Targeting estrogen receptor alpha in breast cancer for novel therapies resistance mechanisms and future directions

Abstract

Estrogen receptor α (ERα) is a key therapeutic target in ER-positive breast cancer. Structurally, ERα contains activation function (AF) domains that drives breast cancer proliferation, metastasis, and drug resistance through classical genomic, non-genomic, and ligand-independent signaling pathways. Clinically approved and investigational drugs targeting Erα include selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators/degraders (SERDs), and novel strategies such as proteolysis-targeting chimeras (PROTACs). Current research focuses on overcoming endocrine resistance via combination therapies targeting mutations in ESR1, the gene encoding ERα, non-genomic signaling pathways, and the tumor microenvironment, which may advance precision medicine in breast cancer. This article summarizes recent advances in ERα-targeting inhibitors and their therapeutic implications, to provide potential precision therapeutic strategies for breast cancer patients with ER positive.

Introduction

Breast cancer is a malignant tumor of the mammary epithelium [1]. About 15.2% of newly diagnosed cases are ductal carcinoma in situ (DCIS), defined as abnormal proliferation of epithelial cells confined within the ductal-lobular system of the breast. DCIS is a non-invasive form of breast cancer (stage 0) and usually has a good prognosis, with an overall recurrence rate after treatment of about 20% [2–4]. In contrast, invasive ductal carcinoma (IDC) is characterized by stromal invasion, and once DCIS progresses to invasive carcinoma, however, tumor cells can detach and spread through lymphatic and blood vessels, causing metastasis and potentially life-threatening disease [5, 6]. The occurrence, development, and metastasis of breast cancer are extremely complex processes. Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are key biomarkers for breast cancer treatment and prognostic assessment [7]. Among all patients with breast cancer, about 70% of breast cancers are ER-positive, which often have a good prognosis and are sensitive to endocrine therapy [8–10].

Among ER-positive breast cancers, the main intrinsic subtypes are Luminal A and Luminal B. Luminal A tumors show high ER/PR expression, are HER2-negative and have low proliferative activity, whereas Luminal B tumors are ER-positive with increased proliferative activity and/or HER2 overexpression. Luminal A tumors generally have a more favorable prognosis [11]. According to the ESMO(European Society for Medical Oncology) clinical practice guidelines for early breast cancer, most patients with Luminal A tumors receive endocrine therapy alone. Chemotherapy is added only when the tumor burden is high, such as involvement of ≥ 4 lymph nodes or tumor stage ≥ T3. For Luminal B (HER2-negative) disease, chemotherapy followed by sequential endocrine therapy is usually recommended. Patients with Luminal B (HER2-positive) tumors should receive chemotherapy combined with anti-HER2 therapy, followed by endocrine therapy. In patients who cannot tolerate chemotherapy, endocrine therapy combined with anti-HER2 therapy may be used as an alternative regimen [12]. Estrogen receptor alpha (ERα) binds to estrogen and activates downstream gene transcription, thereby regulating cell proliferation and differentiation [13]. Through this mechanism, the estrogen receptor signaling pathway plays a central role in endocrine therapy for breast cancer, and its activity is closely linked to tumor growth, invasion and metastasis.

The structure and characteristics of ERα

Domain architecture of ERα

As a steroid receptor in the nuclear receptor family, ER is mainly divided into two subtypes, ERα and ERβ, which can bind to estrogen and subsequently regulate gene expression [14, 15]. ERα is encoded by the ESR1 gene located on chromosome 6 [16], while ERα and ERβ have a common structural system [17–21]. The primary structure is composed of six domains that is, the A/B domain (NH2 terminus), the C domain (DNA binding domain, DBD), the D domain (hinge region), and the E/F domain (ligand binding domain, LBD) [16] (Fig. 1).

Fig. 1.

Structure of Estrogen receptor α (ERα)

The transcriptional activation domains of ERα are responsible for its function as a transcription factor, which can be divided into the ligand-independent transcriptional activation domain, activation function-1 (AF-1), and the ligand-dependent transcriptional activation domain, activation function-2 (AF-2). The ligand-independent one, AF-1, located in the A/B domain, can activate gene transcription in the absence of estradiol, or even in the presence of selective estrogen receptor modulators(SERMs) [16, 22]. AF-1 interacts with coregulators and participates in transcriptional activation [23, 24]. In ERα, this domain is very active in stimulating reporter gene expression from a variety of estrogen response element (ERE) reporter gene constructs in different cell lines, while the activity of the AF-1 domain of ERβ was negligible under the same conditions [25]. Another significant difference between ERα and ERβ is their unique response to the synthetic antiestrogens, such as tamoxifen, raloxifene, and ICI-164,384. On ERE-based reporters, these ligands are partial 17β-estradiol (E2) agonists for ERα but pure E2 antagonists for ERβ [26–28]. The ligand-dependent transcriptional activation region, AF-2, is located in the E/F region. When a ligand (such as estrogen) binds to the LBD, the transcriptional activation function of AF-2 is activated, thereby regulating gene expression [29]. AF-1 and AF- 2 together are very important for the interaction with coregulators [23, 30].

DBD contains two zinc finger structures, corresponding to the C segment domain, which is responsible for recognizing specific DNA sequences and ERE motifs (GGTCAnnnTGACC) [31], as well as for forming homodimers, essential for the transcriptional activity of ERα [32]. The binding site in DBD provides a molecular basis for drug screening and design. Another domain, LBD, corresponds to the E/F segment domain. The E segment includes a pocket region that binds to estrogen compounds, to form a high-affinity binding with E2 [33]. In addition, the LBD region is involved in binding co-regulators (co-activators and co-repressors) and molecular chaperone proteins, responsible for receptor dimerization and other functions. LBD and DBD form the ERα dimerization interface [34]. DBD and LBD contain nuclear export signals (NES), which are important for nuclear export [35], while the C-terminal domain of the F domain is the region with the least homology among members of the nuclear receptor family. This domain is involved in ERα stability, dimerization, protein-protein interactions, and tamoxifen-mediated transactivation [36–39]. The hinge domain corresponds to the D segment domain, important for the combined action of AF-1 and AF-2. AF-1 and AF-2 work synergistically through indirect AF-1/2 interaction via TIF2 (SRC-2) to enhance ERα transactivation [23, 24]. This domain contains a nuclear localization signal (NLS) that is important for nuclear migration [40], which is also required for ERα to bind to mRNA in the cytoplasm [41].

ERα splice variants

Alternative splicing of ERα gives rise to multiple isoforms with distinct structures and functions, including the classical full-length Erα66 [42, 43] as well as a series of N-terminally or C-terminally truncated and exon-skipping variants.

ERα36 is an isoform cloned and characterized by Wang et al.. with a molecular weight of approximately 35.7 kDa [44]. It lacks both the AF-1 and AF-2 transactivation domains but retains the DNA-binding domain, part of the ligand-binding domain and the dimerization domain, and is predominantly localized in the cytoplasm and at the plasma membrane, supporting a dual role in genomic and non-genomic signaling [45–47]. The ERα36 transcript originates from an alternative promoter located upstream of the first intron of the ERα66 gene, giving rise to exon 1′, while exons 2–6 are shared with ERα66. Its C-terminus consists of a unique stretch of 27 amino acids that replaces the 138 C-terminal amino acids encoded by exons 7–8. Three putative N-myristoylation sites are present near the N-terminus and are thought to contribute to membrane localization [48, 49]. ERα36 itself has little intrinsic transactivation capacity and can antagonize ERα66-mediated genomic transcription. In contrast, it efficiently triggers rapid non-genomic signaling at the plasma membrane, rapidly activating the MAPK/ERK cascade and promoting cell proliferation [44]. Silencing ERα36 abolishes rapid ERK activation and other non-genomic estrogen responses, whereas silencing ERα66 does not, indicating that ERα36 is a key receptor for these rapid pathways. Retrospective cohort data suggest that about 40% of breast tumors show high membrane/cytoplasmic ERα36 expression, which correlates with poor response to tamoxifen and with HER2 expression [49]. Another analysis of breast tumors linked high ERα36 mRNA levels to the upregulation of multiple metastasis-associated markers, increased metastatic propensity and tamoxifen resistance, with opposite prognostic associations in PR-positive versus PR-negative patients, suggesting an interaction with PR status [50]. Signaling dominated by ERα36 is largely insensitive to tamoxifen and to fulvestrant [44]. Moreover, in ERα36-positive breast and endometrial cancer cells, both classes of antiestrogens act as agonists at ERα36, activating MAPK/ERK and PI3K/AKT and producing a biphasic dose-response, with low doses stimulating and high doses inhibiting proliferation [51–54]. Structurally, the ligand-binding domain of ERα36 lacks helices H9-H12 found in ERα66 [48]. the H12 domain is critical for fulvestrant induced receptor degradation [55, 56]. Consistent with this, fulvestrant does not promote ERα36 degradation [57, 58], which helps explain why standard antagonists fail to fully suppress ERα36-driven signaling. ERα36 also forms a bidirectional positive feedback loop with EGFR/HER2: in triple-negative and HER2-overexpressing models, growth factor signaling upregulates ERα36 transcription via an AP-1 site in the 5′-flanking region of its promoter [59], while ERα36 stabilizes EGFR at the membrane and enhances HER2 expression [60]. Clinically, tamoxifen can induce concomitant upregulation of ERα36, EGFR and HER2, implicating this loop in acquired resistance [61, 62]. Coupling with the membrane receptor GPER has also been demonstrated: the GPER agonist G1 can directly bind to and activate ERα36 [58], and GPER upregulates ERα36 promoter activity via AP-1 binding sites [57, 58]. These interactions provide an additional mechanism for amplification of non-genomic estrogen signaling and for the emergence of resistant phenotypes.

ERα46 is a 46 kDa N-terminally truncated isoform generated by alternative splicing of exon 1, which lacks the AF-1 transactivation domain present in full-length ERα66 [45, 46]. ERα46 expression has been detected in multiple cell types, including vascular endothelial cells, macrophages and osteoblasts [63–66] ERα46 can form homodimers as well as heterodimers with ERα66 and preferentially participates in ERE-dependent regulation as ERα46/ERα66 heterodimers, thereby replacing ERα66 homodimers. In this context, ERα46 selectively suppresses AF-1-dependent transcription driven by ERα66 while having relatively limited impact on AF-2-dependent transcriptional activation [67]. In various models, ERα46 exhibits antiproliferative and pro-apoptotic effects: in HT-29 colon adenocarcinoma cells, it inhibits cell growth and induces apoptosis [66]. In ERα-positive cells, ERα46 acts as a potent competitive inhibitor of ERα66, whereas in ERα-negative tissues it can confer estrogen-responsive genomic activity [96]. With respect to endocrine resistance, ERα46 has been detected in tamoxifen-resistant breast cancer cell lines and in colorectal tumor samples [68]. In tamoxifen-resistant breast cancer cells, ERα46 levels are reduced, and re-expression of ERα46 suppresses proliferation and decreases the transcriptional activity of ERα66 target genes [68]. In MCF-7 cells, ERα46 inhibits cyclin D1 promoter activity [69], and in MDA-MB-231 cells it reduces basal transcription of pS2, an effect that is relieved by estrogen [70]. Together, these findings support a role for ERα46 as a negative regulator of estrogen-driven proliferation and endocrine resistance.

ERα30 is a truncated isoform encoding a 271-amino-acid protein of approximately 30 kDa. It retains the AF-1 domain, the DNA-binding domain and part of the hinge region, but completely lacks the ligand-binding domain and AF-2, and acquires a unique 10-amino-acid C-terminal sequence. When overexpressed in ER-α66(-)/PR(-)/HER2(-) MDA-MB-231 cells, ERα30 can be detected by an antibody recognizing amino acids 2-185 of ER-α66 and significantly enhances cell proliferation, colony formation, migration and invasion, suggesting that it may use its retained AF-1/DBD to drive transcription of specific target genes that promote malignant behavior in breast cancer cells [71]. Previous studies have shown that some ER-negative patients still derive benefit from antiestrogen therapy [72], and ER variants have been proposed to facilitate progression toward a more invasive, antiestrogen-insensitive phenotype [69, 73]. In the study of ER-α30, this isoform reduced ER-α66 and PR protein expression, linking ER-α30 to the remodeling of ER/PR-negative breast cancer biology and to endocrine resistance [71]. However, this hypothesis currently lacks direct confirmation from drug intervention or gene-silencing experiments, and further functional studies are needed.

ESR1 mutations

Mutations in the ligand-binding domain (LBD) of ERα are strongly enriched under the selective pressure of endocrine therapy and represent a major mechanism of endocrine resistance in metastatic breast cancer. ESR1 mutations occur in less than 1% of primary breast cancers, but their frequency can rise to about 60% in metastatic tumours after treatment with aromatase inhibitors (AIs) [74, 75]. Most of these mutations cluster in the LBD, because this domain forms the direct binding site for oestrogens and anti-oestrogens. Its hydrophobic pocket and surrounding α-helices, especially helix 12 (H12), finely control receptor conformational switching and transcriptional activity. Anti-oestrogens inhibit co-activator recruitment by disrupting the proper folding of H12. Under sustained anti-oestrogen treatment, mutations that weaken drug binding or promote a persistently “active-like” conformation gain a selective advantage [76]. Studies have shown that ESR1 mutations can promote rapid proliferation and invasion of breast cancer cells by reshaping epithelial-mesenchymal transition [77, 78].

The most common ERα mutations in breast cancer are Y537S and D538G. Both are located in the N-terminal portion of H12 within the LBD, followed by less frequent variants such as Y537N, Y537C and L536Q [79]. Y537S lies in the N-terminal regulatory loop region of H12 in the ERα LBD. After mutation, S537 forms a stable hydrogen bond with N351. This interaction remodels the H11-12 loop and locks H12 in an active position. It is noteworthy that the surface-exposed Y537S mutation has only a limited effect on the overall LBD structure [80]. This may explain why Y537S still responds to antagonists in functional studies [81]. In the absence of oestrogen, the mutant receptor maintains a highly active conformation, with enhanced co-activator binding and reduced proteolytic degradation [82, 83]. In the presence of oestrogen, Y537S still drives higher transcriptional output than the wild-type receptor [84], indicating a hyper-activated response to ligand stimulation. The mutant receptor also shows increased binding to FOXA1 and GREB1, which enhances cancer cell motility [85]. Transcriptomic analyses using CRISPR-Cas9-based allele-specific models further show that Y537S strengthens tumour-associated signalling pathways, such as p53 and mTORC1, reflecting broad transcriptional rewiring and network-level adaptation [88]. D538G is similar to Y537S in that it stabilises the active conformation of H12 and induces ligand-independent transcriptional activation. However, the two mutants differ markedly at the transcriptional level. After oestrogen treatment, 416 genes show significantly increased expression in D538G-mutant cells, whereas only 12 genes are upregulated in Y537S-mutant cells [87]. This difference is also evident in functional assays. D538G-mutant cells exhibit stronger invasive capacity [88]. When treated with tamoxifen or fulvestrant, D538G-mutant cells are less resistant than Y537S-mutant cells [89]. Another study reported that patients carrying Y537S have shorter overall survival than those with D538G [90]. These conformational changes in the above mutants reduce inhibitor binding, promote co-activator recruitment and increase proteolytic stability, thereby influencing endocrine therapy resistance in vitro.

ERα RNA binding

ERα is generally considered to act mainly as a DNA-binding transcription factor. Recent studies, however, have revealed that it may also function as an RNA-binding protein. This new concept provides an additional perspective for understanding endocrine resistance. Steiner et al. demonstrated from a structural perspective that the extended DNA-binding domain (DBD) of ERα itself constitutes a functional RNA-binding domain (RBD) and exhibits a specific preference for defined RNA secondary structures [91]. Xu et al. showed that ERα can directly bind multiple classes of mRNAs, with a preferential localization to the 3′ untranslated region (3′UTR) where it recognizes conserved sequence motifs. These findings indicate that ERα possesses non-canonical RBD properties and sequence-specific RNA recognition capacity. Notably, the RNA-binding activity of ERα is separable from its classical DNA-binding-dependent transcriptional function. Mechanistically, ERα relies on an RNA-binding domain in its hinge region to bind with high specificity to the 3′UTRs of stress-responsive mRNAs, including X-box binding protein 1 (XBP1), myeloid cell leukemia 1 (MCL1) and eukaryotic translation initiation factor 4 gamma 2 (eIF4G2). In the cytoplasm, ERα cooperates with the RNA 2’,3’-cyclic phosphate and 5’-OH ligase RtcB (RtcB) to promote inositol-requiring enzyme 1 (IRE1)-mediated non-canonical splicing of XBP1 mRNA. In parallel, ERα enhances the translation efficiency of MCL1 and eIF4G2 mRNAs. Through these actions, ERα fine-tunes, at the post-transcriptional level, the expression of key effector proteins involved in the integrated stress response and the unfolded protein response, thereby enabling ER-positive breast cancer cells to survive and acquire resistance under multiple stress conditions induced by tamoxifen. At the cellular level, deletion of the RBD markedly impairs the proliferative capacity of ER-positive breast cancer cells and suppresses tumour growth in mouse xenograft models [92]. At present, large cohort studies that systematically evaluate the association between the expression of Erα-mediated post-transcriptional targets and patient prognosis or response to endocrine therapy are still lacking. In addition, the temporal and spatial coordination and integration of the DNA-binding and RNA-binding functions of ERα remain to be fully elucidated. Future studies should dissect, in models that more closely reflect the clinical setting, how ERα integrates transcriptional and post-transcriptional regulatory networks, and should assess the safety and efficacy of therapeutic strategies targeting ERα-dependent stress-responsive translational programmes, such as the IRE1-XBP1 pathway, the eIF4F complex or MCL1 [93–96], for overcoming endocrine resistance.

Activation mode of ERα signaling pathway

Classical genomic pathway of ERα

Absence of E2, ERα is normally bound to a multiprotein inhibitory complex in the cytoplasm of target cells, or shuttles between the cytoplasm and nucleus [97]. When binding to its ligand, ERα undergoes a ligand-specific conformational change, leading to separation from the chaperone complex, dimerization, and ultimately stimulation of the receptor transcriptional domain [98–100] followed by release from the heat shock protein complex, HSP90, to which it is bound [101, 102]. HSP90 is a molecular chaperone that protects unbound ERα from degradation [103]. It is shown that ERα without ligand is a short-acting protein with a half-life of only 4–5 h [104]. After forming an ERα homodimer, it binds to specific DNA response elements, EREs, on its target genes, performing genomic signaling of ERα [97]. Direct or indirect interaction with the general transcriptional machinery needs accessory proteins, such as SRC-1, TRAP220, AIB1, CBP/p300, PGC-1, p68 RNA helicase, SRA, and GRIP1 [105] The AF-2 domain consists of an amphipathic helix, which has the functional properties of both hydrophilic and hydrophobic regions in an α-helix [106], which is required for ligand-dependent transcriptional activity and is critical for interaction with members of the SRC accessory factor family [105, 107]. AF-1 is also capable of recruiting accessory factors, which may be similar or different from AF-2. In most cases, AF-1 and AF-2 act synergistically, but in some cells, they also function independently [108].

Through direct genomic actions of the ERα, the expression of genes containing EREs is altered. ERα bound to DNA enhances the organization of the pre-initiation complex and facilitates chromatin disruption at the ERE by recruiting auxiliary factor proteins [105, 109]. The activity of histone acetyltransferases (HATs) can promote nucleosome repositioning, chromatin opening, and interact with the general transcription machinery centered on RNA polymerase II. For example, members of the p160 coactivator family, SRC-1 and ACTR, contain LXXLL amino acid motifs that can bind to the AF-2 region of ligand-activated ERα, serving as a platform for further recruitment of other HATs. These HATs promote transcription of ERα target genes by acetylation of histones, loosening the chromatin structure [110–112].

ERα can also regulate the expression of genes lacking ERE in the promoter by interacting with other transcription factors (such as AP-1 and SP1) [113–115]. ERα regulates the transcription of target genes by forming a complex with Fos and Jun and binding to the AP-1 site. In addition, ERα can also bind to the transcription factor Sp1 to form an ERα-Sp1 complex, and further bind to GC-rich promoter sequences to guide transcriptional activity. This mechanism shows that ERα not only acts directly on genes dependent on ERE, but also acts on target genes that are not associated with classical ERE through transcription factor mediation. At the AP-1 site, ERα exhibits transcriptional activation after binding to estrogen, while ERβ inhibits transcription when binding to estrogen, reflecting the functional specificity of ER subtypes in gene regulation [116, 117].

At the clinical level, molecular therapies that target auxiliary factors that mediate chromatin remodeling (such as SRC-1 and p300) or interrupt ERE-related pathways may become an important direction in anti-tumor strategies. A class of p300/CBP PROTAC degraders has been reported that can inhibit the abnormal activation of specific enhancers by degrading p300/CBP, thereby downregulating oncogene expression and having a potential inhibitory effect on the proliferation of various tumors [118]. Future research should further focus on the regulatory complexity of the ER signaling pathway and its interaction with the tumor microenvironment and explore more precise treatment methods (Fig. 2).

Fig. 2.

Activation Mode of ERα Signaling Pathway

Activation mode of ERα signaling pathway: Classical genomic pathway: Estrogen (E2) diffuses into the cytoplasm and binds to ERα, inducing conformational changes and dissociation from heat shock proteins (HSPs). The receptor dimerizes, undergoes phosphorylation, translocates to the nucleus, and either binds directly to Estrogen Response Elements (EREs) on target gene promoters to initiate transcription or associates with DNA-bound transcription factors (e.g., AP-1, Sp1) at Serum Response Elements (SREs). Ligand-independent activation pathway: In the absence of E2, growth factors (e.g., EGF, IGF) bind to their respective receptors (EGFR, IGFR), triggering kinase cascades (MAPK/PI3K/AKT). These kinases directly phosphorylate ERα, enabling it to dimerize and regulate gene expression in a ligand-independent manner. Non-genomic pathways: Membrane-bound ERα initiates rapid signaling via interactions with adaptor proteins, activating second messengers and kinase cascades. This leads to immediate cytoplasmic effects or indirect transcriptional regulation of genes lacking EREs through the activation of other transcription factors.

Non-genomic pathways of ERα

The non-genomic effects of estrogen show remarkable diversity and importance in different tissues. For example, estrogen activates nitric oxide synthase (eNOS) in endothelial cells and regulates vascular tone through the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways, thereby exerting a biphasic regulatory effect to protect blood vessels [119]. Similarly, in bone tissue, E2 directly acts on osteoblasts and osteoclasts by activating the MAPK pathway, participating in anti-apoptosis and promoting cell proliferation, thereby supporting bone formation and preventing bone loss [120]. In breast cancer, estrogen plays a significant role in cell cycle regulation and carcinogenesis through non-genomic signals. The upstream initiation of non-genomic ER signals involves the Ras-Raf-MEK-MAPK pathway, activating a series of transcription factors (such as c-Fos and c-Jun) through phosphorylation downstream, and further regulating the expression of genes related to cell proliferation [121–123]. On the other hand, the PI3K-Akt-mTOR pathway also depends on the activation of membrane estrogen receptor (mbER), involved in the growth and drug resistance of E2-dependent tumor cells [124]. It is worth noting that the abnormal activation of these signaling pathways is of great significance in the formation of resistance in endocrine therapy and is a key direction of current anti-estrogen therapy research.

Among the signals initiated by mbER, Ras-Raf-MEK-MAPK is first activated by guanine nucleotide binding protein (Ras), which in turn activates Raf and MEK proteins, and finally phosphorylates MAPK [125–128]. This cascade signal is widely present in different types of cells and triggers biological effects on cell proliferation and survival by regulating downstream effector molecules, such as transcription factors c-Jun and c-Fos [121–123]. Another parallel pathway, the PI3K-Akt-mTOR pathway, plays a more direct role in cell survival and metabolic regulation. The activation of PI3K depends on the tyrosine kinase, SRC, and other cooperating molecules. This direct protein-protein interaction further promotes the activation of downstream Akt signals. Akt regulates mTOR in a dependent manner and plays a key role in cell growth and metabolism [124]. In breast cancer cells, this abnormally highly activated signal may be an important basis for cancer cell proliferation and treatment resistance.

The localization of ERα on the plasma membrane and its non-genomic activities are often dependent on post-translational modifications, including mechanisms such as palmitoylation and methylation. For example, palmitoylation of cysteine 447 of ERα significantly increases the hydrophobicity of the receptor, allowing it to anchor in the lipid raft region [129]. Lipid rafts are cholesterol-rich plasma membrane regions where ERα interacts with speleothem-1 and promotes cell proliferation by activating downstream effector molecules such as cyclin D1 [124, 127, 129]. Another important post-translational modification is the methylation of arginine 260 of ERα, catalyzed by arginine methyltransferase 1 (PRMT1), which is a necessary condition for ERα to form a complex with SRC and PI3K [130]. This complex not only stabilizes signal transduction under estrogen stimulation, but also provides a direct bridge between membrane receptors and intracellular signaling networks. Interestingly, the process of ERα methylation is time-dependent, which can only be detected 5 to 15 min after ligand stimulation [116]. In particular, in MCF-7 breast cancer cells, insulin-like growth factor 1 (IGF-1) rapidly induces ERα methylation through PRMT1 and further promotes the binding between ERα and IGF-1 receptor (IGF1R). This cascade regulatory mechanism forms a closed-loop association between ER and the growth signals of cancer cells [134]. In addition, ERα tyrosine 537 (Tyr537) phosphorylation is also an important condition for its interaction with SRC, which promotes the activation of the MAPK pathway and continuously drives the expression of downstream genes and the signaling network of cell proliferation [131]. Therefore, the fine regulation mechanism of ERα provides a dynamic and flexible response mode for non-genomic signal transduction pathways. Importantly, the precise analysis of the interaction between ER and SRC, and PI3K may open up new prospects for the personalized treatment of endocrine resistance.

Ligand-independent activation pathway of ERα

In the absence of E2, ERα can also achieve its biological function through the signaling pathway of polypeptide growth factors, such as IGF-1 and epidermal growth factor (EGF). It is found that EGF signaling can effectively mimic the physiological regulatory effects of E2 on female genitalia, and this effect has been confirmed in vivo studies [132, 133]. In particular, when E2 levels are low, such as during perimenopause or certain pathological conditions, these growth factor pathways can still maintain the activity of ERα [134]. This suggests that ligand-independent signaling mechanisms may be a compensatory mechanism to maintain biological functions.

In the ligand-independent activation process, the AF-1 domain is the core regulatory region because it can be directly activated by cell signals. Under the action of EGF or IGF signals, the serine 118 (S118) site of AF-1 is phosphorylated, and then the signal effect is further amplified through the MAPK pathway [135, 136]. Importantly, S118 phosphorylation promotes the binding of ERα to coactivators (such as p68 RNA helicase and SRC-1), accelerating the initiation of target gene transcription [102, 135]further enhances the transcriptional activity of ERα by inducing the phosphorylation of coactivators such as SRC-1 and AIB1 [141].

In addition, post-translational modification of ERα also plays an important role in ligand-independent activation [116]. Among them, phosphorylation is one of the most critical modifications. S118, S167, S305, and tyrosine 537 (Tyr537) on ERα, the key sites of phosphorylation, can significantly promote the transcriptional activity of ERα by enhancing the binding of ERα to co-activators [120, 132, 137, 138]. Moreover, acetylation, as another important post-translational modification, regulates the functional activity of ERα. Acetylation of lysine 266/268 (K266/268) can stimulate the transcriptional function of ERα, while acetylation of lysine 302/303 (K302/303) inhibits its activity [136], showing the complexity of bidirectional regulation.

The function of ERα is not only reflected in the transcriptional regulation at the genomic level, but also includes non-genomic signaling. E2 requires the assistance of the MAPK pathway to regulate the transcriptional activity of the AP-1 binding site of cyclin D1, and is dependent on the formation of the ERα/SRC/PI3K complex [139, 140]. This intersection of genomic and non-genomic pathways further reveals the complexity of E2 target gene regulation [141]. However, compared with genomic signals, non-genomic effects play a more unique role in tumor pathology. Studies have shown that the non-genomic effects of ERα may be closely related to the resistance of breast cancer to endocrine therapy and the poor prognosis [142–144]. In particular, during disease progression, ERα transforms from a dimer to a monomeric state, accompanied by a decrease in genomic activity and an increase in non-genomic signals [145]. This transformation further suggests that non-genomic pathways may be an important mechanism of cancer progression and treatment resistance.

So targeted inhibitors targeting specific phosphorylation sites (such as S118 and Tyr537) may limit the excessive activity of ERα and have application prospects in the treatment of estrogen receptor-positive breast cancer [146–149]. More importantly, the correlation between non-genomic signals and endocrine resistance in breast cancer provides new ideas for overcoming treatment resistance.

Mechanisms of Estradiol‑Dependent ERα mRNA regulation

In addition to the diverse genomic, non-genomic and ligand-independent mechanisms described above, the intensity and duration of ERα signalling are also determined by estradiol mediated regulation of ERα mRNA abundance. In hormone responsive normal tissues and ER positive breast cancer cells, E2 activated ERα directly binds the ESR1 promoter and distal enhancers in a manner that is specific to tissue and cell type, thereby modulating ESR1 transcription and establishing autoregulatory feedback loops [150].

Autoregulation of ERα and its E2-dependent expression dynamics differ between tissues. Using semi-quantitative RT-PCR and in situ hybridization in normal human endometrium, Matsuzaki et al.. and Witek et al.. showed that ERα mRNA is most abundant in glandular and stromal cells during the early-mid proliferative phase, and then declines markedly during the mid-late secretory phase [151, 152]. In human endometrium, E2 activates ERα, which binds to the ESR1 promoter and adjacent chromatin regulatory regions in endometrial cells. This binding increases ESR1 transcription and ERα protein expression. It establishes a signal amplification loop during the follicular phase, in which cellular sensitivity to E2 gradually increases.

Ellison et al. used MCF-7 breast cancer cells to demonstrate that E2 imposes negative feedback on ERα mRNA transcription. This feedback is mediated by Erα-Sin3A-dependent chromatin remodelling at the proximal ESR1 promoter. E2 reduces both nascent ESR1 transcripts and total ESR1 mRNA. This reduction coincides with specific recruitment of the E2-ERα complex to the proximal ESR1 promoter A, with enrichment of the Sin3A/HDAC co-repressor complex in this region. It is accompanied by H3K14 deacetylation, an increase in H4K20me3 and a marked loss of RNA polymerase II occupancy [150]. Similarly, in ER + breast cancer cells, ligand-activated ERα is recruited to four distal enhancers (E1-E4) located more than 120 kb upstream of ESR1. These enhancers are enriched for oestrogen response elements (EREs) and are co-occupied by the pioneer factors FOXA1 and GATA3, the co-activator p300 and RNA polymerase II. E2 treatment strongly increases recruitment of p300 and RNA polymerase II to enhancers E1-E4, indicating that they act as hubs for E2-dependent transcriptional regulation [153–156]. Notably, E2-dependent autoregulation of ERα transcription is cell-type specific in breast cancer. Read et al.. reported that E2 stimulation markedly decreases ERα mRNA in MCF-7 cells, but instead induces ERα mRNA in T-47D cells [157]. In parallel, E2 promotes ERα protein turnover and degradation in tumour cells, thereby weakening subsequent ER signalling output [158]. Population data from primary breast tumours show a strong correlation between ESR1 mRNA and ERα protein levels [159]. Together, these findings support a model in which E2-activated ERα occupies distal ESR1 enhancers and remodels their transcriptional activity, while simultaneously driving ERα protein degradation. Through this combined mechanism, E2 exerts bidirectional control over ERα mRNA expression in different breast cancer cell lines, thereby limiting the strength and duration of oestrogen signalling.

The drugs targeting ERα and their applications

According to the mechanism of drug effects on ER expression and activity, drugs targeting ERα are mainly divided into four types, that is SERMs, selective estrogen receptor degraders (SERDs) (Table 1), proteolytic targeting chimeras (PROTAC) degraders, and siRNA drugs. SERMs affect ER by reducing its activity, SERDs degrade ER or reduce ER expression [160, 161], and PROTAC degraders directly induce ER degradation by utilizing the ubiquitin proteasome protein degradation system in the human body [162].

Table 1.

Selective Estrogen receptor modulators (SERMs) and selective Estrogen receptor degraders (SERDs)

Drug	Type​	Phase	References
Tamoxifen	SERM	approved	[189]
Raloxifene	SERM	approved	[184]
Toremifene	SERM	approved	[185–187]
Bazedoxifene	SERM	approved	[188]
Fulvestrant	SERD	approved	[189, 190]
Elacestrant	SERD	approved	[191]
Afimoxifene	SERM	Phase III	[192]
TAS-108	SERM	Phase II	[193]
Lasofoxifene	SERM	Phase III	[194]
Arzoxifene	SERM	Investigational	[195]
Ormeloxifene	SERM	Investigational	[196]
Pipendoxifene	SERM	Investigational	[197]
Droloxifene	SERM	Investigational	[198]
Icaritin	SERM	Investigational	[199]
Acolbifene	SERM	Investigational	[200]
Enclomiphene	SERM	Investigational	[201]
ICI-164,384	SERD	Phase I	[202]
AZD-9496	SERD	Investigational	[203]
GDC-0810	SERD	Investigational	[204]
Camizestrant	SERD	Investigational	[205]
Giredestrant	SERD	Investigational	[206]
Imlunestrant	SERD	Investigational	[207]

Traditional endocrine therapies, including SERMs and SERDs, significantly improve outcomes for ER-positive breast cancer. However, acquired resistance is prevalent and limits long-term efficacy [163, 164]. Resistance primarily stems from abnormalities within the ER pathway itself, including ERα deletion or mutation, ligand-independent activation induced by the truncated variant ERα36, and abnormal transcriptional regulation resulting from co-activated SRC 3 upregulation and post-translational modifications such as phosphorylation and methylation [165–167]. Bypass interactions with growth factor receptor pathways constitute another core mechanism, particularly centered on HER2 and the PI3K/AKT/mTOR axis. HER2 amplification/over-expression can activate parallel survival signaling, reduce ER expression, and form bidirectional feedback with ER-inhibiting one pathway can reactivate the other. This interaction is further amplified by integrin-FAK and receptor tyrosine kinases like EGFR and IGF1R [168, 169]. Beyond genomic effects, cytoplasmic ER can directly interact with membrane receptors like HER2 and IGF1R, rapidly activating PI3K/AKT and MAPK cascades to sustain survival in estrogen-deprived conditions [170]. Rewiring of cell cycle and apoptosis programs similarly contributes to resistance. Upregulation of Cyclin D1, CDK4/6, MYC, and Cyclin E1 circumvents ER constraints, while elevated BCL-2/BCL-XL and downregulated BAX/BIK facilitate apoptosis evasion [171–173]. The tumor microenvironment further diminishes endocrine therapy efficacy by activating STAT3, NF-κB, and PI3K/AKT signaling through IL-6 and TNF-α secreted by TAMs and CAFs [174, 175]. Epigenetic alterations including DNA methylation, histone modifications, and miRNA dysregulation also influence ER expression and activity, promoting estrogen-independent transcription [176, 177]. Stress and alternative receptor axes are also involved. For instance, IRE1-XBP1-mediated endoplasmic reticulum stress enhances ER activity and triggers NF-κB survival signaling, while upregulation of the androgen receptor provides an alternative proliferative pathway when ER is restricted [178–180]. Additional bypasses include FGFR1/2-mediated estrogen-independent proliferation via MAPK and PI3K pathways alongside FOXA1 reprogramming [181], Notch and NF-κB promote stemness and inflammatory signaling, while elevated fatty acid synthase (FASN) supports lipid-mediated carcinogenic signaling-its inhibition restores ER degradation and re-sensitizes tumors. Wnt/β-catenin jointly drives resistance phenotypes by reducing ER expression and enhancing stemness and proliferation [180, 182]. Regarding metabolic reprogramming, HK2 is significantly upregulated in tamoxifen-resistant MCF-7 cells, suggesting enhanced glycolysis is closely associated with resistance acquisition [183].

Selective Estrogen receptor modulators (SERMs)

Tamoxifen

Tamoxifen is the earliest and most widely used SERM, which was originally developed as a contraceptive, but it failed to achieve its original purpose due to its effect of promoting ovulation [189]. It was not until 1971 that tamoxifen was first used to treat advanced breast cancer and showed certain effects in clinical studies [208]. Early studies failed to distinguish the tumor characteristics of ER + patients, resulting in the effect of tamoxifen being underestimated [189].

By 1980, the first study showed that the combination of tamoxifen and chemotherapy could improve the survival rate of patients with early breast cancer [209]. Soon, a meta-analysis confirmed the effectiveness of tamoxifen for early breast cancer [210]. Tamoxifen has gradually developed into an important drug for the treatment of breast cancer due to its good clinical efficacy, and it has also promoted the development of SERM drugs [211]. In the clinic, tamoxifen is used to treat early and advanced estrogen receptor-positive (ER-positive or ER+) breast cancer in premenopausal and postmenopausal women [212]. Five years of adjuvant treatment have been shown to significantly reduce the 15-year recurrence and mortality rates of breast cancer patients, and some studies have further recommended a 10-year course of treatment for long-term benefits [213, 214]. Tamoxifen is also approved by the FDA for the prevention of breast cancer in high-risk women and for reducing the occurrence of contralateral breast cancer [215]. The STAR study showed that tamoxifen was superior to raloxifene in reducing the risk of breast cancer, and the risk of uterine cancer and thrombosis was slightly lower than that of raloxifene [216, 217]. Although it may increase the risk of endometrial cancer, tamoxifen has long been the first choice for adjuvant treatment of breast cancer in current studies due to its low adverse reactions and control of breast cancer recurrence and mortality [218].

Interestingly, Tamoxifen is a tissue-specific SERM that exhibits estrogen antagonist or agonist effects in different tissues. In breast tissue, tamoxifen behaves as an estrogen receptor antagonist that can block ER binding to DNA and inhibit estrogen-dependent gene expression, while in the uterus and liver, it mainly performs estrogen- [219]. Tamoxifen has a low direct affinity for ER and mainly relies on the active form after metabolism to take effect. However, its metabolites, such as endoxifen and afimoxifene, have a high affinity for ER [220], significantly enhancing the anti-estrogen effect [221, 222]. In bone tissue, tamoxifen behaves as an estrogen receptor agonist, which is related to its estrogen-mimicking effect and is believed to be the reason for its prevention of osteoporosis [223, 224]. The tissue-selective action of tamoxifen reveals the unique mechanism of SERMs and opens a new era for the clinical application of selective hormone-modulating drugs. It not only demonstrates the unique advantages of breast cancer treatment but also provides important ideas for the prevention and treatment of diseases such as osteoporosis [225]. As one of the earliest SERMs introduced into clinical practice, tamoxifen’s new insights into molecular biology have promoted the further development of SERM drugs and have far-reaching significance for the treatment of a variety of diseases, including breast cancer.

Raloxifene

Raloxifene is a benzothiophene SERM. Initial studies have shown that it can increase bone mineral density and reduce the concentrations of total cholesterol and low-density lipoprotein cholesterol in serum, which was first approved for the prevention of postmenopausal osteoporosis in 1997 [184]. In a comparative study, raloxifene and tamoxifen had similar efficacy in preventing breast cancer, but raloxifene was proven to be safer than tamoxifen [226]. It was subsequently approved to reduce the risk of breast cancer in specific high-risk postmenopausal populations [226].

The function of raloxifene on ER is also tissue-specific, showing estrogen-like agonism in bones and liver, but antagonism in breast and uterus, thus having both bone protection and anti-estrogen tumor protection effects [227]. Its affinity for ERα is close to that of estradiol, and it is a partial agonist for ERα, while it is a pure antagonist for ERβ [26, 228–230]. In addition, it is also an agonist of G-protein coupled estrogen receptor (GPER), which may be related to its metabolic and cardiovascular effects [231, 232]. Raloxifene exhibits an anti-estrogen effect in the breast, which can reduce breast density and thus reduce the risk of breast cancer [227, 233].

Raloxifene is an oral alternative that does not require special gastrointestinal administration requirements [234], which is used for the prevention and treatment of postmenopausal osteoporosis, but its first-line status is lower than that of bisphosphonates [235]. The MORE study showed that raloxifene can reduce the overall risk of all types of breast cancer by 62%, invasive breast cancer by 72%, and ER-positive invasive breast cancer by 84% [236]. In the STAR trial, its efficacy in preventing non-invasive breast cancer was approximately 78% of tamoxifen effect [237]. However, it is worth noting that women with estradiol levels below 2.7 pg /mL already have a low natural risk, and the benefits of medication are limited [236]. Compared with tamoxifen, raloxifene is slightly inferior in reducing the risk of breast cancer, but it has less stimulating effect on the endometrium and a lower risk of endometrial cancer [238, 239]. The incidence of estrogen-related adverse reactions such as breast pain and vaginal bleeding is low. Although the risk of venous thromboembolism is increased, it is still lower than tamoxifen [227, 239, 240]. Clinical utilization of raloxifene should be weight after evaluating individual thrombotic and cardiovascular risks, due to its adverse reactions as hot flashes, lower limb cramps and thrombotic events [227, 236].

Toremifene

Toremifene, a close derivative of tamoxifen, can also exhibit tissue-specific mixed agonist and antagonist effects on ER, with estrogen-like effects in bones, liver, and uterus, and anti-estrogen effects in breast tissue [241]. Therefore, it is approved for the treatment of metastatic breast cancer in postmenopausal women with estrogen receptor-positive or unknown status [242]. Preclinical experiments have shown that it can inhibit the growth of ER-positive breast cancer cells [243, 244]. Although toremifene and tamoxifen have similar side effects, such as sweating, nausea, vomiting, dizziness, vaginal discharge, and vaginal bleeding, toremifene has lower hepatotoxicity and higher safety, which may be related to its inability to induce DNA adduct formation in the liver [245, 246]. It also has better performance in terms of bone density and lipid metabolism [246].

In addition, although the efficacy, safety, and tolerability of toremifene have been proven, its therapeutic utilization is still relatively limited, which may be related to its failure to significantly surpass tamoxifen. Studies have shown that the efficacy of toremifene is about one-third of tamoxifen [247]. Therefore, compared with tamoxifen, toremifene may be more suitable for patients who require long-term treatment due to its lower toxicity, while providing additional benefits in improving bone density and reducing abnormal lipid metabolism.

Selective estrogen receptor degraders (SERDs)

Fulvestrant​

Although both SERD and SERM drugs are developed to target the ER, their molecular mechanisms are different. SERM only inhibits AF-2 while retaining some activity of AF-1, thus showing a “selective” characteristic of coexistence of antagonism and excitation in different tissues. In contrast, SERD blocks ER dimerization in three dimensions by virtue of the long side chains at the 7α and 11β sites [248]and promotes rapid ubiquitination and degradation of the receptor, completely inactivating both AF-1 and AF-2 [249]. As a result, the transcription of almost all ER target genes is shut down, and tumor cells lose their growth advantage relying on ER [250, 251]. Its development laid the foundation for the emergence of a new type of anti-estrogen drug and promoted further research on SERD-type “pure anti-estrogen” drugs. Currently, at least 6 SERDs are undergoing or have completed preclinical research.

Fulvestrant is the first approved SERD drug, developed through rational drug design and approved for medical use in 2002 [189, 190]. The main indication of fulvestrant is the treatment of hormone receptor-positive (HR+) metastatic breast cancer or locally advanced unresectable breast cancer [252–254]. A 2017 Cochrane review showed that the safety and efficacy of fulvestrant as a first-line or second-line endocrine therapy are comparable to existing treatment options [255]. In large-scale clinical trials, the incidence of venous thromboembolism (VTE) was only 0.9%, showing good safety [255]. It can be used as a monotherapy or in combination with abemaciclib or palbociclib for patients with ER+/HER2- advanced or metastatic breast cancer whose disease has progressed after endocrine therapy [256].

Currently, fulvestrant can only be administered by injection, which has inspired the research of oral SERD drugs (such as brilanestrant and elacestrant ) and promoted the exploration of a new drug class, selective androgen receptor degraders (SARDs) [249]. Studies have also shown that oral prodrugs of fulvestrant (such as ZB716) are under development, which may further optimize its ease of use [257, 258]. In the future, with the development of oral SERD drugs, the application potential of fulvestrant may be further expanded.

Elacestrant

Elacestrant is an oral SERD drug that was approved for medical use in 2023 [191]. Elacestrant is indicated for patients with ER + and/or PR-positive, HER2-negative metastatic breast cancer who are postmenopausal women or adult men and have disease progression after failure of at least one other endocrine therapy [259, 260]. Elacestran significantly prolonged progression-free survival (PFS) compared with standard of care (SOC). This benefit was reflected in both the overall population and patients with ESR1 mutations. In addition, as a second-line or third-line regimen, Elacestran is not only well tolerated but also can bring both statistically and clinically significant improvements in PFS, demonstrating its therapeutic potential in [261].

Elacestrant is an antagonist that specifically acts on ERα and can also induce ER degradation [262]. Elacestrant exhibits a weak partial agonist effect at low doses and an antagonist effect at high doses [263]. Compared with fulvestrant, orally administered elacestrant has a higher bioavailability and can more easily cross the blood-brain barrier and enter the central nervous system, with greater therapeutic potential for patients with brain metastases of breast cancer [259]. Studies have shown that this therapy is also suitable for patients whose tumors contain missense mutations in the ESR1 gene and are resistant to other endocrine therapies [264].

Other novel ERα-targeted drugs

Due to the property of PROTAC ER degraders to directly induce ER degradation, this type of drug can achieve a more thorough ER signaling pathway blocking effect. PROTAC ER degraders are a class of “bifunctional small molecules” that carry a ligand specifically binding to ER at one end and another ligand recruiting specific E3 ubiquitin ligases at the other end. The two ends are connected by a flexible chemical skeleton, linker [265, 266] (Table 2). When PROTAC enters the cell, it can simultaneously form a “POI-PROTAC-E3” ternary complex with the ER and the E3 enzyme. Under the spatial close action of the complex, the E3 enzyme covalently transfers the polyubiquitin chain to the ER. The ubiquitinated ER is then recognized and completely degraded by the 26 S proteasome. The same PROTAC molecule can continue to recruit new ER molecules after leaving the degraded ER, achieving substoichiometric, highly efficient, and sustainable protein level elimination, thereby providing a new pharmacological means to overcome ER overexpression or drug-resistant mutations [267, 268].

Table 2.

Proteolysis targeting chimeras (PROTACs) targeting ER

Compound	E3 ligase	E3 ligand	Reference​
ARV-471	CRBN	Lenalidomide	[269]
ERD-308	VHL	VHL032 derivative	[270]
ERD-148	VHL	VHL032 derivative	[271]
PROTAC-2	SCF	IκBα​​​	[272]
PROTAC-B	VHL	HIF-1 alpha	[273]
SNIPER(ER)-3	CIAP	Bestatin amide	[274]
SNIPER(ER)-87	XIAP	LCL161 derivative	[275]

ARV-471 is an oral PROTAC degrader, developed for ER+/HER2- locally advanced or metastatic breast cancer [265, 276], and is currently undergoing a Phase II clinical trial (NCT04072952) [269]. As a PROTAC drug, ARV-471 can simultaneously bind to ER and E3 ligase, recruit ER to the ubiquitin-proteasome system, and achieve efficient degradation. In ER + breast cancer cell lines, its ER degradation half-effective concentration (DC50) is only 1.8 nM. It downregulates estrogen-regulated genes, inhibits the proliferation of estrogen-dependent cells such as MCF-7 and T47D, and can effectively degrade the common ESR1 mutants Y537S and D538G [284]. In addition, when combined with CDK4/6 inhibitors, ER levels can be further reduced, and a significant tumor growth inhibition rate can be achieved, showing a significant synergistic effect [269]. Although the complete chemical structure of ARV-471 has not yet been disclosed, it is known to be a “binary” PROTAC, with an ER ligand end and a patented E3 ligase ligand end, connected in the middle by an adjustable linker. Importantly, the research team has systematically optimized the linker length, lipid solubility, and oral accessibility to achieve nearly complete ER degradation in vivo [277]. The first phase I dose-escalation trial (30–360 mg QD) showed that the ER degradation rate in 12 paired biopsy samples reached a maximum of 90%, and was not affected by whether the ER was mutated. Among the 34 evaluable patients, the clinical benefit rate was 41%, of which 3 of the 21 patients who had previously failed ≥ 5 lines of treatment had partial remission and 1 had significant stability (lesion reduction > 50%) [278]. The most common adverse reactions were nausea (24%), fatigue (12%), and vomiting (10%), most of which were grade 1, with only 2 cases of grade 3 events and no grade 4 or higher related toxicity [278]. Ongoing phase Ib and phase II studies are evaluating its extended efficacy in combination with palbociclib and as a single agent, respectively. A phase III randomized controlled trial has also been planned for direct comparison with fulvestrant or letrozole [278].

ERD-308 is an orally administrable small-molecule PROTAC degrader targeting ERα designed and synthesized by Hu et al. [270]. It is composed of an ER antagonist fragment and a VHL-1 E3 ligase ligand assembled through a linker, and is intended to treat ER + breast cancer [270]. As a PROTAC drug, ERD-308 binds to ERα at one end and recruits the VHL complex at the other end, so that the target protein is efficiently degraded by the proteasome after ubiquitination. Early structure-activity studies have shown that the ER ligand of ERD-308 is derived from a raloxifene derivative [279]. Its design is based on the ERα-raloxifene cocrystal structure, using the N, N-diethylamino site exposed to the solvent surface as a linker anchor, while optimizing the linker length and polarity to improve solubility and degradation efficiency, thereby obtaining the most active PROTAC currently available. Preclinical studies have shown that ERD-308 can degrade more than 80% of ER proteins in MCF-7 cells within 1 h and achieve complete degradation within 3 h; while fulvestrant requires 24 h to achieve 90% degradation, reflecting the advantage of rapid kinetics [270].

ERD-148 is another VHL-mediated ERα degrader discovered during the structure-activity relationship (SAR) study of the [270, 271]. One end of ERD-148 is derived from a raloxifene derivative selectively binding to ERα, while the other end recruits the E3 ligase VHL to mark the receptor as a ubiquitinated substrate and promote its rapid degradation by the proteasome. This mechanism also applies to the common drug-resistant mutants Y537S and D538G [271]. In vitro pharmacodynamics, ERD-148 inhibited proliferation of ER-dependent MCF-7 wild-type and Y537S and D538G mutants with IC50 as 0.8 nM, 10.5 nM, and 6.1 nM, respectively, and could significantly downregulate ERα protein and ER regulatory gene GREB1 mRNA levels at ≥ 10 nM, with targeting specificity [271]. Further studies have shown that 17β-estradiol can reverse the anti-proliferative effect of ERD-148, indicating that it still retains ER antagonist activity. At the same time, no obvious toxicity was observed in estrogen-independent cells such as MDA-MB-468 and MDA-MB-231 even when the concentration reached IC90 or above, indicating that the safety window is relatively wide [271]. Although no in vivo or clinical data have been reported, its efficient degradation at the cellular level and low off-target toxicity lay the foundation for subsequent animal models and clinical evaluations [271].

Small interfering RNAs (siRNAs) are short double-stranded RNA molecules that mediate RNA interference (RNAi), and therapeutically validated or clinically developed siRNA molecules have emerged as an important class of nucleic acid drugs. The FDA has approved two antisense oligonucleotides (ASO) [280], which can block the translation of corresponding proteins by degrading specific mRNAs. Currently, aptamer-siRNAs targeting HER2/HER3 and EGFR/HER2/HER3 have been prepared in vitro [281, 282]. In preclinical studies, HER2/HER3 aptamer-siRNA has been verified to significantly reduce target protein levels in vitro, proving the feasibility of the delivery strategy. The siRNA targeting ER has also been proposed as an auxiliary tool for SERD to increase the depth of receptor degradation. Currently, a delivery system similar to mRNA vaccines is being explored to solve the problem of in vivo drug administration [283, 284]. As expected, siRNA has a high degree of sequence specificity and can theoretically achieve “point-kill” inhibition, and has potential advantages due to its small-dose catalytic effect. However, there are also safety risks such as serum instability, immunogenicity, and off-target mRNA binding, which need to be overcome through chemical modification and targeted delivery [285, 286].

Conclusion

In-depth exploration of ERα and its associated signaling cascades has unveiled novel therapeutic avenues for breast cancer treatment. The AF-2 pocket, a critical structural domain governing ERα’s transcriptional activity, has emerged as a promising target for drug development. Preclinical studies have demonstrated that AF-2-targeting small-molecule inhibitors exhibit significant potential in suppressing hormone-resistant tumors, particularly those harboring acquired resistance to conventional endocrine therapies. A major challenge in ERα-targeted therapy is the emergence of endocrine resistance, which is closely related to ESR1 mutations. Structure-guided drug design has enabled the development of selective antagonists tailored to these resistant mutants, offering a precision medicine approach to overcome therapeutic failure. This strategy not only facilitates real-time treatment optimization through molecular biomarkers but also identifies novel targets for next-generation ERα inhibitors.

However, monotherapeutic interventions often fail to fully circumvent resistance mechanisms due to tumor heterogeneity and compensatory pathways. Consequently, combinatorial regimens, integrating ERα degraders (e.g., PROTACs), CDK4/6 inhibitors, or PI3K/mTOR pathway blockers, have gained traction as a means to enhance efficacy and delay relapse. Such multimodal strategies, grounded in mechanistic synergy and patient stratification, represent a paradigm shift in metastatic breast cancer management.

Abbreviations

ERα

Estrogen receptorα

AF

Activation function

SERMs

Selective estrogen receptor modulators

SERDs

Selective estrogen receptor downregulators/degraders

PROTACs

Proteolysis-targeting chimeras

ER

Estrogen receptor

PR

Progesterone receptor

HER2

Human epidermal growth factor receptor-2

DBD

DNA binding domain

LBD

Ligand binding domain

AF-1

Activation function-1

AF-2

Activation function-2

ERE

Estrogen response element

E2

Estradiol

NES

Nuclear export signals

NLS

Nuclear localization signal

HSP90

Heat shock protein 90

HATs

Histone acetyltransferases

eNOS

Endothelial nitric oxide synthase

MAPK

Mitogen-activated protein kinase

PI3K/Akt

Phosphatidylinositol 3-kinase/akt

mbER

Membrane estrogen receptor

PRMT1

Protein arginine methyltransferase 1

IGF-1

Insulin-like growth factor 1

IGF1R

IGF-1 receptor

EGF

Epidermal growth factor

GPER

G-protein coupled estrogen receptor

VTE

Venous thromboembolism

PFS

Progression-free survival

SOC

Standard of care

DC50

Half-effective concentration

SAR

Structure-activity relationship

RNAi

RNA interference

ASO

Antisense oligonucleotides

Author contributions

Liu J, Wu BX and Wu HT designedthis study; Wu BX, Wu HT, Lan YZ, Chen WJ, Liu JW and Yu XN searched the publications; Wu BX, Wu HT, Lan YZ, Chen WJ, Liu JW, Yu XN, and Liu J interpreted the results, constructed the structure of the review, and prepared the tables; Wu BX and Wu HT prepared the draft of the manuscript; Wu BX and Wu HT prepared the figures; Liu J revised the manuscript critically; All authors read and approved thefinal manuscript.

Funding

Supported by the National Natural Science Foundation of China, No. 82273457; the Natural Science Foundation of Guangdong Province, No. 2023A1515012762 and Science and Technology Special Project of Guangdong Province, No. 210715216902829.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.