreover, TRPS1—a gene linked to tricho-rhino-phalangeal syndrome and frequently amplified in breast cancer—was found to bind directly to H3K9me3-marked heterochromatic replication origins, acting as a non-canonical reader of repressive chromatin. TRPS1 also interacts with the replication machinery and recruits the anaphase-promoting complex/cyclosome, an E3 ligase that targets Geminin for degradation. Overexpression of TRPS1 results in uncontrolled origin refiring and cancer genome amplification, which confer therapeutic resistance to breast cancer cells.^[86] Looking ahead, integrating higher-order chromatin architecture with transcriptomics, epigenomics, and spatial omics may provide a more comprehensive view of how epigenetic mechanisms govern diverse chromatin-related events throughout breast cancer progression.

Epigenetic Therapies for Breast Cancer

Epigenetic regulation is reversible. Drugs targeting dysregulated epigenetic signaling in cancer ideally can restore normal cancer cell function with minimal effects on normal organs or tissues. Epigenetic anti-tumor medicines currently on the market are used primarily in hematologic malignancies and are categorized into four groups: DNA methyltransferase (DNMT) inhibitors, HDAC inhibitors, isocitrate dehydrogenase inhibitors, and EZH2 inhibitors, among which HDAC inhibitors are the most actively tested in breast cancer treatment. Magnesium valproate is the first HDAC inhibitor used in locally advanced breast cancer.^[87,88] The combined application of magnesium valproate and hydralazine, a DNMT inhibitor, has also shown promising results when added to conventional neoadjuvant or adjuvant regimens for locally advanced breast cancer.^[88,89] Romidepsin, vorinostat, and panobinostat—approved by the U.S. Food and Drug Administration or the European Medicines Agency for hematologic malignancies—have likewise demonstrated efficacy in breast cancer.^[90–92] Tucidinostat (chidamide) received approval from the National Medical Products Administration in China for postmenopausal patients with HR⁺/HER2⁻ advanced breast cancer.^[93,94] Compared with exemestane alone, combining exemestane with entinostat (another HDAC inhibitor) significantly improves progression-free survival in patients with HR⁺/HER2⁻ advanced breast cancer who have progressed after endocrine therapy.^[95] Overall, HDAC inhibitors appear to provide additional therapeutic benefit in breast cancer when combined with chemotherapy, radiotherapy, or immunotherapy.^[96,97] In recent years, the role of bromodomain and extra-terminal (BET) family proteins—particularly BRD4—in breast carcinogenesis has attracted increasing attention. BRD4 is a reader of acetylated lysine residues on histones. Its overexpression in breast cancer leads to aberrant activation of super-enhancers that drive expression of proliferation-promoting oncogenes such as c-myc.^[98] BET inhibitors, including GSK525762, ZEN-3694, AZD5153, mivebresib, RO6870810, ODM-207, and INCB057643, are currently being evaluated in several ongoing clinical trials for advanced breast cancer that is refractory to or intolerant of standard treatments.^[99–104] Epigenetic drugs currently being tested in clinical trials for breast cancer are summarized in Table 3.

Table 3.

Epigenetic drugs used in breast cancer.

Drug	Target molecules	Applied tumor types	Combined therapy	Phase(s)	NCT Nos.	Recruitment status	References
Hydralazine	DNMT1/3a/3b	Locally advanced breast cancer	Magnesium valproate based on conventional neoadjuvant or adjuvant chemotherapy	II	NCT00395655, NCT00404508	Terminated (NA), completed	[87,89]
Magnesium valproate	HDAC class I	Locally advanced breast cancer	Hydralazine, doxorubicin, and cyclophosphamide	II	NCT00395655, NCT00404508	Terminated (NA), completed	[87,89,105]
Tucidinostat (chidamide)	HDAC class I	ER+ advanced or metastatic breast cancer that progressed after endocrine therapy	Exemestane	III, NMPA approved	NCT02482753	Completed	[93,94,106]
Entinostat (SNDX-275)	HDAC class I	ER+ advanced breast cancer that progressed after endocrine therapy	Exemestane	II	NCT00676663	Completed	[95,107]
		Hormone therapy-resistant stage IV ER+ breast cancer	Tamoxifen and pembrolizumab	II	NCT02395627	Terminated (insufficient efficacy in an unselected patient population)	[108–110]
		Metastatic breast cancer	Monotherapy	–	–	–	[111]
Romidepsin	HDACs	Advanced breast cancer	Gemcitabine	I	–	–	[112,113]
Panobinostat	HDACs	HER2− locally recurrent or metastatic breast cancer	Monotherapy	II	NCT00777049	Completed	–
Vorinostat	HDACs	Hormone therapy-resistant stage IV ER+ breast cancer	Tamoxifen and pembrolizumab	II	NCT04190056	Terminated (change in practice patterns)*	–
Mivebresib (ABBV-075)	BRD2/4/T	Locally advanced or metastatic solid tumors, including breast cancer	With or without venetoclax	I	NCT02391480	Completed	[99]
AZD5153	BRD4	Relapsed/refractory malignant solid tumors, including TNBC	Monotherapy or in combination with olaparib	I	NCT03205176	Completed	[100]
GSK525762 (molibresib)	BRD2/3/4	HR+/HER2− advanced or metastatic breast cancer	Fulvestrant	I/II	NCT02964507	Terminated (meeting protocol defined futility)	[101]
RO6870810 (TEN-10)	Pan-BET	Advanced solid tumors that were refractory to or intolerant of standard treatments, including breast cancer	–	I	NCT01987362	Completed	[102]
ODM-207	Pan-BET	Advanced HER2− breast cancer	Monotherapy	I	NCT03035591	Completed	[103]
INCB057643	BRD4	Advanced solid tumors that were refractory to or intolerant of standard treatments, including breast cancer	Monotherapy	I/II	NCT02431260	Terminated (by the sponsor because of pharmacokinetic variability)	[104,114]
ZEN-3694	Pan-BET	TNBC without germline mutations of BRCA1/2	Talazoparib	II	NCT03901469	Terminated (interim futility)	–
		ER+ breast cancer	CDK4/6 inhibitors	–	–	–	[115]
		Metastatic or unresectable breast cancer that was refractory to standard treatments	Abemaciclib	I	NCT05372640	Recruiting	–
Tazemetostat	EZH2	Advanced or metastatic solid tumor harboring ARID1A mutation, including breast cancer	Monotherapy	II	NCT05023655	Recruiting	[116]

ARID1A: AT-rich interaction domain 1A; BET: Bromodomain and extra-terminal motif proteins; BRCA 1/2: Breast cancer gene 1/2; BRD4: Bromodomain-containing protein 4; CDK4/6 inhibitors: Cyclin-dependent kinase 4/6 inhibitors; DNMT: DNA methyltransferase; ER: Estrogen receptor; EZH2: Enhancer of zeste homolog 2; HDAC: Histone deacetylase; HER2−: Human epidermal growth factor receptor 2 negative; HR+: Hormone receptor positive; NCT: National clinical trial; NMPA: National Medical Products Administration; –: Not available.

In general, epi-drugs alone are unlikely to provide sufficient efficacy or specificity to benefit patients with breast cancer. Instead, they tend to produce stronger therapeutic effects when combined with chemotherapy, endocrine therapy, or targeted therapy regimens.^[117] Careful patient selection, guided by mechanistic studies, is essential to maximize the benefits of epi-drug treatment. It is also worth noting that epigenetic therapies are not limited to chemical inhibitors of epigenetic enzymes. Antisense oligonucleotides (ASOs)—short, synthetic strands of nucleotides designed to bind RNA molecules in a sequence-specific manner—can potentially target aberrantly expressed lncRNAs in breast cancer.^[118] Preclinical investigations using ASOs against dysregulated lncRNAs such as LINC02568^[57] and LINC02273^[65] have shown encouraging efficacy, underscoring the promise of ASO-based strategies for lncRNA-driven breast cancers. In addition, rapidly advancing epigenome-editing technologies offer new opportunities for more precise intervention in aberrant chromatin events. Following the pioneering CRISPR interference (CRISPRi)^[119] and dCas9-VP64^[120] systems, a variety of dCas9-based platforms—including dCas9-SAM, dCas9-SunTag, and dCas9-Casilio^[121]—have been developed to improve editing efficiency. The therapeutic potential of CRISPRa/CRISPRi approaches has been demonstrated in vitro in breast cancer cell line models.^[122,123] Moreover, CRISPR-Cas9–based systems have been applied to modulate the expression of oncogenic lncRNAs, such as HOTAIR and XIST, showing promising efficacy in cancer treatment.^[124] Beyond breast cancer, CRISPR-Cas9–mediated editing of the BCL11A enhancer has already entered phase I/II clinical trials for pediatric transfusion-dependent β-thalassemia,^[125] highlighting the translational potential of these approaches. With continuous improvements in delivery strategies and editing efficiency, these emerging technologies may soon expand the therapeutic landscape for breast cancer.

Despite these promising advances, the clinical application of epigenetic drugs is still hindered by major challenges, including intolerable toxicity, unexpected off-target effects, and drug resistance. HDAC inhibitors provide a representative example. Most currently developed HDAC inhibitors use a hydroxamic acid moiety as their zinc-binding group. However, hydroxamates have a tendency toward non-specificity, and the resulting toxic side effects on multiple organs—including the cardiac, hematologic, and gastrointestinal systems—often limit their therapeutic potential in cancer treatment.^[126] Beyond non-selective inhibition of different HDAC subclasses, the non-enzymatic activities of HDAC inhibitors may also contribute to adverse effects, such as fever and fatigue caused by cytokine release.^[127] Furthermore, recent studies have shown that HDAC inhibitors can feedback-activate leukemia inhibitory factor receptor (LIFR) signaling in breast cancer, reducing their effectiveness as monotherapies. Mechanistically, HDAC inhibition increases histone acetylation at the LIFR promoter, which recruits the bromodomain protein BRD4 and drives upregulation of LIFR expression. This, in turn, activates the JAK1–STAT3 signaling pathway. Importantly, treatment of breast cancer cells with JAK1 or BRD4 inhibitors has been shown to sensitize them to HDAC inhibition, suggesting that combined targeting of HDAC with JAK1 or BRD4 may represent a promising therapeutic strategy.^[128] Finally, as with other anti-tumor agents, resistance to epigenetic drugs can emerge from the intrinsic heterogeneity and plasticity of tumor cells, often leading to low efficacy or relapse after an initial response.

Several approaches may help overcome these challenges. Careful selection of patients suitable for epi-drug treatment is critical to achieving maximum therapeutic effect. Rapidly advancing high-throughput sequencing technologies and predictive models can aid in patient stratification, helping to identify subgroups most likely to respond to specific epi-drugs.^[129] For instance, a comprehensive multi-omics analysis—encompassing genomic, transcriptomic, epigenomic, proteomic, and phosphoproteomic profiling—was conducted to identify distinct molecular and epigenetic features across four TNBC subtypes: basal-like 1, basal-like 2, mesenchymal (M), and luminal androgen receptor (LAR). The study revealed that PRC2-mediated epigenetic repression contributes to immune evasion in PD-L1–negative mesenchymal tumors, highlighting PRC2 inhibitors as a potential combinatorial strategy for TNBC therapy.^[130] Nevertheless, this approach requires refinement to better align with the economic realities of patients. Another promising direction involves designing highly selective zinc-binding groups to replace the conventional hydroxamate moiety in HDAC inhibitors, thereby reducing non-specificity while maintaining drug potency.^[131] In addition, because poor water solubility and short circulation lifetimes partly account for the limited success of epi-drugs in solid tumors, novel drug delivery systems are under active development. These include cell membrane–cloaked biomimetic nanoparticles and epi-drugs conjugated with receptor-binding peptides, both of which aim to improve therapeutic efficacy.^[132,133] We propose a strategic outline for patient selection and application of epigenetic therapies in breast cancer [Figure 3].

Figure 3.

Strategic outline for patient selection and application of epigenetic therapies in breast cancer. (A) Current breast cancer diagnosis is based on histological grading and molecular subtyping, primarily assessing the expression of ER, PR, HER2, and the Ki-67 proliferation index. The major subtypes—Luminal A, Luminal B, HER2-enriched, and TNBC—display distinct biological characteristics and are treated with different therapeutic strategies. (B) Application of epigenetic therapies in breast cancer. Major classes of epigenetic drugs include inhibitors of HDACs, DNMTs, BET proteins, and EZH2. Beyond small-molecule inhibitors, emerging approaches such as ASOs and CRISPR/dCas9-based epigenome editing are under active investigation. ASOs act by binding specific lncRNA transcripts and inducing their degradation via RNase H, thereby modulating downstream gene expression. CRISPR/dCas9-based systems employ catalytically inactive Cas9 fused to epigenetic effector domains, guided by specific gRNAs, to target genomic loci and install or erase epigenetic marks with high precision. ASO: Antisense oligonucleotide; BET: Bromodomain and extraterminal domain; ChT: Chemotherapy; dCas9: Dead CRISPR-associated protein 9; DNMT: DNA methyltransferase; ER: Estrogen receptor; ET: Endocrine therapy; EZH2: Enhancer of zeste homolog 2; HDAC: Histone deacetylase; HER2: Human epidermal growth factor receptor 2; LN: Lymph node; IncRNA:Long non-coding RNA; PR: Progesterone receptor; RNaseH1: Ribonuclease H1; TNBC: Triple-negative breast cancer.

Conclusions and Perspectives

Over the past decades, tremendous progress has been made in epigenetics, both theoretically and technologically. Many groundbreaking discoveries in this field have used breast cancer as a disease model.^[134–136] The mechanisms and regulators involved in breast cancer are certainly not limited to those discussed above. One emerging yet underexplored epigenetic pathway involves chemical modifications of mRNA, such as N6-methyladenosine (m6A), which confer post-transcriptional regulation of the broader epigenetic landscape in breast cancer. m6A modifications can promote tumor progression either by enhancing oncogenic signaling pathways, such as mammalian target of rapamycin^[137] and PI3K/Akt,^[138] or by facilitating mRNA degradation.^[139] Several recent reviews provide comprehensive summaries of RNA modifications in breast cancer progression and therapy.^[140–142] The biophysical environment of cells can also influence chromatin dynamics through intracellular compartmentalization. Recent work shows that phosphorylated HDAC6 undergoes liquid–liquid phase separation in TNBC cells—but not in other breast cancer subtypes. This phospho-HDAC6–induced aberrant chromatin architecture disrupts chromatin accessibility, histone acetylation, RNA polymerase II elongation, and transcriptional programs. By contrast, nexturastat A, a specific disruptor of phospho-HDAC6 liquid–liquid phase separation, restores chromatin architecture and effectively suppresses TNBC growth.^[143] Importantly, epigenetic regulation is not confined to primary tumor cells. At every stage of breast cancer progression, tumor-infiltrating immune cells—such as tumor-associated macrophages,^[75] cancer-associated fibroblasts,^[67] epithelial cells,^[144] and even astrocytes in the remote metastatic organ^[145]—undergo dynamic epigenetic reprogramming similar to that seen in cancer cells. For instance, the co-opted evolution of brain metastatic cells within the microenvironment induces high expression of Cdk5 and suppression of major histocompatibility complex I, thereby promoting brain metastasis of breast cancer.^[145] Moving forward, delineating the epigenetic circuits governing TME plasticity and exploring combinatorial therapeutic strategies that incorporate epigenetic modulators to constrain this adaptability may improve the durability of treatment responses.

The comprehensive epigenetic landscape of breast cancer can be further unraveled through the integration of cutting-edge multi-omics methods. Single-cell multi-omics platforms, such as single-cell-NanoHi-C^[146] and single-cell-Nano sequencing-cleavage under targets and tagmentation (scNanoSeq-Cut&Tag),^[147] offer high-resolution profiling of chromatin architecture and chromatin modifications, enabling more precise mapping of heterogeneous tumor cells and immune cells within the TME. At the same time, imaging-based approaches such as MERFISH^[148] provide spatial information on gene expression and cellular interactions, linking intratumoral heterogeneity with tissue architecture. In addition, plasma-based multi-omics—including transcriptomics, metabolomics, and proteomics—holds promise for uncovering molecular crosstalk between metabolic disorders and cancer, reinforcing the view of cancer as a systemic disease shaped by complex environmental and pathophysiological factors.^[149] Ultimately, we hope that mechanistic studies in epigenetics will continue to evolve from the bench to the bedside, empowering scientists and clinicians to design more effective treatment regimens to combat breast cancer and improve patient outcomes.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82273155 and 82188102).

Conflicts of interest

None.