English

• 1. Introduction

  The incidence of breast cancer is very high and slowly increasing in women, accounting for 32% of new cases in women according to the 2024 cancer statistics [1]. According to World Health Organization statistics, breast cancer has the second highest mortality rate in the world. The breast cancer mortality rate in China is also at a relatively high level. Thus, it is evident that the prevention and treatment of breast cancer are of great importance for China and the world.

  Breast cancer is a heterogeneous disease which is affected by different factors. The epidemiology of breast cancer has been widely and constantly investigated over the decades. The global incidence and mortality of breast cancer had been increased and fluctuated over the past few decades. Geographic and demographic disparities varied significantly in different areas [2]. Race and ethnicity should also be taken into account which influenced the epidemiology of breast cancer. In addition, high body mass index (BMI) is the most significant risk factor in Chinese women breast cancer occurrence [3]. Currently, breast cancer is categorized into four subtypes according to clinical features and molecular markers: Luminal A, B, human epidermal growth factor receptor-2 (HER2) and triple negative breast cancer (TNBC). There are many pathogenic signaling pathways in breast cancer that can be targeted as anti-breast cancer therapy [4-6]. From 2010 to 2020, the Food and Drug Administration (FDA) approved 30 breast cancer treatment options, some of which are mainly combined with other targeted drugs [7]. Some were approved by the FDA for clinical application, for example, standard endocrine therapies include aromatase inhibitor (AIs; letrozole, anatrozole, exemestane, etc.), selective estrogen receptor modulators (SERM; tamoxifen, etc.), selective estrogen receptor degraders (SERD; fulvestrant, elacestrant, etc.). In recent years, cell cycle-dependent kinase 4/6 (CDK4/6) inhibitors such as palbociclib, ribociclib, abemaciclib combining with endocrine therapy are served as a standard treatment approach for estrogen receptor (ER) positive breast cancer and significantly improved progression-free survival (PFS) and overall survival (OS) in such patients. Other promising molecular targets include epithelial growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF1R), bromodomain-containing protein 4 (BRD4) and phosphatidylinositol-3 kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR) signalling pathways [8-10].

  Among these therapeutic targets, ER is a major target and is overexpressed in about 70% of ER positive breast cancer patients. The contemporary view considered ER as a pivotal pathological factor in the development and progression of breast cancer [11,12]. ERα exerts its biological effects mainly through binding to estrogen, so ERα is considered an important target for breast cancer [13].

  Although endocrine therapy is prevalent for breast cancer for decades, the relapse and resistance are also increasing in breast cancer. Therefore, it is necessary to discover new targets and to develop combination therapy to overcome endocrine therapy resistance of breast cancer progression.

  2. Endocrine therapy and the development of drug resistance

  2.1 Endocrine therapy

  Aberrant signaling of ER is an important reason for the development of ER positive breast cancer. 17β-Estradiol (E2)-ER signaling axis is the main focus of endocrine therapy. In addition, proteolysis targeting chimeras (PROTACs), complete estrogen receptor antagonist (CERAN) and selective estrogen receptor covalent antagonist (SERCA) are emerged as promising therapeutic strategies for breast cancer therapy (Figs. 1 and 2) [14].

  Figure 1

  

  Figure 1.  Effects of endocrine therapy on ER signaling.

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  Figure 2

  

  Figure 2.  Endocrine therapy drugs approval timeline.

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  SERMs are defined as drugs that demonstrated ER agonism or antagonism in a tissue dependent manner [15]. SERMs inhibit breast cancer proliferation primarily by competitively binding ER with estrogen, and their most typical drug is tamoxifen. Tamoxifen was originally developed as a failed oral contraceptive drug, but Jordan et al. developed tamoxifen as a long-term adjuvant therapy and made tamoxifen as an effective endocrine drug for pre-menopausal breast cancer patients [16]. However, tamoxifen is a partial antagonist, with agonist activity present in some tissues. This tissue-selective partial agonist activity is related to tamoxifen that can induce different ER conformational change, which resulted in recruitment of diverse co-regulatory factors. Depending on the different co-regulatory activities, tamoxifen can behave as an antagonist or a partial agonist [17]. Successful research on tamoxifen has given rise to various SERMs such as toremifene, which can be used in postmenopausal patients. Raloxifene can be used for osteoporosis in postmenopausal women to reduce the risk of breast cancer development. Lasofoxifene is another SERM compound with regard to its action with ER but with higher bioavailability compared to tamoxifen. Initially, lasofoxifene was used for the treatment of osteoporosis but with subsequent studies, it was found to decrease the breast cancer incidence without increasing the risk of uterine cancer. The ELAINE-2 (NCT04432454) phase 2 trial [18] investigated the combination of lasofoxifene and abemaciclib and the result showed that lasofoxifene and abemaciclib have significant antitumor activity with acceptable safety and tolerability estrogen receptor 1 (ESR1)-mutated, ER positive metastatic breast cancer. Therefore ELAINE-3 (NCT05696626) phase 3 trial was initiated to evaluate lasofoxifene and abemaciclib versus fulvestrant and abemaciclib.

  Estrogens (primarily E2 and estriol (E3)) are generated by the aromatase P450 enzyme through the conversion of androgens to estrogens [19]. Aromatase inhibitors (AIs) have been discovered to inhibit E2 production. The most commonly used drugs are letrozole or anastrozole as nonsteroidal agents, and exemestane as the steroidal drug. AIs have been approved as standard of care therapy for postmenopausal women and they usually showed better outcome of PFS and OS compared to tamoxifen in such patients [20]. Current combination therapy strategies, particularly those involving CDK4/6 inhibitors, mTOR inhibitors, displayed improved survival rate when compared to AIs alone [21].

  Since SERMs and AIs are resistant after prolonged use, SERDs have emerged to overcome drug resistance. Fulvestrant, a representative SERD, is the marketed drug for ER positive breast cancer. It is ER antagonistic to all tissues and an antagonist that induces proteasome-dependent ER degradation [22]. Fulvestrant antagonized ER transcriptional activity through slowing down the intranuclear mobility of ER, increasing turnover via the ubiquitin-proteasome pathway in ER signaling [23]. Because of the low oral utilization of fulvestrant, many oral SERDs were developed. Recently, elacestrant (RAD1901), an oral SERD which is discovered by Radius Health company, is approved for ER positive breast cancer (NCT02338349). Elacestrant promotes ER turnover, antagonizes ER, and disrupts downstream signaling [24]. In 2023, elacestrant was approved by FDA for ER positive, ESR1-mutated advanced breast cancer in the U.S. [25]. The approval of elacestrant has encouraged a number of ongoing clinical oral SERDs, such as camizestrant (AZD9833) which is now in a phase Ⅲ clinical trial to study camizestrant and a CDK4/6 inhibitor (NCT04711252, NCT04964934) for breast cancer. Giredestrant (GDC-9545) is currently in a phase Ⅲ clinical investigation designed to evaluate giredestrant in combination with palbociclib versus letrozole in combination with palbociclib (NCT04546009). Immunestrant (LY3484356) is in phase Ⅲ clinical trial to study immunestrant with standard hormone therapy (NCT05514054). However, some oral SERDs had been discontinued in clinical trials, other oral SERDs are potential treatment options for ER positive breast cancer. Elacestrant, the first oral SERD on the market, is a very important milestone.

  As biotechnology continues to evolve and update, PROTAC has gained the interest of many researchers. PROTAC is a bifunctional molecule that recruits specific proteins of interest to E3 ubiquitin ligases, resulting in their ubiquitination and degradation [26]. Currently, there are several oral PROTACs in clinical trials, such as ARV-471, an ER PROTAC, which can promote the interaction between ERα and E3 ligase, leading to ubiquitylation of ER followed by degradation through the proteasome [27]. ARV-471 is the most advanced PROTAC compound and it is in phase Ⅲ clinical trial (NCT05654623). The other is AC0682, discovered by Accutar Biotechnology, Inc., can induce ER degradation in many ER positive breast cancer cell lines [28]. Unfortunately, a clinical phase Ⅰ study (NCT05489679) of AC0682 for ER positive breast cancer was terminated. Although AC0680 program has been discontinued, the Accutar Biotechnology, Inc. has entered clinical phase Ⅰ with another compound, AC699 (NCT05654532), an orally potent and selective ER degrader with different mechanism from fulvestrant. The phase Ⅰ study of AC699 is currently ongoing and the preliminary results show that AC699 has good efficacy as well as safety, which is expected to lead to further phase Ⅱ studies. Utilization of PROTAC as a therapeutic approach is a promising strategy, representing a novel anti-cancer option [29].

  As with PROTAC development, SERCA and CERAN were developed and utilized in order to improve the effectiveness of ER suppression/degradation. SERCA antagonizes ER, inactivating wild-type and mutant ER by covalent binding of a cysteine (C530). The ongoing phase Ⅰ clinical evaluations include H3B-6545 as single agent (NCT04568902) and with palbociclib as combination therapy (NCT04288089) for both ER positive breast cancer. CERANs can inhibit ER transcriptional activity, resulting in pure antagonism [30]. OP-1250 is a representative CERAN compound which is in phase Ⅰ/Ⅱ clinical studies (NCT04505826).

  In summary, oral SERDs and novel ER antagonists are a promising and a major source of drug discovery for ER positive breast cancer. While these agents have a positive effect on ESR1 gene mutations, there are still endocrine resistance mechanisms that are not related to ligand-binding domain mutations. Thus, studying the mechanisms of endocrine resistance and combining them with other targeted drugs to overcome endocrine resistance are an important direction for the development of novel ER ligands.

  2.2 Endocrine resistance

  Although many breast cancer patients had benefited from endocrine therapy, the main difficulty is overcoming resistance to endocrine therapy [31]. Here are some of the potential reasons which may contribute to endocrine resistance.

  2.2.1   Loss and mutation of ER

  The growth and differentiation functions of ER positive breast cancers are dependent on E2 pathway. Thus, ER is critical for endocrine therapy and reducing its expression is hypothesized as a major cause of de novo resistance and acquired resistance [32]. Epigenetic modulators are thought to be a key driven force for endocrine resistance such as DNA methylation, chromatin remodeling, miRNA regulation [33]. Histone deacetylase (HDAC), an epigenetic regulator, is thought to inhibit ER expression thereby causing endocrine therapy resistance [34]. Mutations are usually considered as the preferred reason to cause drug resistance. ESR1 (ER encoding gene) mutations were first reported two decades ago [35]. However, the ligand binding domain (LBD) mutations of ER were recognized as a common reason of the resistance in ER positive breast cancer. These mutations are frequently observed in about 20% of patients after AIs treatment [36]. Y537 and D538 are the most common mutations which allowed activation without binding of E2 and resulted in a consecutive active conformation of ER [33].

  2.2.2   Post-translational modifications of ER

  The post-translational modifications are important regulators of ER, which included phosphorylation, methylation, acetylation, total methylation, ubiquitination and palmitoylation [31]. In conclusion, the ER is affected by several post-translational modifications that can alter ER signaling and downstream transcriptional activity thereby allowing breast cancer cells to acquire drug resistance, and whether some specific sites of modification can be used as targets for subsequent therapy remains to be investigated [31].

  2.2.3   Receptor tyrosine kinase (RTK) mutations

  HER2 amplification reduces sensitivity to anti-estrogen therapy primarily [37] by activating alternative survival pathways such as PI3K-AKT and mitogen-activated protein kinase (MAPK), and HER2 activating mutations are observed in resistant metastatic breast cancers [10]. EGFR mutations have been found in 1.7% of resistant breast cancers (including both de novo and acquired resistance) [10]. Amplification of the fibroblast growth factor receptor 1 (FGFR1) gene was found to be associated with intrinsic endocrine resistance in ER positive metastatic breast cancers [9]. In addition to FGFR1 driven signaling, nuclear FGFR1 can interact with ER to promote cell proliferation in the absence of E2, thereby enhancing drug resistance (Fig. 3) [38].

  Figure 3

  

  Figure 3.  Activation of HER2, EGFR, FGFR and other RTKs promotes endocrine resistance.

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  2.2.4   Mutations in PI3K pathway and MAPK pathway

  PI3K signaling pathway mutations are frequently observed in ER positive breast cancer. In preclinical models, abnormal activation of the pathway initiated endocrine resistance [39]. In addition, the MAPK signaling pathway (including nuclear factor 1 (NF1), Rat sarcoma viral oncogene homolog (RAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and mitogen-activated protein kinase 1 (MAP2K1) are often mutated in metastatic breast cancer, especially in NF1. The abnormal behaviors of NF1 were associated with de novo and acquired drug resistance [40]. CtDNA analysis of AIs resistant breast cancer showed RAS mutations are in above 15% patients [41]. Therefore, intervention of MAPK pathway may overcome endocrine resistance [42].

  2.3 CDK4/6 inhibitor treatment resistance

  The application of CDK4/6 inhibitors had changed the treatment landscape for ER positive breast cancer and became one of the new treatments for breast tumors in the last 20 years. The pivotal study of CDK4/6 inhibitors is a combination strategy with endocrine therapy for breast cancer. Currently, the combination therapy serves as the frontline therapy for ER positive breast cancer. However, it will inevitably develop resistance of CDK4/6 inhibitors and the next section focuses on the resistance mechanisms generated by CDK4/6 inhibitors [43].

  2.3.1   Deletion and mutation of retinoblastoma protein (Rb)

  Deletion of Rb1 is one of the potential resistance mechanism of CDK4/6 inhibitors since Rb1 is the major phosphorylated target of the CDK4/6 complex and cyclin D [44].

  2.3.2   Ectopic overexpression of cyclin E

  In addition to Rb1 deletion, ectopic overexpressions of cyclin E1 and E2 can lead to other pathways through activation of CDK2 and promote resistance to in vitro endocrine therapy and palbociclib monotherapy.

  2.3.3   Activation of CDK2

  CDK2 activation is currently considered as a main reason of CDK4/6 inhibitors resistance. The main mechanism of drug resistance is that E2F activates proteins such as CDK2 to produce the cyclin E and CDK2 complex and then phosphorylates Rb, forming positive feedback and promoting protein and DNA synthesis. This activation causes the cells to develop CDK4/6 resistance.

  2.3.4   CDK6 overexpression

  Overexpression of CDK6 may lead to resistance to CDK4/6 inhibitors. The overexpression of CDK6 can lead to decreased ER expression and subsequent anti-estrogen drugs resistance, which may result in resistance of combinational therapy.

  2.3.5   Altered upstream oncogenic signaling

  Exome-wide analysis of CDK4/6 inhibitor-resistant tumor specimens revealed a wide range of potential mechanisms of resistance, including upstream abnormity in ERBB2, AKT1, RAS and FGFR2.

  2.3.6   FAT atypical cadherin 1 (FAT1) inactivating mutations

  Rare inactivating mutations in the FAT1 gene had been found to induce drug resistance, leading to elevated CDK6 expression that develops CDK4/6 inhibitor resistance.

  The endocrine therapy and other target drug resistance had a significant negative impact on breast cancer treatment and impaired the therapeutic efficacy. To prolong the duration of effective treatment and to prevent the drug resistance and metastasis, the combination targets should be explored to enhance the efficacy of endocrine therapy or different target drugs [45-47].

  The resistance mechanism which most likely occurred is CDK4/6 resistance. The CDK4/6 resistance will result in activation of CDK2, followed by phosphorylation of Rb and activation of proliferative signaling pathways. Therefore, CDK2 and CDK2/4/6 inhibitors are required to overcome CDK4/6 resistance. In addition, the ER+/HER2− breast cancer is mostly likely a cold tumor with less expression of programmed death-ligand 1 (PD-L1). However, the PD-L1 expression is associated to ESR1 mutations which may lead to endocrine resistance.

  Targeting ER signaling and immune checkpoints may overcome these resistances. Furthermore, epigenetic modifications may also lead to endocrine resistance. Recently, the histone acetyltransferases 6 (KAT6) inhibitors are in phase Ⅰ/Ⅱ clinical trials for advanced/metastatic breast cancer [48,49].

  3. Combination therapy associated with ER

  3.1 Targeting ER and CDK4/6

  The cell cycle is driven through CDKs, such as CDK4 and CDK6, and their dysregulation is closely associated with tumorigenesis and progression [50]. In particular, cyclin D and CDK4/6 complex activity is essential in estrogen-induced tumor cell proliferation. Cyclin D1 can bind directly to ERα, promoting target gene transcription. The direct binding of cyclin D1 with ER can increase binding of the complex to estrogen response element sequences, therefore increasing ER-mediated transcription [51]. In addition, the cyclin D1 mediated activation of ER was observed, demonstrating the abnormal cell cycle signaling in ER positive breast cancer. As a result, the overexpression of cyclin D1 may be a potential resistance mechanism of ER positive breast cancer (Fig. 4) [51,52]. In recent years, anti-cancer therapy targeting the cell cycle has been shown to be a reasonable option in combination with endocrine therapy. Palbociclib, ribociclib and abemaciclib as the three main CDK4/6 inhibitors were approved and had been successfully used with endocrine therapeutic agents for ER positive advanced breast cancer.

  Figure 4

  

  Figure 4.  Hormonal regulation of cyclin D1-CDK4/6 axis.

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  In 2005, Fry et al. discovered palbociclib (1) through a series of structural modifications with high CDK4/6 selectivity. The structure and activity relationship (SAR) analysis revealed that the addition of C6 substituents can increase the activity of compounds, with the activity sequence being COCH₃ > COOC₂H₅ > Br > H. And when there is an acetyl group on C6, adding C5 methyl will lead to an increase in potency (2 vs. 1). When the C6 acetyl group is directed by adjacent methyl groups outside the plane defined by the pyridine[2,3-d]pyrimidin-7-one ring, it will form additional interactions with the protein. 2-Aminopyridine is an essential group. Although the substitution of 2-aminopyridine with aniline increased the potency of the compound towards CDK4/D (5 vs. 1), the selectivity for CDK4/D vs. CDK2/A was decreased. SAR analysis of piperazine substituents showed that lipophilic groups can increase the activity of compounds (Fig. S1 and Table S1 in Supporting information). Compounds 7, 8, 9, and 1 seem to be comparable in terms of their efficacy and selectivity towards CDK4/D, as well as their activity in cells. However, after further pharmacokinetic characterization, compound 1 has better solubility and up to 56% oral bioavailability, so it was identified as a candidate for cancer treatment. Further clinical studies led to approval of palbociclib [53].

  Palbociclib is a potent and selective orally available CDK4/6 inhibitor which showed preferential activity for preclinical models of luminal breast cancer. Therefore, an attempt to combine palbociclib with endocrine therapy was initiated for this breast cancer subtype.

  The TREnd randomized phase Ⅱ clinical trial (NCT02549430) showed that combination of endocrine therapy with palbociclib significantly improved PFS and had a lower incidence of side effects with the combination than with palbociclib alone [54]. The occurrence of adverse effects with palbociclib was less than that with palbociclib alone. The PALOMA-1 phase Ⅱ trial (NCT00721409) using palbociclib and an AI inhibitor letrozole improved PFS in contrast to letrozole alone in advanced ER positive breast cancer [55]. Based on these results, in February 2015 FDA approved letrozole with palbociclib for ER positive advanced breast cancer in postmenopausal patients. Another phase Ⅱ clinical trial, PALOMA-2 (NCT01740427), validated the findings of PALOMA-1, had a significantly higher median PFS compared to those in the placebo combined with letrozole group [56]. The phase Ⅲ trial PALOMA-3 (NCT01942135) compared the efficacy of fulvestrant in combination with palbociclib versus placebo, and had a higher median PFS compared to those in the placebo combined with fulvestrant group, identifing palbociclib combined with fulvestrant as an alternative approach for the patients with ER positive breast cancer [57,58].

  Although the combination of endocrine agents with CDK4/6 inhibitors has therefore become a first/second-line treatment option for ER positive breast cancer in recent years, the prolonged use is associated with drug resistance and several clinical trials are exploring alternative treatment sequence, different combination strategies to overcome resistance, thus expanding the scope of CDK4/6 inhibitors in the management of breast cancer.

  3.2 Targeting the ER and PI3K/AKT/mTOR pathways

  The PI3K/AKT/mTOR pathway is regarded as an essential regulator in cell growth, survival and proliferation. It was also associated with endocrine resistance for ER positive breast cancer. Genetic mutations are observed in ER positive breast cancer cells, particularly in PIK3CA and AKT1 genes. These mutations can result in overactivation of the PI3K/AKT/mTOR signaling pathway, allowing cell survival and proliferation despite estrogen deprivation [59]. The ER and PI3K/AKT/mTOR pathways can crosstalk through direct or indirect interactions (Fig. 5) [60]: Firstly, activation of estrogen non-dependent ER transcriptional activity can promote cell proliferation, followed by activating the estrogen pathway to trigger the synthesis of many components of the PI3K/AKT/mTOR pathway [61]. Therefore, combined inhibition of PI3K/AKT/mTOR and ER pathway is a promising strategy to treat endocrine resistant breast cancer.

  Figure 5

  

  Figure 5.  Targeting PI3K/AKT/mTOR pathway.

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  3.2.1   Targeting ER and PI3K

  Buparlisib is an orally available class Ⅰ pan PI3K inhibitor against both wild type and mutant PI3K [62]. The BELLE-2 phase Ⅲ trial (NCT01610284) using buparlisib and fulvestreant treatment showed that compared to fulvestreant treatment alone, the combination of buparlisib and fulvestreant was effective in improving PFS, and thus the combination of PI3K inhibitors with endocrine therapy is effective in endocrine resistant breast cancer. However, for pan PI3K inhibitors, the toxic side effects are relatively high, thus in this case, it is necessary to discover a selective PI3K inhibitor, for example an α-specific PI3K inhibitor, to enhance safety and efficacy [63]. In 2013, Caravatti et al. discovered alpelisib (11) as an α-specific PI3K inhibitor from the 2-aminothiazole class. SAR showed that the proline portion is an active essential group and that substitution by azetidine leads to a slight decrease in the inhibitory effect on PI3Kα. The authors attribute this to the fact that after the reduction of the side chain size to 4 atoms, the compound loses a favorable van der Waals contact with the side chain imidazole ring of PI3Kα residue H855. Further methylation of the proline portion, conformational reversal or even removal can lead to loss of selectivity or significant decrease in activity. Subsequently, to increase potency, one of the methyl groups of the tert‑butyl group is changed to trifluoromethyl group, and the activity of the series of compounds is further increased (Fig. S2 and Table S2 in Supporting information). Compared to the compounds with no substitution (12) and chlorine substitution (13) at the R1 site, 11 showed good pharmacological data and was finally selected as a clinical candidate in the following pharmacokinetic experiments [64]. In a randomized SOLAR-1 phase Ⅲ clinical trial (NCT02437318), the safety was significantly improved in alpelisib and fulvestrant combination therapy in the PIK3CA-mutated ER positive breast cancer [65]. However, the drug-drug interactions and the bioavailability became unpredictable when these two drugs are combined with the CDK4/6 inhibitor ribociclib, thus the three-drug combination may not be suitable for follow-up studies [66]. There are also clinical phase Ⅰb studies that have combined alpelisib in combination with letrozole and found that this combination was safe and had reversible toxicity [67].

  In conclusion, although there are various types of PI3K inhibitors, selective PI3K inhibitors are preferred since they have less toxicity compared to those pan PI3K inhibitors. At present, the development of PI3K inhibitors still needs to be studied, and the toxic side effects of combination therapy are relatively greater than those of other targets, so how to reduce toxic side effects and develop more selective PI3K inhibitors is an important direction for the future.

  3.2.2   Targeting ER and AKT

  MK-2206, a variant pan-AKT inhibitor, was found in preclinical studies to significantly induce apoptosis and intensely enhance the onset of apoptosis in breast cancer cells in combination with fulvestrant, followed by a clinical phase Ⅰ study that showed that combination therapy of MK-2206 with anastrozole, a third-generation aromatase inhibitor, was well tolerated and the major grade 2 and higher side effects were rash and hyperglycemia, but that the treatment did not exceed the expectations of standard endocrine therapy [68]. Subsequently, after determining the optimal dose through phase Ⅰ, a phase Ⅱ clinical study was conducted (NCT01344031, NCT01776008) [69]. Unfortunately, this phase Ⅱ clinical study did not yield good results and MK-2206 may not increase the efficacy in PIK3CA-mutated ER positive breast cancer compared to anastrozole alone and therefore was not further studied in such patient population. Therefore, subsequent studies may need to continue with further modifications on AKT inhibitors.

  3.2.3   Targeting ER and mTOR

  mTOR signaling is important in tumor genesis and therefore targeting mTOR is a potential method to treat breast cancer. RAD001 (Everolimus) can target mTOR and has strong antitumor activity. Thus, in 2018, Royce et al. conducted a BOLERO-4 phase Ⅱ study of everolimus (NCT01698918) and endocrine therapy for ER positive breast cancer. The researchers combined everolimus and letrozole as a first-line treatment and found that the combination significantly improved FBS compared to letrozole alone, although median overall survival was not achieved [70]. A year later, Tesch et al. combined everolimus with the steroidal aromatase inhibitor exemestane in the clinical IIIB 4EVER trial (NCT01626222), and the combination resulted in significantly improvement in PFS in contrast to exemestane alone and was also effective in more advanced and severe pre-treatment patients [71]. Thus, the combination of everolimus with endocrine therapy became an alternative option for ER positive breast cancer.

  Despite the good therapeutic efficacy of everolimus, there is a negative feedback loop downstream of mTORC1 that can reactivate tumors and lead to enhanced IGF1R-dependent AKT activity, which can develop into drug resistance in the long run. Thus, in recent years, dual mTORC1/2 inhibitors have been developed that target the mTORC1-associated feedback loop. AZD2014 is another mTOR inhibitor that targets both the mTORC1 and mTORC2. In 2015, Guichard et al. studied the combination of AZD2014 with fulvestrant in ER positive breast cancer and the result demonstrated that it induced tumor growth inhibition in several xenograft models [72]. In 2019, Pancholi et al. investigated the effects of the dual mTORC1/2 inhibitor vistusertib and fulvestrant combination in ER positive endocrine resistant breast cancer by examining proliferative activity, cell signaling, cell cycle, and effects on ER-mediated transcription, as well as the patient-derived xenograft (PDX) model. The results showed that the combination of vistusertib and fulvestrant showed synergistic effects and delayed tumor progression after cessation of treatment in two ER positive endocrine resistant PDX models [73]. Sapanisertib is an oral and highly selective mTOR inhibitor with dual targeting mTORC1 and mTORC2. In a phase Ⅱ clinical trial, combining sapanisertib and fulvestrant in 141 enrolled patients, PFS was 3.5 months with fulvestrant alone and 7.2 months (once daily) and 5.6 months (once a week) with the combination, which significantly enhanced PFS, but with a corresponding toxicity was also increased and thus not suitable for further development [74].

  In 2022, Ruhainee and collaborators derived andrographolide from Andrographis paniculata, a naturally occurring plant, and evaluated its potential anti-neoplastic properties against breast cancer cells. They employed a variety of methods including the MTT assay, real-time quantitative reverse transcription PCR (qRT-PCR), and Western blot (WB) experiment to evaluate the impact of andrographolide. The study revealed that andrographolide is capable of interacting with both ER and the mTOR pathway, leading to an impaired proliferation of MCF-7 and MDA-MB-231 cells. Moreover, andrographolide was observed to trigger cell apoptosis by suppressing B-cell CLL/lymphoma 2 (Bcl-2) and upregulating Bax within these cell lines. In MCF-7, the compound can inhibit cell proliferation via ERα, PI3K, and mTOR downregulations (Fig. 6). Interestingly, the therapeutic efficacy of andrographolide in MCF-7 cells was found to be comparable with the performance of fulvestrant [75]. Consequently, the identification and development of novel dual-action molecules that can target both ER and the PI3K-mTOR-AKT signaling cascade allow further investigation for their potential use for the treatment of breast cancer.

  Figure 6

  

  Figure 6.  Mechanism of action of andrographolide on MCF-7 cells.

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  The above studies revealed that targeting ER and PI3K/AKT/mTOR pathway can improve the therapeutic outcome of breast cancer patients and overcome endocrine resistance. Thereafter, the inhibitors that targeting these two signaling pathways had entered clinical studies.

  3.3 Targeting the ER and HDAC

  HDACs regulate a variety of cellular events including transcription, cell cycle progression and differentiation by removing the acetyl groups from histones and non-histones. The HDAC family has now been found to regulate estrogen and progesterone mediated signaling. Acetylation has been found to be a key mediator that regulates ER transcription and turnover (Fig. 7) [76]. HDACs are key enzyme family which controls the acetylation state of protein lysine residues. HDAC can regulate the ER signaling by recruiting HDAC1 and metastasis-associated protein 1 (MTA1) to activate DNA methyltransferase DNMT3B [77]. HDAC3 stabilizes ERα mRNA to maintain its expression, while HDAC6 interacts with ERα AF2 domain via its deacetylase domain, regulating ERα membrane localization and signaling [78,79]. Therefore, the HDAC inhibitors are an alternative option for ER positive breast cancer and combining endocrine therapy with HDAC inhibitors may show promise for overcoming endocrine resistant breast cancer.

  Figure 7

  

  Figure 7.  Cross-talk of HDAC inhibitors and ER ligands. (ⅰ) Effect of HDAC inhibitors on ERα expression and acetylation. (ⅱ) Effect of HDAC inhibitors on ERα transcriptional activity. (ⅲ) Effect of HDAC inhibitors on p21^WAFI/CIP1 expression.

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  In 2015, Munster et al. investigated the effect of HDAC inhibitor and tamoxifen combination therapy on tamoxifen-resistant breast cancer. First, the team obtained tamoxifen-resistant cells from MCF-7 and T47D by sequential administration of 4-OH tamoxifen, named TAMR^M and TAMR^T respectively. They found that both cell lines showed accelerated growth and reduced sensitivity to endocrine deprivation. Then, the team used the combination of the HDAC inhibitor abexinostat (PCI24781) with tamoxifen to treat TAMR^M cell, and the result showed that the combination of abexinostat and tamoxifen resulted in approximately 54% of TAMR^M cell death and cell proliferation inhibition, reversing the upregulated expression of Bcl-2, c-Myc, and p21. Thus, this study suggested that combining HDAC inhibitors with endocrine therapy is effective in tamoxifen resistant ER positive breast cancer [80].

  In 2015, Zhou et al. developed a series of oxabicycloheptene sulfonate-histone deacetylase inhibitor (OBHS-HDACi) compounds 17–26 targeting ER and HDAC, which have good binding affinity and antagonist activity for ERα (Fig. S3 in Supporting information). Most of the compounds showed greater antitumor potency and selectivity against MCF-7 cell compared to tamoxifen, furthermore, all compounds were nontoxic to healthy VERO cells (half maximal inhibitory concentration (IC₅₀) > 100 µmol/L), whereas vorinostat (SAHA) and tamoxifen were somewhat toxic to VERO cell (IC₅₀: 4.1 ± 0.19 and 15.1 ± 5.21 µmol/L), with compound 18 had the highest binding affinity for ERα and ERα/ERβ selectivity up to 28 and also exhibited partial ERα antagonist activity in transcriptional analyses as well as antitumor activity comparable to that of 4-OHT in MCF-7 cells (IC₅₀: 19.1 ± 4.06 and 15.6 ± 1.77 µmol/L) and strong inhibition of HDAC1 and HDAC6 (IC₅₀: 0.107 and 8.34 µmol/L). These results may be attributed to the scaffold of OBHS reinforcing the steric hinderance with helix 11. Furthermore, compound 21 was found as the most potent HDAC1 inhibitor (IC₅₀: 22 nmol/L), which was 2-fold stronger than the approved drug SAHA (IC₅₀: 47.4 nmol/L) [81].

  The SERDs/HDAC inhibitors, derived from tetrahydroisoquinoline (THIQ)-hydroxamate hybrids, were designated and synthesized by Xiang and colleagues (Fig. S4 in Supporting information). These THIQ-hydroxamate hybrids demonstrated notably potent inhibition of HDAC6 and suppressed the proliferation of MCF-7 cells, with compound 27 as the most potent one. Compound 27 proved to be the most effective HDAC6 inhibitor, preferentially targeting HDAC6 (IC₅₀: 62.8 nmol/L), compared to an IC₅₀ of 582.4 nmol/L in HDAC1, and an IC₅₀ exceeding 1000 nmol/L in HDAC4. Moreover, compound 27 was identified as the most powerful agent for promoting the degradation of ERα (IC₅₀: 90.8 ± 6.4 nmol/L), and it displayed the most excellent antiproliferative activity with a half maximal growth inhibition concentration (GI₅₀) value of 0.17 µmol/L, outperforming both SAHA (GI₅₀: 1.25 µmol/L) and fulvestrant in MCF-7 cell lines. Therefore, 27 was selected for the next pharmacological tests. By WB and qRT-PCR, 27 was found to be able to concentration-dependently degrade ERα and antagonize E2-induced expression of growth regulating estrogen receptor binding 1 (GREB1) and progesterone receptor (PGR) in MCF-7 cells, and then flow analysis revealed that 27 could induce apoptosis more efficiently than fulvestrant, and interestingly, 27 was also able to inhibit the proliferation in ER negative breast cancer cells.

  Furthermore, compound 27 showed better antitumor effects in MCF-7 xenograft models than drug combinations (fulvestrant and SAHA) without significant toxicity by promoting ERα degradation and increasing α-tubulin acetylation [82].

  Due to the better effect of 27 on ERα degradation and HDAC inhibition, in 2023, Xiang et al. optimized and synthesized a series of THIQ-hydroxamic acid bifunctional conjugates based on compound 27. Among them, 30 (Fig. S4) showed the highest antiproliferative activity on MCF-7 cells (IC₅₀: 1.96 µmol/L) and the highest inhibition of HDAC6 with an enzymatic IC₅₀ of 25.96 nmol/L, which was slightly higher than that of the 27 (IC₅₀: HDAC6, 63.03 nmol/L; MCF-7, 4.38 µmol/L). Similar to 27, 30 was able to concentration-dependently degrade ERα and inhibit HDAC6, and was able to reduce mRNA levels of GREB1 and PGR and trigger apoptosis in MCF-7 and MDA-MB-231 cells. Furthermore, 30 was not agonistic to endometrial cells and was able to display ER-independent effects through inhibition of HDAC6 signaling pathway. This study thus demonstrates a dual functional compound targeting ER degradation and HDAC6 inhibition in ER positive breast cancer [83].

  Studies of HDAC inhibitors with endocrine therapy had also entered clinical studies. Vorinostat, an HDAC inhibitor, was investigated in a phase Ⅱ clinical study by Munster et al. in 2011 for the combination with tamoxifen for the treatment of hormone-resistant breast cancer. The safety of the combination was evaluated and it showed that the combination of tamoxifen and vorinostat was well tolerated by patients [84]. Because of the promising in vitro antitumor effect of the subtype-selective HDAC inhibitor chidamide (tucidinostat) and exemestane, the combination was employed in 2019 in clinical ACE phase Ⅲ trial (NCT02482753). The results showed that exemestane and tucidinostat significantly improved PFS compared to exemestane alone in metastatic ER positive breast cancer after initial endocrine therapy. Grade 3–4 hematologic adverse reactions appeared in the two-drug co-administration group, even though these adverse reactions can develop, the combination may still be a novel treatment approach for the patients [85]. In 2022, Xu et al. performed a meta-analysis to investigate the safety and efficacy of exemestane with HDAC inhibitors in contrast to exemestane alone in advanced settings. The result showed that the combination group exhibited significantly prolonged PFS and most of the toxicities were asymptomatic and manageable. Thus, the use of endocrine therapy in combination with HDAC has clear efficacy in advanced ER positive breast cancer after first line therapy [86].

  3.4 Targeting the ER and bromodomain and extraterminal domain (BET)

  Breast cancer is generally caused by aberrant patterns of gene expression and epigenetic alterations. Bromodomain and BET protein is an epigenetic machinery that contributes to read the histone acetylation and can regulate the gene expression involved in carcinogenesis. The amplification or overexpression of BET had been found in breast cancers, thus targeting BET has important clinical implications.

  In breast cancer tissues, the expressions of BRD2, BRD3, and BRD4 are frequently observed, whereas BRDT is less detected. It has been noted that BRD2 and BRD4 are either amplified or overexpressed in about 12% and 17% of cases when examining various subtypes of breast cancer. Furthermore, BRD4 has been recognized as a pivotal gene in the progression of estrogen-driven breast cancer. It showed that tamoxifen resistance is attributed to BET protein BRD3/4 through recruiting the histone H3K36 methyltransferase wolf-hirschhorn syndrome candidate 1 (WHSC1) to the ESR1 gene which positively regulating its expression. WHSC1 and ERα are overexpressed in breast cancer and can form a positive feedback loop. The N-terminal region of BRD3/4 can interact with WHSC1 and plays a crucial role in tamoxifen resistant settings. BRD3/4 can recruits WHSC1 and read the acetylated lysines in the histone tail of the ESR1 promoter region, increasing ER signaling pathway transcription through H3K36 methylation (Fig. 8) [87]. BET Protein can also regulate the signaling of ERα: BRD4 recruits CDK9/p-TEFb to release paused RNA Polymerase Ⅱ (Pol Ⅱ) at promoter regions, enabling transcriptional elongation of ERα target genes (CCND1, TFF1) [88]. In ESR1-mutant (e.g., Y537S) breast cancers, BET inhibitors disrupt BRD4 localization at super-enhancers, suppressing ERα-driven transcriptional programs [89].

  Figure 8

  

  Figure 8.  Role of BRD protein in breast cancer.

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  JQ1, a permeable small molecule, functions as an inhibitor of BRD4 by competitively binding to the acetyl lysine recognition sites of bromodomains. Its competitive binding mechanism displaces BRD4-related oncoproteins from chromatin, eliciting antiproliferative responses in BRD4-dependent cell lines and PDX models. Further studies confirmed that, JQ1, a BET protein inhibitor, can significantly inhibit the breast cancer cell growth including TamR breast cancer cells. Additionally, combined in vivo experimental studies of JQ1 and fulvestrant demonstrated that their synergistic use could more potently reduce ERα levels and impede tumor growth. These findings indicate that epigenetic proteins BRD3/4 and WHSC1 excert essential functions in the regulation of ER signaling, presenting them as promising targets for the treatment of tamoxifen resistant breast cancer [90].

  GS5829 is another oral BET inhibitor. The efficacy of GS5829 with fulvestrant versus fulvestrant alone was investigated in a randomized phase Ⅰb/Ⅱ clinical trial (NCT02983604). Unfortunately, this study was terminated early for toxicity reasons and the phase Ⅱ portion of the study was not conducted. GSK525762 is a pan BET inhibitor which blocks the binding of BET family proteins to acetylated histones. A phase Ⅰ study of fulvestrant with GSK525762 in participants with ER positive advanced breast cancer (NCT02964507) was initiated thereafter.

  Although current clinical studies of BET protein inhibitors in ER positive breast cancer have not been successful primarily due to their toxicity management. Thrombocytopenia was reported as the main dose-limiting toxicity in clinical trials. Other side effects such as fatigue, gastrointestinal disorders and anemia are most commonly occurred in patients who received the treatment [91,92]. However, targeting BET and combining it with endocrine therapy may be beneficial in tamoxifen resistant settings and well-tolerated BET inhibitors need to be developed to maximize clinical efficacy without exceeding toxicity thresholds.

  3.5 Targeting the ER and lysine specific demethylase 1 (LSD1)

  In addition to HDAC, it has been shown that breast cancer development is also strongly associated with histone demethylation [93]. Histone demethylation is mainly regulated by histone demethylase (HDM) and it is regarded as an important part of epigenetics. Two classes of histone demethylases have been identified: the first class is the LSD which includes two isoforms, LSD1 and LSD2 [94]. The second group is histone demethylases containing the Jumonji structural domain [95]. LSD1 is overexpressed in breast cancer, therefore targeting LSD1 may be a new target for drug development for breast cancer treatment. LSD1 is a substrate of protein kinase A (PKA) and is an important mediator of ER signaling pathway. Surprisingly, ERα engages not only LSD1, but its partners of the CoREST corepressor complex and the molecular chaperone Hsp90. The recruitment of Hsp90 to promote ERα transcriptional activity and stimulate breast cancer cell growth [96].

  Against endocrine resistance, dual-targeted compounds have emerged as an effective strategy. In 2020, Zhou et al. developed a series of dual targeting compounds against ERα and HDM. Primarily based on new compounds synthesized by modifying the OBHS pharmacophore scaffold previously developed in their lab, these novel OBHS-LSD1 inhibitors exhibited good ERα binding affinity and strong inhibitory activity against LSD1. Compared with 4-OH tamoxifen, these compounds exhibited higher anti-proliferative activity against MCF-7 cell line in vitro. Among them, compound 35 showed strong inhibitory activity against LSD1 and MCF-7 cells (IC₅₀: 1.55 and 8.79 µmol/L). The structural analysis of all compounds (31–42) shows that the replacement of pyridine group to the phenyl ring of sulfonate in OBHS can significantly improve LSD1 inhibition. The type and position of substituents (35–39) also affect the activity. The space occupied by substituents on benzene sulfonates had a positive effect on the binding affinity, and the inhibitory activity changed significantly when different naphthalene groups (31 and 32) and coumarin moieties (33 and 34) were employed (Table S3 in Supporting information). Apoptosis analysis of 35 revealed that the effect of the compounds was partially mediated by induction of apoptosis on MCF-7 cells. Furthermore, the docking study of 35 suggested that the appropriate location of phenol group in the main scaffold is important for the binding affinity of ER. The results of molecular docking contribute to the unique dual mechanism of OBHS-LSD1i conjugates and targets [97].

  Although these compounds have not been further investigated on the mechanisms of HDM and ERα, it can be seen that targeting ERα and HDM is effective in breast cancer. Therefore, in the future, attempts can be made to design compounds with better activity so that the mechanism as well as in vivo experiments can be investigated in detail.

  3.6 Targeting the ER and vascular endothelial growth factor receptor-2 (VEGFR-2)

  It is well known that angiogenesis is essential for cell growth and metastasis. Angiogenesis is very important for breast cancer cell growth and distant metastasis. It is usually regulated by the VEGFs and their receptors. Among them, VEGFR-2 is a member of the VEGF/VEGFR signaling and belongs to the receptor protein tyrosine kinase family [98]. Raf-1/MAPK/extracellular signal-regulated kinase (ERK) signaling was activated by phosphorylation of VEGFR-2 and this signaling axis is important for cell proliferation, invasion and angiogenesis. In addition, it has been shown that ER can interact with Raf-1/MAPK/ERK and can be observed in endocrine resistance [99]. However, the efficacy of VEGFR-2 inhibitors as monotherapy for breast cancer is very limited. Thus, some studies have combined VEGFR-2 with ER targets to overcome endocrine resistance in ER positive breast cancer.

  In 2016, Xiang et al. synthesized a series of 6-arylindolylisoquinolones 43–46 (Fig. S5 in Supporting information) as dual target inhibitors of ERα and VEGFR-2 and evaluated them in vitro activities. It was found that these compounds exhibited powerful ERα binding affinity and antagonism, and showed strong VEGFR-2 inhibitory potency. In addition to MCF-7, these compounds also showed excellent anti-proliferative activity against MDA-MB-231, Ishikawa and HUVEC cell lines. Compound 46 is more active than tamoxifen against MCF-7 (IC₅₀: 1.2 and 5.3 µmol/L). And it showed the best inhibitory activity against MDA-MB-231 (IC₅₀: 0.5 µmol/L) and the strongest inhibition of VEGFR-2 (IC₅₀: 0.8 µmol/L), and was therefore selected as the superior compound. Further study investigated the effects of 46 on the progesterone receptor (PGR) and MAPK signaling pathway by RT-PCR and WB in MCF-7 cells. It showed that 46 inhibited the expression of PGR and had significant anti-estrogenic activity. Compound 46 has demonstrated the ability to suppress the activation of VEGFR-2 as well as the Raf-1/MAPK/ERK signaling cascade. It has been shown to inhibit the activation of VEGFR-2 as well as the downstream Raf-1/MAPK/ERK pathway in MCF-7 cells. Consequently, 46 emerges as a potential dual-action compound for novel anti-breast cancer agents. Moreover, these findings pave the way for exploring innovative avenues in the discovery of breast cancer treatments [100].

  Based on the previous research, in 2017, Xiang et al. developed a series of novel SERM-ERα/VEGFR-2 dual-target compounds and investigated their anti-breast cancer proliferative activity. These compounds were based on coumarin scaffold, known for their angiogenesis-inhibiting properties by disrupting endothelial cell growth. In addition, they used a 3,4-disubstituted-2H-chromen-2-one as a backbone to probe the biological effects of the two coumarin-terminal groups (R1 and R2) (47–51) as well as hydrogen-bonding of medium-sized tertiary amine side-chain moieties (52–55). Most of these compounds exhibited superior inhibitory effects against MCF-7 and Ishikawa cells than compounds BL-18d and tamoxifen, etc., and the SAR study indicated that the piperidine substituent was necessary for activity compared to dimethylamine, diethylamine, and pyrrolidinyl. Retaining the phenolic hydroxyl group on R1 and substitution of R2 with fluorine slightly increased the binding affinity compared to the other groups, the authors concluded that fluorine induces minimal spatial changes and therefore promotes the interaction of ligands with target protein (Table S4 in Supporting information). Therefore, 55, which has the best ER binding affinity (IC₅₀: 2.19 µmol/L) and excellent cytotoxicity for MCF-7 and Ishikawa cells (IC₅₀: 9.54 ± 2.65 and 11.12 ± 2.34 µmol/L), was selected for the subsequent pharmacological studies. In RT-PCR assays, 55 could exert significant anti-estrogenic effects by inhibiting the transcriptional expression of PGR in MCF-7 cells, which is comparable to the properties of SERMs. Further mechanistic studies on it showed that compound 55 could inhibit the migration of MCF-7 cells and block the cell cycle at G0/G1 phase. Unfortunately, there are no in vivo experiments with compound 55. Based on the assessment of in vitro study, VEGFR-2/Raf-1/MAPK/ERK signaling pathway represents a new approach for development of multifunctional drugs targeting ER and VEGFR-2 (Fig. 9) [101].

  Figure 9

  

  Figure 9.  Role of compounds 46 and 55 in targeting ERα and VEGFR-2 initiated signaling pathways.

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  In 2019, Xiang et al. discovered a series of 3-arylquinoline analogs targeting ERα and VEGFR-2 based on the studies done by previous authors, and the study of this series of compounds revealed that compounds 56 and 57 (Fig. S6 in Supporting information) had good ERα binding affinity (IC₅₀: 2.33 and 1.78 µmol/L) with 57 having an ERα binding affinity close to that of the control drug tamoxifen (IC₅₀: 1.56 µmol/L) and strong VEGFR-2 inhibitory activity (IC₅₀: 104 nmol/L for 56 and 86 nmol/L for 57). In addition, both compounds exhibited excellent cytotoxicity against MCF-7 (IC₅₀: 2.8 and 1.8 µmol/L), superior to tamoxifen and raloxifene (IC₅₀: 16.7 and 12.5 µmol/L). Using RT-PCR and flow analysis, compound 57 was found to down-regulate PGR mRNA expression in breast cancer MCF-7 cells and to block the cell cycle in G1 phase. In addition, compound 57 was found to inhibit cell migration which can be considered as a potential anticancer lead compound for further study [102].

  In conclusion, by targeting both VEGF-2 and ERα, the dual-target compound has good anti-proliferative activity in breast cancer, and it can potentially overcome endocrine therapy-induced drug resistance by inhibiting Raf-1/MAPK/ERK and ER signaling pathway (Fig. 9).

  3.7 Targeting the ER and tubulin, MDM2, SRC kinase

  Tubulin is an α/β heterodimeric protein that mediates key pathways in cell division. The polymerization and depolymerization of tubulin is a dynamic process that maintains a certain balance under normal physiological conditions. Disruption of this balance affects essential cellular functions and results in disruption of normal mitotic spindle formation and therefore inhibiting cell division and cell cycle. Many microtubulin-binding compounds, for example paclitaxel and vincristine, were being used clinically in a variety of different cancers [103]. In 2014, O'Boyle et al. synthesized a series of β-lactams 58–65 and evaluated their binding to ER and antiproliferative activity. SAR showed that the substituent group in the core β-lactam structure is critical, and the introduction of the α-(hydroxyaryl)-methyl substituent increased the antiproliferative activity while maintaining the ER binding affinity compared to the phenyl (60)- versus diphenyl (61)-substituted compounds. Removal of the substituent group (59) or introduction of basic pyrrolidine substituent (65) altered the core β-lactam structure and led to loss of ER binding activity, whereas substitution and deletion of methoxy groups on the other phenyl rings (63 and 64) significantly decreased antiproliferative activity (Fig. S7 and Table S5 in Supporting information). Thus compound 62 with a trimethoxyaryl ring and introduction of α-(hydroxyaryl)-methyl substituent was identified as the compound with the best antiproliferative activity with IC₅₀ values of 0.008 µmol/L (ERα) and 0.015 µmol/L (ERβ). In antiproliferative assays, the most potent β-lactam 62 was shown to be a dual-target compound designed for tubulin and ER, resulted in complete tubulin depolymerization. Further studies showed that compound 62 can induce apoptosis by decreasing Bcl-2 and Mcl-1 in MCF-7 cells. These compounds represented a new class of ER antagonists that may be used in anticancer or modulation of inflammatory responses. Compound 62 has been found as a lead compound to design new compounds with higher affinity for ER and tubulin [104].

  In 2023, Zhou et al. developed a series of dual-target compounds targeting ER and tubulin on the basis of a bridged bicyclic structure and selenocyano (SeCN) side chains (66–77). Most of the compounds showed stronger activity in inhibiting breast cancer cell proliferation in contrast to OBHS. SAR study showed that the antiproliferative activity of the compounds was strongly correlated to the length of the side chain of SeCN, and the compounds containing hexyl side chain (68) showed superior cytostatic activity compared to the compounds with octyl side chain (66) and butyl side chain (67). When comparing the antiproliferative activity of the compounds in breast cancer, the inhibition activity of ortho-substituted compounds were better than that of the compounds with meso‑ and para-substituents (68, 69, 72, 74, 75, 77). The introduction of electron-withdrawing groups at the ortho site enhanced the antiproliferation of the compounds (70–72, 76, 77). The stronger the electron-withdrawing group implied the better proliferation inhibition of the compounds (70–72) (Fig. S8 and Table S6 in Supporting information). In MCF-7 and T47D cell lines, compounds 72 (IC₅₀: 0.09 ± 0.02 and 2.82 ± 0.17 µmol/L) and 77 (IC₅₀: 0.06 ± 0.02 and 2.56 ± 0.23 µmol/L) showed better antiproliferative effects than OBHSA (IC₅₀: 0.23 ± 0.03 and 6.92 ± 0.19 µmol/L) and fulvestrant (IC₅₀: 0.12 ± 0.04 and 1.76 ± 0.06 µmol/L). Similarly, 72 and 77 were more antiproliferative than fulvestrant in drug resistant cell lines (including mutant ERα cell lines (T47D^D538G and T47D^Y537S) and Tam-resistant cell line LCC2. Further studies revealed that compounds 72 and 77 were able to degrade ERα in MCF-7 cells in a dose dependent manner, with 72 also showing strong degradation in drug-resistant cells. The compounds 72 and 77 were found to inhibit microtubule protein polymerization and disrupt the cellular microtubule network by cell-free microtubule protein polymerization assay and fluorescence. Finally, in vivo tumor inhibition assays revealed that 72 and 77 significantly inhibited tumor growth and showed low toxicity in MCF-7 and LCC2 xenograft models [105]. p53 can be activated by different genotoxic stimulation and can initiate a transcriptional program. The p53 mutations are relatively rare in ER positive breast cancer, but there is an increased occurrence of main dysregulated p53, MDM2 and MDM4. MDM2 is considered as an ER transcriptional target, where MDM2 can directly interact with ER (Fig. 10) [106].

  Figure 10

  

  Figure 10.  Schematic diagram of the feedback loop and signal crosstalk between MDM4, MDM2, ERα and p53.

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  In 2020, Portman et al. used the MDM2 inhibitor NVP-CGM097 (78) (Fig. S9 in Supporting information) alone and in combination with fulvestrant or palbociclib for evaluation for ER positive breast cancer. The antitumor effects of combination therapy on p53 wild-type and mutant ER positive cell lines (MCF-7, ZR75–1, T47D) as well as endocrine and CDK4/6 resistant breast cancer cell lines were evaluated. The use of endocrine therapy or CDK4/6 inhibitors with MDM2 inhibitors has a synergistic effect. It achieves this effect by increasing antagonism of cell cycle progression. At the same time, combination therapy induces senescence in fulvestrant or palbociclib resistant cell lines and significantly inhibits tumor growth in fulvestrant resistant models [107]. Thus, the use of MDM2 inhibitors with endocrine therapy may be effective for treating ER positive breast cancer by promoting the pro-apoptotic effects of p53 and dual targeting cell cycle pathways.

  In breast cancer, Src is a key component of estrogen-induced non-genomic MAPK and matrix metalloproteinase activation as well as estrogen-mediated cell proliferation and cell cycle progression [108]. Resistance to tamoxifen has been found to be accompanied by elevated Src kinase activity, which resulted in an invasive phenotype. Saracatinib is a known inhibitor of Src kinase. In 2006, Costello et al. discovered a novel subseries of C-5-substituted anilinoquinazolines from which discovered saracatinib (79). The R1 site is well tolerated by a variety of substituents, with tetrahydropyran-4-yloxy proving the optimal for the ribose pocket and providing additional affinity for the enzyme active site. SAR has shown that increased potency can be maintained by selecting a basic, flexible side chain bearing solubilizing moiety for the R2 site, and that a range of branched alkyl chains such as (N-methylpiperazin-4-yl)propoxy (79–84) are similarly broadly potent to tolerate the cyclic nucleus. However, the study of the pattern of aniline substitution is extremely important in increasing the potency of the inhibitor, with bulky chlorobenzodioxole substituents affecting the deflection of the tetrahydropyran-4-yloxy in the opposite direction of the hydrophobic pocket. Replacement of the benzodioxane structure (88) by the benzodioxole structure (79) through a ring expansion strategy would instead greatly reduce compound activity. The authors concluded that the introduction of the chlorine atom increases the lipophilic interaction of the compound with alanine residues within the c-Src kinase selective pocket (Ala403) (84 vs. 85), whereas removal decreases the binding affinity (Fig. S10 and Table S7 in Supporting information). Combined with the above SAR studies on the quinazoline core, saracatinib (79) was found as the optimal compound among the series, with an IC₅₀ value of 2.7 nmol/L for c-Src enzyme inhibition [109]. Given its favorable physicochemical properties, selectivity, pharmacokinetics and preclinical model activity, saracatinib (79) was further developed and subjected to clinical studies.

  In 2009, Hiscox et al. investigated the combination effect of Src inhibition and anti-estrogen therapy in endocrine resistant breast cancer. The result showed that long-term treatment of MCF-7 and T47D with tamoxifen or the Src inhibitor saracatinib resulted in the development of cellular resistance and resulted in decreased antiproliferation activity. However, when saracatinib was combined with tamoxifen in MCF-7 and T47D cells, the Src activity was reduced, the phosphorylation of adherent spot kinase was inhibited and the invasive behavior in vitro was completely abolished. In addition, the combination treatment significantly inhibited the expressions of c-myc and cyclin D1 and inhibited cell proliferation and the following drug resistance, with all cancer cells disappearing after 12 weeks. These data suggested that the combination of drug-targeted Src kinase with anti-hormonal therapy effectively prevented the resistance, indicating the potential clinical benefit of Src inhibition as a clinical treatment [110]. In 2011, Chen et al. also investigated the effect of endocrine therapy in combination with Src inhibitors on endocrine resistant cells and further explored the mechanism. Their study found that the combination of saracatinib and ER-blocking drugs could cause cycle arrest in breast cancer cells by increasing p27. The results demonstrated that the combination was more effective in increasing p27 expression, decreasing Ki67 expression, and inhibiting the MDA-MB-361 xenografts growth compared to the two drugs alone. The combined inhibition of ER and Src postponed the occurrence of resistance in vivo, indicating the effectiveness of combination therapy.

  In conclusion, the discovery of new combination targets with endocrine therapy can effectively enhance the antiproliferation of breast cancer and can effectively alleviate or even overcome the drug resistance that occurs with endocrine therapy alone.

  3.8 Targeting the ER and nuclear factor kappa B (NF-κB)

  NF-κB, a widely expressed class of transcription factors, has been considered a key modulator of the mammalian immune and inflammatory response for many years. Recently, it has been found to be associated with chemoresistance. Activation of NF-κB, particularly the binding of the p50 subunit of NF-κB to DNA is a potential prognostic biomarker that identifies a high-risk subgroup of ER positive breast cancer patients which will experience early recurrence. Furthermore, preliminary preclinical studies suggest that in these aggressive ER positive breast cancer cell lines, therapeutic strategies to prevent or interrupt NF-κB activation can recover the sensitivity to endocrine therapy such as tamoxifen [111]. The pathway interrelationship between NF-κB and ER can be expressed as follows (Fig. 11): When cells are stimulated by appropriate extracellular signals, IκB kinase (IKK) complexes consisting of IKKα and IKKβ catalytic subunits and IKKγ regulatory subunits are activated, and these activated complexes phosphorylate IκB, thereby regulating different genes expression that are important mediators of inflammation and survival in tumor progression [111,112].

  Figure 11

  

  Figure 11.  Known intracellular signaling mechanisms upstream and downstream of NF-κB activation.

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  Since activation of NF-κB drives oncogene transcriptional programs that may result in endocrine resistance, targeting the NF-κB pathway can be used to overcome endocrine resistance through combination therapy. In 2022, Zhou et al. synthesized novel OBHS-RES compounds that target the dual inhibitory activity of ERα and NF-κB. Most OBHS-RES compounds showed additional nitric oxide (NO) inhibition compared to tamoxifen. The length of the R2 benzenesulfonate portion balanced the antiproliferative activity of the conjugates with NO inhibition in MCF-7 cells when R1 retained the resveratrol analogue unit, but overall, conjugates with NF-κB inhibitor units in the R2 benzenesulfonate portion (92–95) showed superior pharmacological properties. The compounds with the pterostilbene moiety had better antiproliferative activity than those with resveratrol moiety, so the para-substituted 92 (vs. 94) was finally selected for further pharmacological studies (Fig. S11 and Table S8 in Supporting information). Compound 92 showed good antiproliferative effect in MCF-7 cells (IC₅₀: 3.7 ± 0.56 µmol/L) compared to 4-OHT (IC₅₀: 9.5 ± 1.06 µmol/L), and compound 92 also showed good anti-inflammatory effect (NO inhibition IC₅₀ value of 0.44 ± 0.23 µmol/L and 95% inhibition at 10 µmol/L). Subsequent in vivo experiments in a BALB/C nude mouse MCF-7 breast cancer model showed that compound 92 was more effective than tamoxifen. Using these types of isomeric 3D ligands to explore the compliance of the 92 structure to ER specificity revealed that one of the enantiomers had better biological activity than the other [113]. Thus, OBHS-RES conjugates provide a new strategy for the development of effective antiproliferative and anti-inflammatory agents for the treatment of ER positive breast cancer.

  3.9 Targeting the ER and activating oxidative stress

  The Increased levels antioxidant proteins are associated with tamoxifen resistance. It has also been found that tamoxifen resistant MCF-7 cell line had higher levels of antioxidant proteins than normal MCF-7 cell line due to the activation of Nrf2/ARE [114]. Therefore, inducing intracellular oxidative stress may be a strategy to improve the efficacy of tamoxifen treatment.

  Reactive oxygen species (ROS) levels are very important for cell growth, and low to moderate levels of ROS can promote tumor cell growth, but when ROS levels increase dramatically to generate oxidative stress, which leads to cancer cell death, it has been shown that the accumulation of oxidative stress and ROS will lead to redox imbalance and eventually to breast cancer cell death [115]. Therefore, increasing ROS levels or inhibiting the antioxidant system may be a promising strategy for breast cancer.

  Hexokinase (HK), a crucial enzyme in the glucose metabolic pathway, facilitates the conversion of glucose into glucose-6-phosphate through phosphorylation. The enzyme is notably abundant in various types of cancerous tumors. In breast cancer, ER has been observed to specifically modulate the expression levels of enzymes involved in glycolysis and to trigger a variety of signaling cascades that are pivotal for metabolic processes, cellular multiplication, and the overall survival of the cell [116]. Thus, in 2022, Deng et al. synthesized a series of dual-target compounds targeting HK1 and ERα that were able to exhibit moderate ERα binding affinity, SAR showed that the introduction of the PhSe group significantly improved the antiproliferation activity of all compouns. The stereoisomers 99, 102, and 104 showed more effective antiproliferative activity in MCF-7 cells than 96–98. After determining the PhSe configuration, the types of substituents (99–102 and 104) and positions (97 vs. 102) on the R ring affect the activity of the compound. For compounds 100, 101, and 103, chlorine substitution produces higher efficacy than bromine substitution, methyl substitution (97 and 102) exhibits higher activity than methoxy substitution (96 and 99), but naphthalene ring substitution (104) has the best activity (Table S9 in Supporting information). 104 for ERα positive cancer cells exhibit the best cell inhibitory activity (IC₅₀: 0.05 and 2.67 µmol/L for MCF-7 and LCC2 cells) and selectivity (selectivity index > 1200), so it was further selected for biological activity research. HK1 has been shown to be a direct target of compound 104. Further mechanistic studies demonstrated that compound 104 could target mitochondria bound HK1 and promote ROS production, leading to mitochondrial damage and thus inducing apoptosis, while targeting ERα could have a synergistic effect (Fig. 12). In vivo studies demonstrated that compound 104 had a bioavailability of 23.20% and it exhibits better tumor-suppressing effects compared to tamoxifen in MCF-7 and LCC2 xenograft models. The findings from these in vivo and ex vivo experiments indicate that compound 104 could be a novel anti-breast cancer therapy, potentially addressing the challenge of drug resistance [117].

  Figure 12

  

  Figure 12.  Mechanism diagram of compound 104.

  DownLoad: Full-Size Img PowerPoint

  Thioredoxin reductase (TrxR), a crucial enzyme in cellular redox homeostasis, has been identified as a promising target for cancer therapy. Overexpression of TrxR was observed in breast cancer and associated with the prognosis and survival rates [118]. Studies have indicated that both Trx and TrxR can be regulated by ER and E2. It is suggested that the ER and Trx system may synergistically modulate redox equilibrium and cellular proliferation [119]. In 2023, Lu et al. synthesized a series of dual-target SERD-NHC-Au(I) compounds that can target TrxR and ERα pathways. Compound 105 showed anti-proliferative activity against MCF-7 (IC₅₀: 1.05 ± 0.15 µmol/L) compared to the positive drug auranofin (IC₅₀: 3.10 ± 0.24 µmol/L) and had no agonistic effect on Ishikawa cells (IC₅₀: 0.45 ± 0.12 µmol/L). Subsequently, compound 105 was found to degrade ERα by WB and was found to inhibit intracellular TrxR enzyme activity but not degrade TrxR protein by measuring intracellular TrxR cell enzyme activity and WB. Further mechanistic studies demonstrated that compound 105 could target TrxR and promote ROS production, leading to mitochondrial damage and thus inducing apoptosis. Meanwhile, it can significantly downregulate ER level and block ER downstream signaling pathways. Moreover, it can trigger immunogenic cell death (Fig. 13). This suggested that compound 105 could be a promising compound for ER positive breast cancer [120].

  Figure 13

  

  Figure 13.  Representation of complex 105 dual targeting ER and TrxR with underlying mechanism of ICD effects.

  DownLoad: Full-Size Img PowerPoint

  Therefore, targeting ERα with activation of oxidative stress for synergistic therapy can effectively overcome endocrine resistance and can be used to treat breast cancer in the future by targeting the targets that can lead to elevated ROS. Although such dual-target compounds are not currently in clinical studies, they remain a potential direction for the treatment of this breast cancer subtype.

  3.10 Targeting the ER and aromatase

  In recent years, ERα/aromatase dual-target PROTAC degraders have demonstrated significant potential in overcoming endocrine-resistant breast cancer. Zhou et al. designed a series of novel dual-target PROTAC molecules by combining ERα/aromatase ligands with E3 ligase ligands, achieving synchronous degradation of ERα and aromatase. Among these, compound 106 exhibited excellent anti-proliferative activity, effectively inhibiting MCF-7 cell growth while showing potent efficacy against ERα-mutant cells. Mechanistic studies revealed that 106 degraded target proteins via the ubiquitin-proteasome system, downregulated the oncogene MYC, and induced G2/M phase cell cycle arrest. In vivo experiments further confirmed that 106 suppressed tumor growth with low toxicity. Additionally, molecular docking analysis demonstrated a unique binding mode of 106 to ERα and aromatase, providing a structural basis for its efficient degradation capability. This study validates that ERα/aromatase dual-target degradation synergistically regulates estrogen synthesis and signaling pathways, offering a novel therapeutic strategy to combat endocrine resistance and highlighting its substantial clinical translation potential (Fig. S12 in Supporting information) [121].

  4. Conclusion and perspective

  ER positive breast cancer is the most prevalent subtype and although the use of endocrine therapy is effective in treating these patients, drug resistance inevitably occurs with endocrine therapy, and thus many targeted drugs can be used in combination with endocrine therapy to overcome endocrine resistance, and some drug combinations are currently ongoing in phase Ⅱ/Ⅲ clinical trials. This review summarizes the studies on the combination of some molecular targeted drugs with endocrine therapeutic agents and dual target compounds in ER+ breast cancer.

  At present, CDK4/6 inhibitors combined with endocrine therapy has become the first-line therapy for ER positive advanced breast cancer, which indicates that combination therapy is very effective for breast cancer management. However, endocrine drugs and targeted drugs will inevitably produce drug resistance, and the resistance mechanisms are diverse, so how to choose to use different drug combination strategies for combination targeted therapy to achieve long-term control of the disease may be a future research direction. With the development of science and technology, there are already technologies for identifying effective drug combinations, such as the use of CRISPR-CAS9-based high-throughput loss-of-function gene screening technology to find reasonable and efficient drug combinations, and the use of FDA-marketed drugs and compounds in clinical studies to conduct high-throughput screening of drug libraries is also one of the most important ways to find drug combinations, and it is believed that through these technologies, we will be able to better select drugs for combination therapy. It is believed that through these techniques, drugs can be better selected for combination therapy. Therefore, in the future, we should conduct more thorough research on breast cancer targets and select better drug combinations to treat breast cancer patients.

  Therefore, the future research direction may be to explore dual or more targets combination therapy for ER positive breast cancer under the balance of safety and efficacy. The design and optimization of dual-target or even multi-target compounds are gradually improving, and these compounds have better anti-breast cancer effects in vitro studies. The main difficulty of multi-target compounds is to optimize the affinity ratio of the drug to different targets in order to achieve the effect of increasing efficiency and reducing toxicity. The design of multi-targeted drugs should not only ensure the activity and selectivity of the target, but also remove duplicated or unnecessary fragments to reduce the molecules so as not to affect the drug absorption, distribution and other pharmacokinetic problems. It is believed that in the future multi-target compounds will be better developed and applied in the treatment of breast cancer.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Lijuan Liu: Writing – original draft, Visualization, Methodology, Investigation, Data curation. Zhihao Zhao: Writing – original draft, Investigation, Data curation. Feiwan Zou: Validation, Investigation. Wukun Liu: Supervision, Investigation, Conceptualization. Yunlong Lu: Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

  Acknowledgments

  We thank the financial supports of the National Natural Science Foundation of China (No. 82204181), Nanjing University of Chinese Medicine National Natural Science Foundation of China Counterpart Funding (No. XPT82204181), Jiangsu Provincial Health Commission (No. Z2021057), the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine), High level key discipline construction project of the National Administration of Traditional Chinese Medicine-Resource Chemistry of Chinese Medicinal Materials (No. zyyzdxk-2023083), and the Key R&D Program of Jiangsu Province (No. BE2023840). We also thank for the State Key Laboratory of Coordination Chemistry (Nanjing University, China).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111451.

  
  
• 1. [1]

     R.L. Siegel, A.N. Giaquinto, A. Jemal, CA Cancer J. Clin. 74 (2024) 12–49. doi: 10.3322/caac.21820

  2. [2]

     V. Grann, A.B. Troxel, N. Zojwalla, et al., Soc. Sci. Med. 62 (2006) 337–347. doi: 10.1016/j.socscimed.2005.06.038

  3. [3]

     H. Xu, B. Xu, Chin. J. Cancer. Res. 35 (2023) 565–583. doi: 10.21147/j.issn.1000-9604.2023.06.02

  4. [4]

     N. Cancer Genome Atlas, Nature 490 (2012) 61–70. doi: 10.1038/nature11412

  5. [5]

     P. Eroles, A. Bosch, J.A. Perez-Fidalgo, A. Lluch, Cancer Treat. Rev. 38 (2012) 698–707. doi: 10.1016/j.ctrv.2011.11.005

  6. [6]

     Y.S. Sun, Z. Zhao, Z.N. Yang, et al., Int. J. Biol. Sci. 13 (2017) 1387–1397. doi: 10.7150/ijbs.21635

  7. [7]

     S. Arora, P. Narayan, C.L. Osgood, et al., Clin. Cancer Res. 28 (2022) 1072–1086. doi: 10.1158/1078-0432.ccr-21-2600

  8. [8]

     F. Andre, E. Ciruelos, G. Rubovszky, et al., N. Engl. J. Med. 380 (2019) 1929–1940. doi: 10.1056/nejmoa1813904

  9. [9]

     J.M. Giltnane, K.E. Hutchinson, T.P. Stricker, et al., Sci. Transl. Med. 9 (2017) eaai7993. doi: 10.1126/scitranslmed.aai7993

  10. [10]

      P. Razavi, M.T. Chang, G. Xu, et al., Cancer Cell 34 (2018) 427-438.e6. doi: 10.1016/j.ccell.2018.08.008

  11. [11]

      H.J. Burstein, N. Engl. J. Med. 383 (2020) 2557–2570. doi: 10.1056/nejmra1307118

  12. [12]

      A.C. Tecalco-Cruz, I.A. Perez-Alvarado, J.O. Ramirez-Jarquin, L. Rocha-Zavaleta, Cell. Signal. 34 (2017) 121–132. doi: 10.1016/j.cellsig.2017.03.011

  13. [13]

      B.V. Phillips-Farfán, B.G. Quintanar, R. Reyes, A. Fernández-Guasti, Physiol. Behav. 268 (2023) 114237. doi: 10.1016/j.physbeh.2023.114237

  14. [14]

      C.N. Haines, S.E. Wardell, D.P. McDonnell, Essays Biochem. 65 (2021) 985–1001. doi: 10.1042/ebc20200174

  15. [15]

      D.P. McDonnell, S.E. Wardell, C.Y. Chang, J.D. Norris, J. Clin. Oncol. 39 (2021) 1383–1388. doi: 10.1200/jco.20.03565

  16. [16]

      V.C. Jordan, Eur. J. Cancer 44 (2008) 30–38. doi: 10.1016/j.ejca.2007.11.002

  17. [17]

      D.J. Kojetin, T.P. Burris, E.V. Jensen, S.A. Khan, Endocr. Relat. Cancer 15 (2008) 851–870. doi: 10.1677/ERC-07-0281

  18. [18]

      S. Damodaran, C.C. O'Sullivan, A. Elkhanany, et al., Ann. Oncol. 34 (2023) 1131–1140. doi: 10.1016/j.annonc.2023.09.3103

  19. [19]

      E.R. Simpson, S.R. Davis, Endocrinology 142 (2001) 4589–4594. doi: 10.1210/endo.142.11.8547

  20. [20]

      T.V. Augusto, G. Correia-da-Silva, C.M.P. Rodrigues, et al., Endocr. Relat. Cancer 25 (2018) R283–R301. doi: 10.1530/erc-17-0425

  21. [21]

      A. Gennari, F. Andre, C.H. Barrios, et al., Ann. Oncol. 32 (2021) 1475–1495. doi: 10.1016/j.annonc.2021.09.019

  22. [22]

      A.L. Wijayaratne, D.P. McDonnell, J. Biol. Chem. 276 (2001) 35684–35692. doi: 10.1074/jbc.M101097200

  23. [23]

      G. Jane, Z. Wei, H. Marc, et al., Cell 178 (2019) 949-963.e18. doi: 10.1016/j.cell.2019.06.026

  24. [24]

      T. Bihani, H.K. Patel, H. Arlt, et al., Clin. Cancer Res. 23 (2017) 4793–4804. doi: 10.1158/1078-0432.CCR-16-2561

  25. [25]

      S.M. Hoy, Drugs 83 (2023) 555–561. doi: 10.1007/s40265-023-01861-0

  26. [26]

      D.P. Bondeson, A. Mares, I.E.D. Smith, et al., Nat. Chem. Biol. 11 (2015) 611–617. doi: 10.1038/nchembio.1858

  27. [27]

      L.B. Snyder, J.J. Flanagan, Y. Qian, et al., Cancer Res. 81 (2021) 44. doi: 10.1158/1538-7445.am2021-44

  28. [28]

      W. He, H. Zhang, L. Perkins, et al., Cancer Res. 81 (2021) PS18-09. doi: 10.1158/0008-5472.CAN-20-2245

  29. [29]

      M. Bekes, D.R. Langley, C.M. Crews, Nat. Rev. Drug. Discov. 21 (2022) 181–200. doi: 10.1038/s41573-021-00371-6

  30. [30]

      E. Ferraro, E.M. Walsh, J.J. Tao, et al., Cancer Treat. Rev. 109 (2022) 102432. doi: 10.1016/j.ctrv.2022.102432

  31. [31]

      F. Rasha, M. Sharma, K. Pruitt, Mol. Cell. Endocrinol. 532 (2021) 111322. doi: 10.1016/j.mce.2021.111322

  32. [32]

      R. Jeselsohn, G. Buchwalter, C. De Angelis, et al., Nat. Rev. Clin. Oncol. 12 (2015) 573–583. doi: 10.1038/nrclinonc.2015.117

  33. [33]

      A.B. Hanker, D.R. Sudhan, C.L. Arteaga, Cancer Cell 37 (2020) 496–513. doi: 10.1016/j.ccell.2020.03.009

  34. [34]

      X. Yang, D.L. Phillips, A.T. Ferguson, et al., Cancer Res. 61 (2001) 7025–7029.

  35. [35]

      M. Palaniappan, Biomedicines 12 (2024) 2700. doi: 10.3390/biomedicines12122700

  36. [36]

      J. Rinath, Y. Roman, B. Gilles, et al., Clin. Cancer Res. 20 (2014) 1757–1767. doi: 10.1158/1078-0432.CCR-13-2332

  37. [37]

      H. Kurokawa, A.E. Lenferink, J.F. Simpson, et al., Cancer Res. 60 (2000) 5887–5894.

  38. [38]

      L. Formisano, K.M. Stauffer, C.D. Young, et al., Clin. Cancer Res. 23 (2017) 6138–6150. doi: 10.1158/1078-0432.CCR-17-1232

  39. [39]

      T.W. Miller, B.T. Hennessy, A.M. Gonzalez-Angulo, et al., J. Clin. Invest. 120 (2010) 2406–2413. doi: 10.1172/JCI41680

  40. [40]

      F. Bertucci, C.K.Y. Ng, A. Patsouris, et al., Nature 569 (2019) 560–564. doi: 10.1038/s41586-019-1056-z

  41. [41]

      C. Fribbens, I. Garcia Murillas, M. Beaney, et al., Ann. Oncol. 29 (2018) 145–153. doi: 10.1093/annonc/mdx483

  42. [42]

      R.J. Sullivan, J.R. Infante, F. Janku, et al., Cancer Discov. 8 (2018) 184–195. doi: 10.1158/2159-8290.cd-17-1119

  43. [43]

      J. Huang, L. Zheng, Z. Sun, J. Li, Int. J. Mol. Med. 50 (2022) 128. doi: 10.3892/ijmm.2022.5184

  44. [44]

      L.M. Spring, S.A. Wander, F. Andre, et al., Lancet 395 (2020) 817–827. doi: 10.1016/S0140-6736(20)30165-3

  45. [45]

      H. Hu, W. Zhang, L. Lei, et al., Chin. Chem. Lett. 35 (2024) 108765. doi: 10.1016/j.cclet.2023.108765

  46. [46]

      Y. Xu, L. Huang, Y. Bi, et al., Chin. Chem. Lett. 34 (2023) 107719. doi: 10.1016/j.cclet.2022.07.062

  47. [47]

      X. Zhang, X. Liu, L. Qin, et al., Chin. Chem. Lett. 34 (2023) 107618. doi: 10.1016/j.cclet.2022.06.041

  48. [48]

      H. Ikeda, K. Kawase, T. Nishi, et al., Nature 638 (2025) 225–236. doi: 10.1038/s41586-024-08439-0

  49. [49]

      T. Mukohara, Y.H. Park, D. Sommerhalder, et al., Nat. Med. 30 (2024) 2242–2250. doi: 10.1038/s41591-024-03060-0

  50. [50]

      Y.J. Choi, X. Li, P. Hydbring, et al., Cancer Cell 22 (2012) 438–451. doi: 10.1016/j.ccr.2012.09.015

  51. [51]

      R.M. Zwijsen, E. Wientjens, R. Klompmaker, et al., Cell 88 (1997) 405–415. doi: 10.1016/S0092-8674(00)81879-6

  52. [52]

      S.C. Scott, S.S. Lee, J. Abraham, Semin. Oncol. 44 (2017) 385–394. doi: 10.1053/j.seminoncol.2018.01.006

  53. [53]

      P.L. Toogood, P.J. Harvey, J.T. Repine, et al., J. Med. Chem. 48 (2005) 2388–2406. doi: 10.1021/jm049354h

  54. [54]

      L. Malorni, G. Curigliano, A.M. Minisini, et al., Ann. Oncol. 29 (2018) 1748–1754. doi: 10.1093/annonc/mdy214

  55. [55]

      R.S. Finn, J.P. Crown, I. Lang, et al., Lancet Oncol. 16 (2015) 25–35. doi: 10.1016/S1470-2045(14)71159-3

  56. [56]

      R.S. Finn, M. Martin, H.S. Rugo, et al., N. Engl. J. Med. 375 (2016) 1925–1936. doi: 10.1056/NEJMoa1607303

  57. [57]

      M. Cristofanilli, N.C. Turner, I. Bondarenko, et al., Lancet Oncol. 17 (2016) 425–439. doi: 10.1016/S1470-2045(15)00613-0

  58. [58]

      N.C. Turner, D.J. Slamon, J. Ro, et al., N. Engl. J. Med. 379 (2018) 1926–1936. doi: 10.1056/nejmoa1810527

  59. [59]

      S.E. Nunnery, I.A. Mayer, Drugs 80 (2020) 1685–1697. doi: 10.1007/s40265-020-01394-w

  60. [60]

      P. du Rusquec, C. Blonz, J.S. Frenel, M. Campone, Ther. Adv. Med. Oncol. 12 (2020) 1758835920940939. doi: 10.1177/1758835920940939

  61. [61]

      N. Vasan, E. Toska, M. Scaltriti, Ann. Oncol. 30 (2019) x3–x11. doi: 10.1093/annonc/mdz281

  62. [62]

      E. Geuna, A. Milani, R. Martinello, et al., Expert Opin. Investig. Drugs 24 (2015) 421–431. doi: 10.1517/13543784.2015.1008132

  63. [63]

      J. Baselga, S. -A. Im, H. Iwata, et al., Lancet Oncol. 18 (2017) 904–916. doi: 10.1016/S1470-2045(17)30376-5

  64. [64]

      P. Furet, V. Guagnano, R.A. Fairhurst, et al., Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748. doi: 10.1016/j.bmcl.2013.05.007

  65. [65]

      H.S. Rugo, F. Andre, T. Yamashita, et al., Ann. Oncol. 31 (2020) 1001–1010. doi: 10.1016/j.annonc.2020.05.001

  66. [66]

      S.M. Tolaney, Y.H. Im, E. Calvo, et al., Clin. Cancer Res. 27 (2021) 418–428. doi: 10.1158/1078-0432.ccr-20-0645

  67. [67]

      I.A. Mayer, V.G. Abramson, L. Formisano, et al., Clin. Cancer Res. 23 (2017) 26–34.

  68. [68]

      C.X. Ma, C. Sanchez, F. Gao, et al., Clin. Cancer Res. 22 (2016) 2650–2658. doi: 10.1158/1078-0432.CCR-15-2160

  69. [69]

      C.X. Ma, V. Suman, M.P. Goetz, et al., Clin. Cancer Res. 23 (2017) 6823–6832. doi: 10.1158/1078-0432.CCR-17-1260

  70. [70]

      M. Royce, T. Bachelot, C. Villanueva, et al., JAMA Oncol. 4 (2018) 977–984. doi: 10.1001/jamaoncol.2018.0060

  71. [71]

      H. Tesch, O. Stoetzer, T. Decker, et al., Int. J. Cancer 144 (2019) 877–885. doi: 10.1002/ijc.31738

  72. [72]

      S.M. Guichard, J. Curwen, T. Bihani, et al., Mol. Cancer Ther. 14 (2015) 2508–2518. doi: 10.1158/1535-7163.MCT-15-0365

  73. [73]

      S. Pancholi, M.F. Leal, R. Ribas, et al., Breast Cancer Res. 21 (2019) 135. doi: 10.1186/s13058-019-1222-0

  74. [74]

      J.A. Garcia-Saenz, N. Martinez-Janez, R. Cubedo, et al., Clin. Cancer Res. 28 (2022) 1107–1116. doi: 10.1158/1078-0432.ccr-21-2652

  75. [75]

      R. Tohkayomatee, S. Reabroi, D. Tungmunnithum, et al., Molecules 27 (2022) 248.

  76. [76]

      R. Margueron, V. Duong, A. Castet, V. Cavailles, Biochem. Pharmacol. 68 (2004) 1239–1246. doi: 10.1016/j.bcp.2004.04.031

  77. [77]

      T. Kovács, E. Szabó-Meleg, I.M. Ábrahám, Int. J. Mol. Sci. 21 (2020) 3177. doi: 10.3390/ijms21093177

  78. [78]

      Z. Cui, M. Xie, Z. Wu, Y. Shi, Med. Sci. Monit. 24 (2018) 2456–2464. doi: 10.12659/msm.906576

  79. [79]

      J.M. Craig, T.H. Turner, J.C. Harrell, C.V. Clevenger, Endocrinology 162 (2021) bqab036. doi: 10.1210/endocr/bqab036

  80. [80]

      P. Raha, S. Thomas, K.T. Thurn, et al., Breast Cancer Res. 17 (2015) 26. doi: 10.1186/s13058-015-0533-z

  81. [81]

      C. Tang, C. Li, S. Zhang, et al., J. Med. Chem. 58 (2015) 4550–4572. doi: 10.1021/acs.jmedchem.5b00099

  82. [82]

      G. Luo, X. Lin, S. Ren, et al., Eur. J. Med. Chem. 226 (2021) 113870. doi: 10.1016/j.ejmech.2021.113870

  83. [83]

      S. Xiong, X. Wang, M. Zhu, et al., Bioorg Chem 134 (2023) 106459. doi: 10.1016/j.bioorg.2023.106459

  84. [84]

      P.N. Munster, K.T. Thurn, S. Thomas, et al., Br. J. Cancer 104 (2011) 1828–1835. doi: 10.1038/bjc.2011.156

  85. [85]

      Z. Jiang, W. Li, X. Hu, et al., Lancet Oncol. 20 (2019) 806–815. doi: 10.1016/S1470-2045(19)30164-0

  86. [86]

      L. Xu, W. Jiang, W. Li, et al., Exp. Ther. Med. 24 (2022) 575. doi: 10.3892/etm.2022.11512

  87. [87]

      P.G. Alluri, I.A. Asangani, A.M. Chinnaiyan, Cell. Res. 24 (2014) 899–900. doi: 10.1038/cr.2014.90

  88. [88]

      E. García-Oliver, C. Ramus, J. Perot, et al., PLoS Genet. 13 (2017) e1006541. doi: 10.1371/journal.pgen.1006541

  89. [89]

      A.J. Stonestrom, S.C. Hsu, M.T. Werner, G.A. Blobel, Drug Discov. Today Technol. 19 (2016) 23–28. doi: 10.1016/j.ddtec.2016.05.004

  90. [90]

      Q. Feng, Z. Zhang, M.J. Shea, et al., Cell Res. 24 (2014) 809–819. doi: 10.1038/cr.2014.71

  91. [91]

      J. Hilton, M. Cristea, S. Postel-Vinay, et al., Cancers (Basel) 14 (2022) 4079. doi: 10.3390/cancers14174079

  92. [92]

      S.A. Piha-Paul, J.C. Sachdev, M. Barve, et al., Clin. Cancer Res. 25 (2019) 6309–6319. doi: 10.1158/1078-0432.ccr-19-0578

  93. [93]

      E. Hervouet, P.F. Cartron, M. Jouvenot, R. Delage-Mourroux, Epigenetics 8 (2013) 237–245. doi: 10.4161/epi.23790

  94. [94]

      Y. Shi, F. Lan, C. Matson, et al., Cell 119 (2004) 941–953. doi: 10.1016/j.cell.2004.12.012

  95. [95]

      R.J. Klose, E.M. Kallin, Y. Zhang, Nat. Rev. Genet. 7 (2006) 715–727. doi: 10.1038/nrg1945

  96. [96]

      M.A. Bennesch, G. Segala, D. Wider, D. Picard, Nucleic Acids Res. 44 (2016) 8655–8670. doi: 10.1093/nar/gkw522

  97. [97]

      M. He, W. Ning, Z. Hu, et al., Eur. J. Med. Chem. 195 (2020) 112281. doi: 10.1016/j.ejmech.2020.112281

  98. [98]

      H.P. Dhakal, B. Naume, M. Synnestvedt, et al., Histopathology 61 (2012) 350–364. doi: 10.1111/j.1365-2559.2012.04223.x

  99. [99]

      Z. Madak-Erdogan, M. Lupien, F. Stossi, et al., Mol. Cell. Biol. 31 (2011) 226–236. doi: 10.1128/MCB.00821-10

  100. [100]

       Z. Tang, C. Wu, T. Wang, et al., Eur. J. Med. Chem. 118 (2016) 328–339. doi: 10.1016/j.ejmech.2016.04.029

  101. [101]

       G. Luo, X. Li, G. Zhang, et al., Eur. J. Med. Chem. 140 (2017) 252–273. doi: 10.1016/j.ejmech.2017.09.015

  102. [102]

       X. Li, C. Wu, X. Lin, et al., Eur. J. Med. Chem. 161 (2019) 445–455. doi: 10.1159/000502203

  103. [103]

       M. Jordan, Curr. Med. Chem. Anticancer Agents 2 (2002) 1–17. doi: 10.2174/1568011023353859

  104. [104]

       N.M. O'Boyle, J.K. Pollock, M. Carr, et al., J. Med. Chem. 57 (2014) 9370–9382. doi: 10.1021/jm500670d

  105. [105]

       X. Deng, X. Deng, W. Ning, et al., J. Med. Chem. 66 (2023) 11094–11117. doi: 10.1021/acs.jmedchem.3c00465

  106. [106]

       W.M. Swetzig, J. Wang, G.M. Das, Oncotarget 7 (2016) 16049–16069. doi: 10.18632/oncotarget.7533

  107. [107]

       N. Portman, H.H. Milioli, S. Alexandrou, et al., Breast Cancer Res. 22 (2020) 87. doi: 10.1186/s13058-020-01318-2

  108. [108]

       F. Acconcia, C.J. Barnes, R. Kumar, Endocrinology. 147 (2006) 1203–1212. doi: 10.1210/en.2005-1293

  109. [109]

       L.F. Hennequin, J. Allen, J. Breed, et al., J. Med. Chem. 49 (2006) 6465–6488. doi: 10.1021/jm060434q

  110. [110]

       S. Hiscox, N.J. Jordan, C. Smith, et al., Breast Cancer Res. Treat. 115 (2009) 57–67. doi: 10.1007/s10549-008-0058-6

  111. [111]

       Y. Zhou, S. Eppenberger-Castori, U. Eppenberger, C.C. Benz, Endocr. Relat. Cancer 12 Suppl 1 (2005) S37–S46. doi: 10.1677/erc.1.00977

  112. [112]

       S.C. Baumgarten, J. Frasor, Mol. Endocrinol. 26 (2012) 360–371. doi: 10.1210/me.2011-1302

  113. [113]

       W. Ning, Z. Hu, C. Tang, et al., J. Med. Chem. 61 (2018) 8155–8173. doi: 10.1021/acs.jmedchem.8b00224

  114. [114]

       S.K. Kim, J.W. Yang, M.R. Kim, et al., Free Radic. Biol. Med. 45 (2008) 537–546. doi: 10.1016/j.freeradbiomed.2008.05.011

  115. [115]

       A. Ghanem, A.M. Melzer, E. Zaal, et al., Free Radic. Biol. Med. 163 (2021) 196–209. doi: 10.1016/j.freeradbiomed.2020.12.012

  116. [116]

       Z. Wu, J. Wu, Q. Zhao, et al., Clin. Transl. Oncol. 22 (2020) 631–646. doi: 10.1007/s12094-019-02187-8

  117. [117]

       X. Deng, B. Xie, Q. Li, et al., J. Med. Chem. 65 (2022) 7993–8010. doi: 10.1021/acs.jmedchem.2c00525

  118. [118]

       A.K. Rao, Y.S. Ziegler, I.X. McLeod, et al., J. Mol. Endocrinol. 43 (2009) 251–261. doi: 10.1677/JME-09-0053

  119. [119]

       B.J. Deroo, S.C. Hewitt, S.D. Peddada, K.S. Korach, Endocrinology 145 (2004) 5485–5492. doi: 10.1210/en.2004-0471

  120. [120]

       Y. Lu, X. Sheng, C. Liu, et al., Pharmacol. Res. 190 (2023) 106731. doi: 10.1016/j.phrs.2023.106731

  121. [121]

       L. Xin, C. Wang, Y. Cheng, et al., J. Med. Chem. 67 (2024) 8913–8931. doi: 10.1021/acs.jmedchem.4c00196

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  Figure 1  Effects of endocrine therapy on ER signaling.

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  Figure 2  Endocrine therapy drugs approval timeline.

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  Figure 3  Activation of HER2, EGFR, FGFR and other RTKs promotes endocrine resistance.

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  Figure 4  Hormonal regulation of cyclin D1-CDK4/6 axis.

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  Figure 5  Targeting PI3K/AKT/mTOR pathway.

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  Figure 6  Mechanism of action of andrographolide on MCF-7 cells.

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  Figure 7  Cross-talk of HDAC inhibitors and ER ligands. (ⅰ) Effect of HDAC inhibitors on ERα expression and acetylation. (ⅱ) Effect of HDAC inhibitors on ERα transcriptional activity. (ⅲ) Effect of HDAC inhibitors on p21^WAFI/CIP1 expression.

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  Figure 8  Role of BRD protein in breast cancer.

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  Figure 9  Role of compounds 46 and 55 in targeting ERα and VEGFR-2 initiated signaling pathways.

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  Figure 10  Schematic diagram of the feedback loop and signal crosstalk between MDM4, MDM2, ERα and p53.

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  Figure 11  Known intracellular signaling mechanisms upstream and downstream of NF-κB activation.

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  Figure 12  Mechanism diagram of compound 104.

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  Figure 13  Representation of complex 105 dual targeting ER and TrxR with underlying mechanism of ICD effects.

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