ET resistance has been associated with alterations in the ER expression at gene and protein levels or in the expression of CYP19A1 (or ARO1) gene encoding for aromatase. Pre-clinical and clinical evidence supports that ER expression may change during the natural history of disease and under ET exposure, and that, a switch to an ER-negative phenotype results in ET inefficacy. An estimated 20% of ER⁺ BC patients treated with ET lose ER expression over time [3, 12, 60], mainly due to epigenetic and post-transcriptional mechanisms [3, 4, 15, 61]. The first point mutations in the ESR1 gene, which encodes for ERα, were reported two decades ago [62]. However, it was only more recently that these mutations were found to enable hormone-independent ER transcriptional activity, resulting in constitutive ER activity and ET resistance [4, 60, 61, 63–65]. Such mutations mostly occur in the LBD of ESR1 [4, 64, 65] and are detected in 20% of recurrent BCs following long-term treatment with AIs or TAM [63]. Although at a much lower frequency than point mutations, ESR1 amplification and gene fusions have also been reported [63–65].

Next-generation oral SERMs or SERDs (e.g., elacestrant, SAR43985, rintodestrant, ZN-c5, H3B-6545, and ARV-471) are currently in clinical development to target both wild-type and mutant ER as a way to overcome ER mutation-driven resistance to ET [63].

Additionally, ER transcriptional activity can be modulated by different coregulatory proteins and/or transcriptional factors. Coregulatory proteins can be coactivators [e.g., nuclear receptor coactivator 1 (NCoA1), NCoA2, NCoA3] or corepressors [e.g., NCoR1, nuclear receptor subfamily 2 group F member 2 (NR2F2)] and are recruited to bind to ER and respectively enhance or repress the transcriptional activity of specific DNA elements [estrogen response elements (EREs)] located in the promoter regions of different ER target genes. Moreover, transcriptional factors, like AP-1 and SP-1, regulate the transcription of genes that do not contain EREs. Changes in the expression of coregulatory proteins or transcriptional factors critically influence the effectiveness of ET and are also associated with endocrine resistance [3, 6, 12].

Amplification of the CYP19A1 gene is also found in AI-resistant breast tumors (21.5% of AI-treated and < 2% of primary tumors), leading to increased aromatase activity, estrogen availability, and consequent ER signaling [66]. Although irreversibly resistant to AIs, these tumors remain sensitive to FULV, being strong candidates for treatment with SERDs [27, 63].

Overall, any variation in ER expression or signaling may contribute to endocrine resistance and more aggressive phenotypes. The regulation of ER expression is complex and not totally understood as reviewed by Hua et al. [66], requiring further investigation.

Cell cycle and apoptosis regulators play a key role in the proliferation of normal and BC cells. Therefore, it comes with no surprise that deregulation of these proteins is associated with endocrine resistance.

Several studies have shown that the activity of both positive and negative cell cycle regulators can have an impact on BC sensitivity to ET [3, 67, 68]. Increased expression of positive regulators, like c-MYC and cyclins D1 and E1, can activate cyclin-dependent kinases (CDKs), contributing to aberrant cell cycle progression and resistance to ET. Reduced activity of negative cell cycle regulators, like p21 and p27, and inactivation of retinoblastoma (RB) tumor suppressor also enable aberrant cell cycle progression, contributing to endocrine resistance. Additionally, increased expression of anti-apoptotic molecules like BCL-XL and reduced expression of pro-apoptotic molecules like BCL-2-interacting killer and caspase 9 also play a role in ET resistance [3, 4, 12].

Three CDK4/6 inhibitors (CDK4/6i; palbociclib, ribociclib, abemaciclib) are currently approved in combination with standard ET to treat metastatic ER⁺ BC. Clinical studies showed that combined CDK4/6i and letrozole significantly improve progression-free survival (PFS) compared to letrozole alone in advanced ER⁺ BC resulted in the approval of CDK4/6i in combination with an AI in first line treatment of this disease [69–71]. In addition, the combination of a CDK4/6i and FULV has been approved for use following progression on initial AI monotherapy (Table 2, Figures 3 and 4) [20, 72, 73].

The activity of cell cycle and apoptosis regulators can be modulated by tyrosine kinase receptors (TKRs) and transcription factor signaling pathways, with evidence of crosstalk between ER signaling, TKR pathways, cell cycle regulators, and apoptotic molecules towards ET escape.

Comprehensive evidence suggests that endocrine resistance can be driven by increased expression and activity of TKRs and subsequent activation of downstream signaling pathways, such as PI3K/AKT/mTOR and MAPK. Upon aberrant activation of TKRs (most commonly by amplification or mutation), downstream intracellular signal transduction pathways are activated, inducing ER phosphorylation and activation in the absence of estradiol [3, 4, 60, 63].

TKRs are a family of cell membrane receptors with a tyrosine kinase intracellular domain that phosphorylates tyrosine residues of target proteins. They include HER2, EGFR, FGFRs, and IGFR. HER2/ERBB2 amplification has long been known to reduce sensitivity to anti-estrogens in ER⁺/HER2⁺ tumors, with the current standard of care for this BC subtype being a combination of ET and HER2 inhibitors. Treatment of HER2-amplified BC will not be explored, as it is out of the scope of this review but has been recently reviewed by Chow et al. [87]. More recently, HER2-activating mutations have been associated with both intrinsic and acquired ET resistance [88, 89]. Somatic mutations in HER2 are observed in approximately 5% of endocrine-resistant, non-HER2-amplified BCs [90]. Tumors with an incidence of HER2 mutations as high as 10% are further enriched in lobular histology [91]. Combined blockade of HER2 mutations and ER results in synergistic antitumor activity in vitro and in vivo [88, 89], and the combination of neratinib (a tyrosine kinase inhibitor of HER2, EGFR, HER4, and HER3 heterodimers) and FULV has shown promising activity in metastatic ER⁺/HER2-mutated BC [91].

FGFR1–4 constitute a family of four highly conserved TKRs. Deregulation of FGFR has been reported in a variety of human cancers, including BC [92]. FGFR1 amplification is found in approximately 14% of metastatic BCs and is associated with de novo endocrine resistance [93]. FGFR2 genomic and expression alterations can be found in approximately 6% of BCs [94], with FGFR2 associated with endocrine resistance in vitro [95]. Clinical trials combining pan-FGFR inhibitors and ET are ongoing in FGFR1/2-amplified ER⁺/HER2^– metastatic BC. FOENIX-MBC2 TAS-120-201 (NCT04024436) is a phase II trial evaluating the combination of FULV with futibatinib (TAS-120). The triple combination of ET, CDK4/6i, and FGFR inhibitor is also being explored in a phase Ib trial (VICC BRE 16126; NCT03238196). FGFR4 genomic and expression alterations can be found in approximately 7% of BC patients [94]. Recently, FGFR4 overexpression and hotspot mutations have been associated with metastases and endocrine resistance in lobular metastatic BC [96], and a recent study suggested that FGFR4 promotes the transition from a more differentiated luminal phenotype to a highly proliferative and metastatic, endocrine-resistant, HER2-enriched subtype [97]. The combination of ET with an FGFR4-selective inhibitor seems to be an attractive strategy in tumors exhibiting FGFR4 genomic signatures.

The PI3K/AKT/mTOR pathway is one of the most frequently activated pathways in several types of cancer [98] and the link between PI3K/AKT/mTOR pathway deregulation and endocrine resistance in BC is well established [31, 99]. A number of drugs targeting PI3K/AKT/mTOR are currently being investigated in clinical trials in combination with standard therapies as a strategy to overcome acquired resistance in BC. Somatic mutations in PIK3CA are the most common cause of aberrant PI3K/AKT/mTOR pathway activation, prompting the development of various PI3K inhibitors (PI3Ki) with a different selectivity against the four PI3K catalytic subunit isoforms [100]. The selective alpha isoform inhibitor alpelisib (BYL719) was the first oral PI3Ki developed and was recently approved for the treatment of advanced PIK3CA-mutated ER⁺/HER2^– BC progressing on previous ET (Table 2, Figure 4) [85]. AKT is another potential therapeutic target to overcome endocrine resistance, with several AKT inhibitors (AKTi) currently under clinical investigation. The combination of the pan-AKT inhibitor capivasertib (AZD5363) with FULV has been shown to improve PFS in metastatic ER⁺ BC progressing on AIs [101]. This combination may be particularly effective in ER⁺ BC with AKT1 mutations [102, 103]. Notably, it has been recently shown that triple inhibition by FULV, CDK4/6i, and AKTi durably impairs BC cell growth, prevents progression, and reduces metastases of tumor xenografts resistant to the combination of CDK4/6i and FULV or FULV alone [104]. A phase III trial (CAPItello-291; NCT04305496) is currently ongoing to evaluate capivasertib in combination with FULV in metastatic ER⁺ BC following progression on AIs. Furthermore, a phase Ib/III trial is evaluating capivasertib plus palbociclib and FULV in locally advanced, unresectable, or metastatic ER⁺ BC (CAPItello-292; NCT0486266).

Inhibition of mTOR, a downstream target of the PI3K pathway, has also been shown to reduce endocrine resistance. The mTOR complex-1 inhibitor everolimus in combination with exemestane was shown to prolong PFS in advanced ER⁺/HER2^– BC after progression on non-steroidal AIs [82], independently of PIK3CA mutational status [105]. Consistently, patients with AI-resistant metastatic BC have also been shown to obtain clinical benefits from everolimus combined with FULV (Table 2, Figure 4) [83, 84].

RANKL and its receptor RANK belong to the tumor necrosis factor (TNF) superfamily and were identified in the late 1990s as key regulators of osteoclastogenesis [106, 107]. The pivotal role of the RANKL-RANK pathway in bone physiology and pathology led to the development of denosumab, a fully human anti-RANKL monoclonal antibody currently approved for the treatment of bone remodeling diseases, including cancer-induced bone metastases [108, 109].

In the last decade, extensive research stemming from the observation of RANK-expressing RANKL-sensitive cancer cell lines from different tumor types, including BC [110–113], clearly corroborated that the RANKL-RANK pathway is a major mediator of breast physiology and carcinogenesis [107, 114–116] as recently reviewed by our study group [117]. In view of this evidence, inhibition of the RANK pathway emerged as a promising strategy in BC prevention and treatment.

The fact that expansion of RANK-positive mammary basal stem cells is mediated by paracrine RANKL signaling [115] and that RANK-positive BC cases are more common among TNBC subtypes [118–123] established the relevance of the RANKL-RANK pathway mainly in this BC subtype. Accordingly, numerous studies have shown that RANK-expressing TNBC is a more aggressive subtype, as RANKL activates a signaling cascade involving NFκB, AKT (PKB), JNK, extracellular signal-regulated kinase (ERK), proto-oncogene tyrosine-protein kinase Src (Src), and MAPK, increasing migration, invasion, stemness, transformation, epithelial-mesenchymal transition (EMT), anchorage-independent growth, and metastatic ability [117]. It was only recently that our own research [124] and that of Benítez et al. [125] disclosed a link between RANK expression and luminal BC phenotype and carcinogenesis, respectively. In a recent study, our group characterized the phenotype of RANK-overexpressing (RANK OE) luminal BC cell lines, showing that RANK OE cells have a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but are more invasive in vivo [124]. In silico analysis of the transcriptome of human breast tumors confirmed the association between RANK expression and stem cell and mesenchymal markers in ER⁺/HER2^– tumors, leading to the hypothesis that luminal RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistant cancer cells [124].

Prior studies have shown that multiparous mouse mammary tumor virus (MMTV)-RANK mice, with RANK OE in the mammary gland under MMTV, develop spontaneous breast tumors with long latency and only after multiple pregnancies [126], although tumor latency decreases and tumor incidence increases compared to wild-type after carcinogenic protocols [22, 115]. Genetic or pharmacological inhibition of RANK signaling abrogated carcinogenesis in this model and also delayed tumor onset and decreased tumor and metastases incidence in HER2⁺ or polyomavirus-induced BC, MMTV-Neu, and MMTV-polyomavirus middle T antigen (PyMT) mice [127, 128]. In these models, RANK was focally expressed in non-transformed mammary glands but increased in mammary pre-neoplastic lesions and invasive adenocarcinomas. The long latency of MMTV-RANK carcinogenesis was recently found to be due to RANK-driven senescence, which initially delays tumor onset in oncogene-driven models but promotes stemness, luminal-like tumor growth, and metastases in later stages of tumor progression [125, 128]. In accordance with these findings, transcriptomic analysis of human breast tumors from luminal subtype linked RANK expression to senescence [125].

The work of our group has also disclosed that RANK OE cells are more resistant to FULV compared to RANK-low counterparts [124]. This suggests that the RANK pathway may be implicated in intrinsic ET resistance. Interestingly, continuous pathway activation by prolonged RANKL exposure led to a decrease in proliferation and down-regulated ER and PR, with an increase in FULV resistance. This suggests that RANK signaling may also be associated with the induction of acquired resistance to ET due to ER loss. Activation of the RANK signaling pathway leads to an increase in noncanonical NFκB signaling, which plays a key role in the above-mentioned regulation of proliferation of mammary epithelial cells—through the RANKL–RANK–NFκB inhibitor (IκB) kinase α (KKα; IKKα)–IκBα–p50/p65–cyclin D1 axis [129]. Unlike the canonical NFκB pathway that responds rapidly to signals from different receptors, the noncanonical NFκB pathway specifically responds to a small group of receptors, namely lymphotoxin-β receptor (LTβR), B-cell-activating factor receptor (BAFFR) belonging to the TNF family receptor, CD40, and RANK [130].

The RANK–NFκB axis has been associated with increased resistance to the anti-HER2 therapy lapatinib in RANK-positive HER2⁺ BC cells [131]. Moreover, although several signaling pathways crosstalk due to activation of different receptors, lapatinib was not able to counteract RANK-induced NFκB activation in this study. Conversely, it was previously reported that NFκB inhibitors abrogate RANKL-induced EMT, cell migration, and invasion [132].

It is widely accepted and clearly demonstrated that multiple mechanisms are involved in the crosstalk between NFκB and ER. Accordingly, there is compelling evidence of a role for the NFκB pathway in ET resistance, indicating that RANK-mediated NFκB signaling may contribute to ET resistance. This suggests that it may be of particular interest to further study the mechanism of RANK-mediated ET resistance and investigate the efficacy of RANK pathway inhibition—either at the RANKL level or via inhibition of downstream mediators like NFκB—as a mechanism to overcome it (Figure 5).

The NFκB family comprises five inducible transcription factors p65(RelA), RelB, cRel, NFκB1 or p50, and NFκB2 or p52, all of which play critical roles in cell proliferation and survival and inflammatory and immune responses [133–136], being crucial for normal organ development, including of the mammary gland [137].

A growing body of evidence indicates abnormal activation of the NFκB pathway in multiple malignancies, suggesting a putative role for NFκB in tumorigenesis [134, 138–141] and chemotherapy resistance [142].

Members of the NFκB family have a conserved N-terminal Rel homology domain (RHD) [143] that is responsible for dimerization, nuclear translocation, DNA binding, and interaction with IκB. This family of molecules includes multiple proteins, among which IκBα, IκBβ, and IκBε, are the most important NFκB regulators. NFκB molecules exist either as homo or heterodimers, but the most abundant intracellular form is the p50(NFκB1)/p65(RelA) heterodimer. On unstimulated cells, NFκB homo or heterodimers are hijacked in the cell cytoplasm, binding to their IκB inhibitor [143]. Activation of the NFκB cascade occurs either through canonical or noncanonical pathways [144, 145].

The canonical pathway is activated following stimulation by inflammatory cytokines [interleukin-1 (IL-1), lipopolysaccharide (LPS), TNFα, T- and B-cell mitogens], growth factors, viral proteins, and extracellular physical and chemical stress [146, 147]. These ligands activate their receptors and initiate a downstream cascade that ultimately leads to phosphorylation and subsequent ubiquitination of IκBα by a trimeric IKK complex [IKKα, IKKβ, and IKKγ or NFκB essential modulator (NEMO)], enabling nuclear translocation of the p50/p65 heterodimer. Upon translocation, the complex interacts with κB sites to accelerate the transcription of target genes [144, 145, 148–150].

Conversely, in the noncanonical pathway, NFκB-inducing kinase (NIK) phosphorylates and activates IKKα in response to signals from a small group of receptors, including the RANK [151]. Activated KKα then phosphorylates p100, resulting in p52 liberation and promoting p52/RelB nuclear translocation [152, 153].

Contrarily to p50/RelA activation in the canonical pathway, the role of p52/RelB in therapy resistance remains poorly understood. Nevertheless, RelB expression was found to be high in ER⁺ BC cells [154], and inhibition of p52/RelB was shown to be able to reverse ER expression [155].

Regarding ET resistance, comprehensive evidence shows that NFκB regulates a series of genes relevant to endocrine resistance [156–158]. In fact, there seems to be a reciprocal interaction between endocrine treatments and the immune system. Inflammation cytokines like IL-1 and TNFα are higher in metastatic BC patients, where they seem to activate the NFκB pathway and lead to endocrine resistance. Antibodies directed against transcription factors upstream of this pathway have restored sensitivity in cell line models of endocrine-resistant BC [4]. There is also supporting evidence that a dysregulated immune response or excessive inflammation in the tumor microenvironment (TME) could be related to endocrine resistance and could promote BC progression and metastasis [159, 160]. Downstream NFκB-regulated proteins, like BCL-2, cyclin D1, and cytokines IL-6 and IL-8, have been shown to be major players in this process [148, 154]. Additionally, previous studies documented that estrogen withdrawal led to the increased transcriptional activity of p65(RelA) and sustained estrogen-independent tumor growth through upregulation of cyclin D1 and BCL-3 [157]. Oida et al. [161] focused on the role of NFκB in hormone dependency in BC and showed that NFκB inhibition enhanced ER expression and promoted recovery of TAM sensitivity in ER-reduced cell sublines. Another study by Nehra et al. [162] reported p65(RelA) inhibition, either by overexpression of mutant IκB or by synergistic restoration of sensitivity to TAM by the small-molecule NFκB inhibitor parthenolide in resistant MCF-7 cell lines, along with decreased BCL-2 expression and induced caspase-dependent apoptotic cell death in resistant cells. Notably, all effects could be reversed by caspase-8 specific inhibition. In TAM-resistant BC cells, Zhou et al. [134] described the increased transcriptional activity of NFκB and AP-1, which was suppressed after treatment with parthenolide or the proteasome inhibitor bortezomib. Additionally, increased p50/NFκB1, p52/NFκB2, and cRel expression were observed in breast tumors compared with normal surrounding tissues [163].

Owing to the possible putative role of NFκB in BC, targeting the NFκB pathway might be a successful therapeutic strategy. Several inhibitors have been reported to date, still in early developmental stages (Figure 6).

Most of these inhibitors prevent proteasomal degradation of IκB proteins, resulting in NFκB hijacking in the cytoplasm. To accomplish this, the drug has either to inhibit IκB function (e.g., proteasome inhibitors), phosphorylation of IκB proteins (e.g., parthenolide), or IKKα and IKKβ. Selective IKKβ inhibitors in particular have shown exciting results in preclinical models, but also important toxicities, precluding their use in clinical practice [164]. Another drug class that can block NFκB activity is the class of nuclear translocation inhibitors. Regardless of the mechanism, the use of NFκB inhibitors in BC endocrine resistance may help elucidate target drivers of this phenomenon and contribute to restoring ET sensitivity.

It is acknowledged that BC subtypes have different clinical outcomes due to different biological and cellular mechanisms involved in tumor aggressiveness, metastases formation, and treatment response [165]. Increased Notch activity and/or Notch deregulation lead to the transformation of normal breast cells into cancer cells, with differential Notch ligand activation potentially associated with oncogenesis and progression of particular BC subtypes and poor clinical outcomes (Table 3) [166, 167].

The Notch signaling pathway is mediated by one of four Notch receptors (Notch1–4). While Notch1 and Notch2 are ubiquitously expressed throughout development and in adult life, Notch3 and Notch4 are more abundant in vasculature cell subtypes. Notch1 and Notch2 knockouts are embryonically lethal due to multiple organ defects, while Notch3 and Notch4 knockouts are viable but display subtle vascular abnormalities [147]. Five Notch ligands are recognized in mammals: delta-like ligands 1 (DLL1), DLL3, DLL4, and Jagged 1 (JAG1) and JAG2 [166].

Depending on the cancer type, the Notch pathway can be hyper- or hypo-activated, which relies on specific Notch receptors. In some organs, both pathway activation and repression have been observed, depending on the tumor subtype or model system in question [168].

Overexpression of Notch1 and/or JAG1 have been associated with poor overall survival, supporting the role of Notch as a prognostic biomarker in BC [167]. While Notch gain-of-function mutations are present in a limited number of BCs, Notch is overexpressed, activated, and crosstalks with other oncogenic pathways in several breast tumors [165].

Some of the earliest known targets of Notch signaling include transcriptional repressors, such as the hairy/enhancer of split (HES) genes, the HES subfamily members HEY1, HEY2, and HEYL, c-Myc, and cyclin D1 (Figure 7) [167, 168].

The Notch pathway is involved in breast tumorigenesis, enhanced BC stem cell (BCSC) self-renewal and proliferation, promotion of EMT, angiogenesis, and immunomodulation [168, 169]. However, depending on the BC subtype and/or context, Notch can have an oncogenic or tumor suppressor role [166]. Dysregulation of Notch signaling, namely through activating Notch receptor activating mutations, overexpression of ligands and/or receptors, and/or overexpression of target genes, contributes to increased proliferation, cell transformation, and drug resistance in tumors such as breast, multiple myeloma, prostate, T-cell acute lymphoblastic leukemia, among others [167].

Cumulative evidence indicates that cancer stem cells (CSCs) are key drivers of acquired endocrine resistance in ERα⁺ breast tumors. Endocrine-resistant BC shows an increase in BCSCs with Notch3/4 expression [165].

Estradiol has been shown to inhibit Notch activity by affecting the receptor cellular location, and TAM and raloxifene have been shown to block this effect, thereby activating the Notch pathway. Therefore, the Notch pathway can be hyperactivated in resistant BC cells, and this can be abrogated by blocking this pathway [166]. Taken together, this provides a strong rationale for studies combining Notch inhibitors with current BC treatment modalities [165].

Interaction between cellular components of the TME and BC cells also regulates Notch signaling-driven therapeutic resistance in breast tumors. Different cellular components of the TME can induce CSC survival, stemness, and resistance through either transforming growth factor beta (TGF-β)-dependent mechanisms or by releasing soluble factors such as cytokines, chemokines, and growth factors that favor angiogenesis and an immunosuppressive environment. In turn, all these factors augment Notch ligand- and receptor-mediated chemoresistance, endocrine resistance, and radioresistance in breast tumors. Additionally, cancer-associated fibroblasts and tumor-associated macrophages can collaborate via cell-cell interaction to promote endocrine resistance, with Notch signaling potentially contributing to the crosstalk between these two cell types [166].