Nuclear Mechanisms Involved in Endocrine Resistance

Endocrine therapy is a standard treatment offered to patients with ERα (estrogen receptor α)-positive breast cancer. In endocrine therapy, ERα is either directly targeted by anti-estrogens or indirectly by aromatase inhibitors which cause estrogen deficiency. Resistance to these drugs (endocrine resistance) compromises the efficiency of this treatment and requires additional measures. Endocrine resistance is often caused by deregulation of the PI3K/AKT/mTOR pathway and/or cyclin-dependent kinase 4 and 6 activities allowing inhibitors of these factors to be used clinically to counteract endocrine resistance. The nuclear mechanisms involved in endocrine resistance are beginning to emerge. Exploring these mechanisms may reveal additional druggable targets, which could help to further improve patients’ outcome in an endocrine resistance setting. This review intends to summarize our current knowledge on the nuclear mechanisms linked to endocrine resistance.


INTRODUCTION
Breast cancer (BC), a systemic disease characterized by early tumor cell dissemination (1), is the most frequent cancer among women and leading cause of cancer-related death in women worldwide (2). Disseminated BC cells often enter dormancy and may later grow out to a metastatic lesion (3,4). In a metastasis-free state, there are good therapy options to substantially prolong survival of BC patients. BC is a heterogenous disease, requiring subtyping, classically based on immunohistochemistry (IHC), to offer the patient the best possible treatment. The statuses of estrogen receptor a (ERa), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) are routinely examined. The majority of BCs are ERa/PR-positive. Additionally, Her2-positive BCs and triplenegative (ERa-, PR-and Her2-negative) BCs (TNBCs) are found. Subtyping by mRNA expression profiling revealed four major BC subtypes (luminal A, luminal B, Her2-enriched and basal-like) (5), which overlap with the IHC-subtypes. Luminal A and B tumors are mostly ERa-positive BCs, whereby luminal B tumors are more aggressive. Basal-like BCs show commonly features of TNBCs.
Routine treatment options for BC patients include ERa-and Her2-targeting therapies, chemotherapy, surgery and radiation. Besides ERa and Her2 expression, the luminal subtype, tumor grading and lymph node involvement play a role in therapy decision (6). Endocrine therapy is a standard treatment for patients with ERa-positive BCs. Two principal strategies are used in endocrine therapy to block estrogen-dependent ERa activity. One strategy utilizes anti-estrogens to compete with estrogen for binding to the ERa protein. Anti-estrogens are roughly divided in selective ERa modulators (SERMs), such as tamoxifen (TAM) (7,8), and selective ERa downregulator (SERDs), such as fulvestrant (FULV) (9,10). In contrast to SERMs, SERDs are pure ERa inhibitors, induce ERa degradation and prevent ERa from becoming transcriptionally active (9)(10)(11). In the other strategy, estrogen synthesis is blocked by an aromatase inhibitor (AI), such as exemestane, resulting in estrogen deficiency (12). Both strategies are effective for treating ERa-positive BCs.
Endocrine resistance (ENDO-R), the resistance to ERatargeting therapy, is a major obstacle in treatment of ERapositive BCs. In first-line treatment, ENDO-R is observed in approximately half of all ERa-positive BCs (13). Many factors contributing to ENDO-R have been identified. While there are excellent reviews on the mechanisms of endocrine resistance, which primarily focus on signaling pathways, cell cycle regulators, microRNAs and/or mutation in the ERa-coding gene esr1 (14)(15)(16)(17)(18)(19), this review preferentially aims to summarize the currently known nuclear mechanisms that contribute to ENDO-R. Where necessary, event(s) in other cellular compartments that are crucially linked to the nuclear mechanism discussed, are also described.

ERa, THE TARGET OF ENDOCRINE TREATMENT The ERa Protein
There are two estrogen receptors, ERa and ERb (20). While ERb is generally considered to act anti-proliferative, ERa promotes proliferation. Estrogen-activated ERa is a potent stimulator of cyclin D1 expression (21,22), leading to activation of cyclindependent kinases (CDKs) 4 and 6, which in turn phosphorylate retinoblastoma protein to initiate cell cycle entry (23).
Expression of ERa is regulated by transcription of its gene estrogen receptor 1 (esr1) and by proteasome-dependent degradation of the ERa protein (24). Primarily, ERa acts as a transcription factor on estrogen-responsive element (ERE)containing genes by directly binding to its recognition sequence. It is also possible that ERa binds indirectly to DNA by tethering to other transcription factors, such as activating protein-1 (AP-1) (25,26). Two trans-activation domains, transactivation function (AF)-1 and AF-2, allow ERa to interact with the transcriptional machinery, whereby AF-2 mediates estrogen-dependent ERa transcriptional activity (27). Two splice variants of ERa, ERa46 and ERa36, exist, whereby ERa46 does not contain the AF-1 domain and ERa36 lacks both transactivation domains.
The ERa protein can be phosphorylated at many sites, which has an impact on its activity (27). Particularly important are phosphorylations at Ser-118 and Ser-167 in the AF-1 domain. These modifications, which promote ligand-dependent as well as ligand-independent transcriptional activities of ERa, affect the interaction of ERa with transcriptional co-factors, such as CREB (cAMP regulatory element binding protein)-binding protein (CBP) or steroid receptor co-activator (SRC). Phosphorylation at these sites can be triggered by receptor tyrosine kinases (RTKs) through the PI3K/AKT/mTOR/p70S6K and the Ras/Raf/MEK1/ ERK1/2 pathways.
Besides genomic activities, non-genomic activities of ERa have been documented, which leads to the activation of the PI3K/AKT/mTOR/p70S6K and the Ras/Raf/MEK1/ERK1/2 pathways (27). These activities may involve interactions of ERa with PI3K and the non-receptor tyrosine kinase c-Src.

Role of ERa in Endocrine Resistance
Given that ERa is the key transcriptional driver in ERa-positive BC cells, it is not surprising that ERa inhibitors have a tremendous effect on transcription. Exposure of MCF-7 cells to anti-estrogens leads to altered expression of approximately twothirds of 1.8 x 10 4 studied genes (28). Though TAM or FULV induce similar changes in gene expression, it takes different strategies to overcome the inhibitory actions of the two antiestrogens. In the presence of TAM, ERa can still be active in an estrogen-independent manner allowing ERa-based escape mechanisms. Indeed, one study showed that ERa was transcriptionally active in approximately three quarters of BC specimens from patients who relapsed on TAM (29). ERa-based escape mechanisms in TAM resistance include phosphorylation of the ERa protein, overexpression of ERa co-activators, such as SRC-1, a switch to AP-1-responsive gene activation and a shift from genomic to non-genomic ERa activities (25,30). Nevertheless, as shown with MCF-7 cells, TAM resistance coincides with an altered chromatin organization (31). In addition, hundreds of genes are differently expressed in tamoxifen resistant (TAM-R) cells as compared to estrogentreated parental cells.
Like TAM resistance, resistance to AI often occurs with the ERa protein remaining active. A frequent escape mechanism involves a mutation in the AF-2 domain allowing constitutive ERa activation in the absence of estrogen (16,18).
As pure ERa antagonists, SERDs block ERa activity completely (10), requiring cells to find escape routes independent of ERa usage. In fact, in clinical samples, FULV resistance is associated with decreased ERa pathway activity (32). Also, in contrast to TAM-R MCF-7 cells, fulvestrant resistant (FULV-R) MCF-7 cells show almost no response of ERa-regulated genes to estrogen (33). Furthermore, TAM-R sublines are usually sensitive to FULV (34). Nevertheless, in some cases, TAM resistance may be accompanied by FULV resistance (34) suggesting that TAM resistance can also be achieved by ERa-independent mechanisms.
Pre-existing vs. Acquired Endocrine Resistance in Established BC Cell Lines Drug resistance can either happen when cells in the tumor preexist that are intrinsically insensitive to the drug or when cells acquire resistance during treatment. Given the heterogenous nature of tumors (35,36), it is not unlikely that drug-resistant cell clones have spontaneously developed during clonal evolution (37) without having been challenged by a particular drug. Such pre-existing drug-resistant clones would be expected to allow the cancer to rapidly progress under treatment pressure. In fact, a study on patients with ERa-positive advanced BC treated with FULV and the CDK4/6 inhibitor palbociclib revealed that cancers with pre-existing escape mutations reduced progression free-survival significantly and showed "no need" to develop additional escape mutations (38).
Most of our knowledge on mechanisms underlying antiestrogen resistance has come from studies with established BC lines, predominantly MCF-7, T47D and ZR75-1. These cell lines have been established from pleural effusions of metastatic BC patients (39). Their ERa chromatin binding profiles overlap with those of primary BCs with poor outcome confirming the aggressive nature of these cell lines (40). Numerous studies demonstrated that the MCF-7 cell line is a heterogenous population (41)(42)(43)(44)(45), which, when challenged by anti-estrogens, form multiple FULV-R and TAM-R clones, all containing the same DNA aberrations (46). This suggests that the resistant clones all derived from one subpopulation of cells that preexisted in the MCF-7 cell line (46). It would explain why FULV-R clones appear rapidly (within a couple of weeks) when MCF-7 cells are exposed to FULV (45). Importantly, at the time when MCF-7 or other commonly used BC lines were established, endocrine therapy was not available (40). Hence, it is likely that established BC cell lines contain cells that spontaneously became endocrine resistant in the absence of endocrine treatment before the tumor cells have been collected from the patient decades ago. This should be taken into consideration when interpreting the results obtained in resistance studies with established BC cell lines.

Current Targets for Therapy in Endocrine Resistance
The PI3K/AKT/mTOR pathway has become a major focus in ENDO-R research and has stimulated the development of drugs that target this pathway (47,48). PI3K and mTOR inhibitors have been found to be effective drugs to treat patients with an endocrine resistant BC (49,50). More recently, CDK4 and CDK6 have been shown to be appropriate druggable targets in ENDO-R (17,51). Combinatorial treatments with drugs directed to the PI3K/AKT/mTOR pathway and to CDK4/6 are discussed to further improve treatment efficacy (52).
As the second major pathway that is activated by RTKs, the Ras/Raf/MEK/ERK1/2 pathway also contributes to ENDO-R ( Figure 1). Independent of RTKs, this pathway can also be activated by mutations in Ras, Raf or MEK or by downregulation of the Ras inhibitor neurofibromatosis type 1 (NF1) (61).
Dual CDK4/6 inhibitors, such as palbociclib (PD-0332991), in combination with endocrine therapy are currently standard of care for advanced ERa-positive breast cancer (51,(65)(66)(67)(68). Activation of CDK4/6 requires physical interaction with their co-factor cyclin D1 (69), whose level raises upon activation of certain proteins, such as RTKs or ERa (70). High expression of cyclin D1 is associated with poor prognosis in ERa-positive breast cancer (71) and linked to an increased risk of relapse on TAM (72). In FULV resistance, of the two CDKs particularly CDK6 may play a role. Increased expression of CDK6 was reported in FULV-treated MCF-7 cells (73,74). Inhibition of CDK6 suppressed growth of FULV-resistant MCF-7 cells. A high CDK6 level in breast cancer of FULV-treated metastatic patients was found to predict a worse outcome (74).
Deciphering the changes happening in the nucleus upon acquisition of ENDO-R may result in the identification of additional druggable factors in ENDO-R.

TRANSCRIPTION FACTORS
Sequence-specific transcription factors (TFs) are key drivers of gene expression and can have activating or repressive functions. Activating TFs induce gene transcription by binding to promoters and/or enhancers followed by recruitments of coactivators and RNA polymerase (75). Two major types of activating TFs are distinguished: pioneer and settler TFs (76). Pioneer TFs assist loading of settler TFs by initiating chromatin accessibility (Chromatin Accessibility). Both types of TFs are involved in ENDO-R ( Figure 2). They may act as effectors of signaling pathways involved in ENDO-R and/or may reprogram cells from ERa-dependent to ER-independent gene expression.

AP-1
ATF2 and c-Jun are members of the AP-1 family of transcription factors and often form heterodimers (77,78). Impairment of ERa activity can lead to a shift from ERE-dependent to AP-1dependent ERa-induced transcription, involving c-Jun (79). Interestingly, c-Jun activity can be regulated by RTKs, partly through the Ras/Raf/MEK/ERK1/2 and the PI3K/AKT signaling pathways (80) linking c-Jun to RTK-induced ENDO-R. Another study showed that silencing of ATF2 in FULV-R and TAM-R MCF-7 sublines strongly decreased ERa-independent cellular growth and concomitantly increased the level of ERa and the expression of ERa-responsive genes (81). On the other hand, ATF-2 and c-Jun can have opposite effects on genes. For instance, while c-Jun represses, ATF-2 activates PTEN transcription in BC cells, leading to opposing effects of the two AP-1 members on AKT activity (82).
A screen in a chemical library resulted in the discovery of two ATF-2 inhibitors, celastrol (CSL) and acetyl isogambogic acid (AIGA), which both proved to be potent inhibitors of melanoma growth (83). CSL, known as an anti-inflammatory drug, could also be shown to counteract cis-platin resistance of non-small cell lung cancer by inhibiting ATF-2 (84).

E74-Like Factor 5
Elf5, also known as epithelium-specific Ets transcription factor 2 (ESE2), a member of the E26-transformation-specific/E-twentysix-specific sequence (ETS) domain family of transcription factors (85), plays a role in BC progression (86). Elf5 is highly expressed in basal-like BCs, while its expression in luminal BCs is lower than in normal breast tissue (87). However, resistance of MCF-7C cells to FULV and TAM coincides with an increase in Elf5 expression. Ectopic expression of Elf5 in MCF-7 and T47D cells was found to down-regulate ERa and FoxA1 levels and to suppress the expression of ERa-driven genes. Additionally, it induces a gene signature resembling that of basal-like BC cells. It is thought that the Elf5-induced switch from a luminal to a basallike subtype may be one route for ERa-positive BC cells to escape the growth-suppressing effects of anti-estrogens. FIGURE 1 | Mechanisms that induce ENDO-R by deregulation of the PI3K/AKT/mTOR and/or Ras/Raf/MEK/ERK1/2 pathways, two pathways that not only stimulate proliferation by raising cyclin D expression and thereby activating CDK4/6 but also promote survival. A common mechanism involves a higher activity of certain RTKs. This includes higher activities of Her proteins, induced by higher availability of ligands, such as EGF or HRG, or by gain-of-function mutation (Her2). FGFR1 is often amplified (amp) in ENDO-R and requires co-factors FGFR substrate 2 (FRS2) and phospholipase C-g; (PLC-g) to activate the two pathways. IR or IGF1R may contribute to ENDO-R if activated by insulin or IGFs. Higher IGF availability can be achieved by reduced expression of IGF binding proteins (IGFBPs). The expression of the IR/IGF1R co-factor insulin receptor substrate (IRS) may also play a role in ENDO-R. RTK-independent activation of PI3K/AKT/mTOR pathway is commonly caused by a gain-of-function mutation of the gene pik3ca coding for p110a, which together with p85a forms the PI3Ka complex. Dysfunction of PTEN, which prevents AKT activation by blocking the formation of phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) is another way by which this pathway can be upregulated. RTK-independent activation of the Ras/Raf/MEK/ERK1/2 pathway in ENDO-R include gain-of-function mutations in ras, raf or mek-encoding genes as well as dysfunction of NF1, an inhibitor of Ras. Arrows indicate positive, T-shaped symbols negative effects. A green or red star denotes a gain-of function or a loss-offunction mutation/deletion, respectively.

Estrogen-Related Receptor-a
ERRa is an orphan nuclear receptor that shows a high homology to ERa in the DNA binding domain, potentially allowing ERRa to activate ERa-target genes in the absence of estrogen (88). Higher levels of ERRa and lower levels of ERa were found in BC specimens from patients who relapsed on TAM compared to BC specimens from untreated patients (29). Furthermore, in TAM-R and FULV-R MCF-7 cells, ERRa expression is increased, while silencing of ERRa has a stronger inhibitory effect on growth of resistant sublines than it does on the growth of the parental cell line. Moreover, high ERRa expression predicts poor prognosis for TAM-treated patients (29) ( Table 1).
The potential role of ERRa in diabetes has encouraged the development of ERRa inhibitors (102). Specific ERRa-targeted drugs have been generated by preventing the recruitment of the co-activator SRC to the ERRa protein. These drugs were proven to have little effects on ERRa relatives ERRb and ERRg, did not influence ERa activity and were well tolerated when administered to rats. Such inhibitors were also shown to act anti-proliferatively on breast cancer cells in vitro and in vivo (103). They may be potentially useful for treating ERRa-induced ENDO-R.

Forkhead Box Protein A1
Expression of FoxA1 correlates with ERa expression in primary ERa-positive BCs (104). As a pioneer TF, FoxA1 facilitates ERa binding to promoters and enhancers and cooperates with ERa to drive ERa-dependent transcription (105). Most of the ERa binding takes place outside of proximal promoters (106), coinciding with enhanced gene looping allowing recruitment of distal regulatory transcriptional machinery (31). Silencing of FoxA1 results in failure of estrogen to stimulate growth of MCF-7 or ZR75-1 cells, confirming the essential role of FoxA1 in ERa function (105).
Overexpression of FoxA1 leads to transcriptional reprogramming mainly based on higher FoxA1 occupation of so-called super enhancers (107). Super enhancers are clusters of enhancers densely occupied with transcription factors and located in the vicinity of genes important for cell identity (108). Ectopic expression of FoxA1 desensitizes MCF-7 cells to FULV and TAM (92). Furthermore, FoxA1 was found to be overexpressed in TAM-R sublines derived from MCF-7 and BT474 (92), though TAM resistance of MCF-7 cells may also coincide with a lower FoxA1 level (109). FoxA1 is frequently overexpressed in primary BC, which happens more often in luminal B than luminal A tumors (92).
There are contradicting results in terms of the predictive value of FoxA1 overexpression for TAM-treated patients. While higher FoxA1 mRNA levels correlated with poor survival (92), FoxA1 protein overexpression was associated with favorable outcome (93). In 3.7% of primary BCs and even in 7% of lobular BC the FoxA1 gene is mutated (110). These mutations were found to be associated with higher FoxA1 expression and activity.
Among the genes targeted by FoxA1 is anterior gradient 2 (AGR2), the human homologue of XAG-2, a Xenopus laevis protein playing a potential role in neural development (111). AGR2 is a protein disulfide isomerase and involved in protein maturation control in the endoplasmic reticulum (112). In murine mammapoiesis, AGR2 regulates epithelial proliferation and lobuloalveolar development (113). AGR2 is able to upregulate the EGFR ligand amphiregulin (114) and the expression of cyclin D1 (115), being consistent with the finding that, in primary BC, the level of cyclin D1 correlates with that of AGR2 (116).
As shown with MCF-7 cells, AGR2 is important for ERadriven proliferation (112,(116)(117)(118)(119)(120). AGR2-overexpressing MCF-7 cells show a delay in FULV-induced ERa degradation, likely caused by physical interaction of the AGR2 with the ERa protein (121). Silencing of AGR2 increased the sensitivity of ZR75-1 and T47D cells to FULV and TAM, reduced c-Src kinase activity and decreased the level of the anti-apoptotic protein survivin (115). In TAM-R MCF-7 cells, AGR2 is highly expressed while being mainly regulated by FoxA1 independently of ERa (117). If secreted, AGR2 can bind to the membrane receptor LY6/PLAUR domain containing 3 (LYPD3), whose expression is regulated by FoxA1 in cooperation with the transcription factor grainyhead like transcription factor 2 (GRHL2) (122). There is evidence that AGR2, LYPD3, GRHL2 and FoxA1 act together to foster ENDO-R.
Higher AGR2 expression is associated with unfavorable prognosis in BC (117). This holds true also for ERa-positive BC (116), where AGR2 is more abundant (112,123). Furthermore, higher AGR2 expression predicts a weaker response to TAM in primary BC (116,122).
Antibodies against AGR2 and LYPD3 have been found to be effective to suppress growth of TAM-R breast cancer cells in mice (122). Additionally, humanized anti-AGR2 and anti-LYPD3 antibodies are in development. In a pre-clinical trial, an anti-LYPD3 antibody-auristatin conjugate (BAY 1129980) is tested for treatment of LYPD3-expressing non-small cell lung cancer (124).

FoxM1
The FoxM1 gene is transcriptionally regulated by ERa and is important for ERa-driven cellular growth (125). Accordingly, FULV and TAM reduce FoxM1 expression. However, long-term treatment with TAM increases FoxM1 expression in MCF-7 cells (126), while FoxM1 depletion sensitizes TAM-R MCF-7 cells to TAM (125). Among the genes upregulated by FoxM1 are cyclin D1 and ATP-binding cassette super-family G member 2 (ABCG2) (125,126). ABCG2, a transporter protein that pumps drugs out of the cell (127), was found to contribute to anti-estrogen resistance (126). Many genes, including ABCG2, require active ERK2 for FoxM1-dependent transcription linking FoxM1 transcriptional activity to the Ras/Raf/MEK/ERK1/2 pathway. FoxM1 may also be connected to the PI3K/AKT pathway, as overexpression of activated AKT can increase FoxM1 expression (128).
FoxM1 may be suitable as a predictive marker in ENDO-R. Overexpression of FoxM1 in ERa-positive breast cancer was found to correlate with worse prognosis of TAM-treated patients (94,126). Interestingly, a gene signature linked to the protein 14-3-3z, a FoxM1 regulator, is also associated with unfavorable prognosis of TAM-treated patients (129) suggesting that a 14-3-3z-FoxM1 axis can drive ENDO-R.
FoxM1 might be targeted through 14-3-3z, whose activity can be inhibited by small molecules, such as FOBISIN101, or by the peptide inhibitor R18 (130). R18 was found to strongly support the apoptotic effect of TAM on MCF-7 cells (131).

Hypoxia-Inducible Factor 1/2a
Hypoxia stabilizes HIF-1a and HIF-2a proteins allowing them to initiate transcription of numerous genes engaged to ensure survival under hypoxic conditions (132). In cancer, also nonphysiological activation of these transcription factors occur (133). HIF-1a and HIF-2a are involved in tumor progression (134). Among others, they promote metastasis and cancer stem cell activity (Cancer Stem Cells).
Overexpression of HIF-1a or HIF-2a was found to desensitize MCF-7 cells to FULV (135,136). Likewise, exposure to hypoxia reduced ERa expression and FULV sensitivity of various ERapositive breast cancer cell lines (136,137). Also, FULV-R MCF-7 cells showed higher expression of HIF-2a, but not HIF-1a, and could be sensitized to FULV by inhibition of HIF activity.
Interestingly, HIF and ERa share many genes that they can transcriptionally activate (138). Of these, sodium-dependent neutral amino acid transporter 2 (SNAT2) has been linked to FULV resistance. SNAT2 is a transmembrane transporter for short chain neutral amino acids, such as glutamine, and an amino acid sensor (139,140). When overexpressed in MCF-7 cells, SNAT2 induces FULV resistance in vitro and in vivo (138). FULV-R or TAM-R MCF-7 cells can use glutamine instead of glucose for maintaining metabolism (141), which may play a role in SNAT's ability to induce FULV resistance. SNAT2 overexpression was associated with worse outcome in luminal B-type, but not in luminal A-type cancers (138).
The activation of HIF also leads to a disconnect between glycolysis and the tricarboxylic acid cycle, whose maintenance becomes then dependent on glutamate (142). Hence, when HIF is activated, glutamine metabolismus is gaining importance in cancer's energy generation. Therefore, endocrine resistant breast cancer with high HIF activity may be responsive to drugs interfering with glutamine metabolism. A promising druggable target is glutaminase (GLS) which converts glutamine to glutamate (143). The GLS-inhibiting drug CB-839 is now tested in clinical trials (142). In one study, it is combined with paclitaxel to treat TNBCs.

Nuclear Factor kB
The NFкB pathway has been linked to oncogenesis (144) and to ENDO-R (145). The NFкB family of transcription factors include NF-kB1 (p50), NF-kB2 (p52), RelA (p65), RelB and c-Rel, which homo-or heterodimerize to interact with specific DNA binding sites. Upon phosphorylation of the NFкB regulator IkB (inhibitor of NFкB) by IKK (IkB kinase) the NFкB protein is released from the inhibitory complex and translocates to the nucleus to regulate transcription (146). Being a strong activator of cyclin D1 synthesis (22), NFкB may replace ERa in stimulating proliferation when ERa activity is impaired. In a number of FULV-R MCF-7 sublines, increased NFкB (p65, RelB) activity has been noted, whose inhibition resulted in growth-suppressive effects (147)(148)(149)(150). NFкB has also been found to prevent apoptosis of FULV-R MCF-7 cells (150).
In FULV resistance induced by mesenchymal stem/stromal cells (MSCs) or carcinoma-associated fibroblasts (CAFs) the atypical IkB protein B-cell lymphoma-3 (Bcl-3) plays a role, whose expression is associated with poorer survival of TAMtreated patients (90). Bcl-3 can activate NFкB-dependent transcription by binding to transcriptionally repressive p50/p50 and p52/p52 homodimers and "convert" them to activators (151). Bcl-3 is a growth-stimulatory factor in cancer cells (152,153). It may partially act as such by upregulating the expression of c-Myc (154,155), a proliferation-inducing protein which may contribute to ENDO-R (141,156) and by stimulating the expression of sex determining region Y-box 2 (Sox2) (157), a stem cell protein involved in drug resistance (158). In addition, Bcl-3 downregulates the expression of selenoprotein P, plasma 1 (SEPP1) and kelch-like 4 (KLHL4) (90), two genes whose mRNA levels inversely correlate with relapse-free survival of TAMtreated patients. Interestingly, KLHL4 has recently been reported to bind p53 to increase the expression of the cell cycle inhibitor p21 (159). Hence, part of Bcl-3's growthstimulatory activity may be based on its suppressive effect on KLHL4 expression.
A number of drugs interfering with the NFкB pathway have been developed (160). some of which are used in clinical trials (161). For instance, the anti-alcoholismus drug disulfiram, which also inhibits NFк;B activity, is tested in a phase II trial of patients with a Her2-negative BC.

X-Box Binding Protein-1
Unfolded protein response (UPR) is activated in the event of endoplasmic reticulum stress (162). UPR is important for ENDO-R, as it is able to act as prosurvival mechanism by eliminating endoplasmic reticulum stress and by re-installing metabolic homeostasis (163). XBP1 is a key transcription factor involved in regulating UPR and is activated by UPR. Upon UPR initiation, XBP1 is activated by unconventional cytoplasmic splicing resulting in the conversion of XBP1 mRNA coding for the unspliced XBP1(U) form to the mRNA encoding spliced XBP1(S) form. In contrast to the XBP1(U) protein, the longer XBP1(S) protein harbors a transactivation domain allowing XBP1(S) to activate transcription through CREB responsive elements. One important target gene of XBP1(S) is esr1, the gene coding for ERa, another the gene encoding the NFкB transcription factor p65/RelA (164). Overexpression of XBP1 renders MCF-7 cells more resistant to FULV (165), while its depletion reduces cell growth of FULV-R MCF-7 cells by inducing apoptosis (164), likely caused by reduced expression of XBP1-regulated anti-apoptotic protein Bcl-2 (165). Higher ratio of XBP1(S)-to XBP1(U)-mRNA correlates with worse prognosis of patients with ERa-positive BC, while XBP1(U) expression alone predicts better survival (100).
The importance of UPR for drug resistance has planted the idea of inducing an overload of ER stress (166). This could be achieved by certain nanoparticles or by the proteasome inhibitor bortezomib, the latter being already used to treat certain haematopoietic cancers.

Y-Box Binding Protein 1
The transcription factor YB-1, a so-called cold-shock protein, is involved in cellular stress responses (167). By binding to the ERa protein and interfering with its activity (168,169) and by upregulating the expression of EGFR and Her2 (168,170,171), YB-1 induces a shift from ERa-towards EGFR/Her2-driven gene expression. In line with this, in primary BCs, YB1 expression correlates with the expression of EGFR and Her2 and inversely with that of ERa and PR (170,172,173). Higher YB-1 expression is associated with poorer prognosis in BC (174)(175)(176)(177)(178) and predicts a worse outcome of TAM-treated patients (101).
Its role in ENDO-R is further supported by the finding that ectopically expressed YB-1 desensitizes MCF-7 and T47D cells to FULV and TAM (179,180). Lapatinib counteracts the FULV-desensitizing effect of YB-1 confirming the involvement of EGFR and Her2. Silencing of YB-1 reverses the switch from ERa to Her2 expression and re-sensitizes cells to anti-estrogens. Interestingly, in FULV-R cells, YB-1 expression is not upregulated, but its phosphorylation at Ser102 is increased (180). P-Ser102 modified YB-1 has been shown to foster anchorage-independent growth and radiation resistance of BC cells (181,182). Ser102 can be phosphorylated by AKT, p70S6K, and ribosomal S6 kinase (p90RSK) (180). As shown with MCF-7 and ZR75-1 cells, FGFR2-dependent signaling increases the interaction between YB-1 and ERa (169), suggesting also a link between YB-1 and FGFR2.
Interference with YB-1 activity is possible by the novel multikinase inhibitor TAS0612, which targets AKT, p70S6K, and p90RSK and thereby prevents YB-1 phosphorylation at Ser102 and its subsequent transport into the nucleus (180). TAS0612 was shown to efficiently suppress growth of Fulv-R BC cells in vitro and in vivo.

CHROMATIN ACCESSIBILITY
Chromatin accessibility is defined by the ability of DNA-binding factors to access chromatin DNA, which is highly compacted by its interactions with histones and other chromatin-binding factors (183). Chromatin accessibility is vital to active transcription. Only 2-3% of the chromation contains accessible DNA to which 90% of the TFs bind. Besides pioneer TFs, chromatin remodeling complexes, such as switch mating type/ sucrose non-fermenting (SWI/SNF), histone modifiers, histone readers and mediators play an important role in opening up chromatin (75,76). Post-transcriptional modifications (PTMs) of histones play a key role in regulating chromatin accessibility (184). PTMs are regulated by "writing" enzymes that add a modification and "erasing" enzymes that remove a modification (185). For instance, histone acetyl transferases (HATs), such as CBP, acetylate histones, thereby promoting transcription, while histone deacetylases (HDACs) deacetylate histones, thereby repressing transcription. PTMs can be recognized by histone readers. Bromodomain histone readers, such as bromodomaincontaining protein 4 (BRD4), recognize acetylated histones (186). The bromodomain and extraterminal (BET) family of bromodomain histone readers has recently gained attention as a potential target in cancer therapy.
Long-term repression of transcription can be achieved by DNA methylation, leaving an epigenetic mark that can be transmitted to daughter cells. Abnormal de novo DNA methylation in tumorigenesis prevent the activation of key genes involved in terminal differentiation and thereby in inhibition of proliferation (187).
Resistance to FULV, TAM or AI is accompanied by changes in histone PTM and DNA methylation patterns indicating that resistance to these drugs are accompanied by epigenetic reprogramming (188)(189)(190).

AT-Rich Interaction Domain 1A
Among the genes required for the anti-proliferative effects of FULV and TAM is ARID1A, a factor of the SWI/SNF complex BAF (28). It is recruited by FoxA1 to FoxA1/ERa-regulated genes and in turn attracts HDAC1, thereby blocking ERadepending transcription (Figure 2). Loss of ARID1A leads to increased histone 4 acetylation and recruitment of BRD4 to these genes. This allows that these genes can be transcribed even though anti-estrogens are present, which eventually results in FULV and TAM resistance (28).
Higher expression of ARID1A correlates with good prognosis in BC (89). However, mutations in ARID1A gene, found in 5% of primary and 12% of metastatic BCs, are associated with unfavorable prognosis (28). BET-inhibitors, available for therapy of cancer patients (191), may be useful tools to counteract ENDO-R caused by ARID1A dysfunction.

HDACs
Based on their homology to yeast deacetylases, four classes of human HDACs are distinguished: class I, IIa, IIb. III and class IV (192). Originally identified as enzymes that deacetylase histones, HDACs were later found also to modify non-histone proteins. Class I HDACs (HDAC1, -2, -3, and -8) are primarily located in the nucleus and engaged in histone deacetylation. By removing acetyl group from lysines, histones become more positively charged, which strengthens the interaction with the negatively charged DNA. This leads to higher compaction of the chromatin, which is then less available for transcription (185). HDACs are typically recruited by transcriptional repressors.
In ERa-negative breast cancer cells, HDAC1 contributes to the inactivation of the esr1 promoter (193). In addition, independent of its histone-regulating function, HDAC1 binds directly to the ERa protein, thereby further suppressing ERa activity (194). Subsequently, suppression of HDAC activity by an HDAC inhibitor (HDACi) results in re-occurrence of the ERa protein in ERa-negative cells (195,196). Furthermore, inhibition of HDAC3 was shown to reduce the formation of FULV-R MCF-7 colonies (197). Also, knock-down of HDAC2 was found to strongly increase the sensitivity to TAM (198). Treatment of TAM-R MCF-7 cells with HDACi was reported to induce apoptosis as well as autophagy and to reduce cellular growth in vitro and in vivo (199)(200)(201). It has been speculated that alterations in the expression of ERa co-repressors, such as nuclear co-repressor (NCoR) 1 and 2, may play a role in the cytotoxic effect of HDACi on TAM-R BC cells, as these corepressors recruit HDACs (202). Loss of such co-repressors may lead to an epigenetic imbalance of ERa-driven gene activity. Importantly, NcoR1 is lost in more than half of all ERapositive BCs.
In a phase III trial, patients who relapsed on endocrine therapy show a survival benefit when treated with the HDACi tucidinostat in addition to the AI exemestane (96). Hence, there is evidence that HDACs are involved in ENDO-R.

Non-Canonical Histone Variant H2A.Z
Histone variants replace canonical histones at certain places of the chromatin, particularly in transcriptionally active regions of the genome, and thereby locally influence epigenetics (203). Histone variants may allow higher rates of nucleosome turnover and may improve chromatin remodeling at active promoters and enhancers.
There is growing evidence that cancer cells misuse histone variants to foster their proliferative activity. In breast cancer, the mRNA expression of the histone variant H2A.Z correlates with the mRNA levels of cell cycle proteins, including cyclins (204). H2A.Z may be of particular importance for ERa-driven breast cancer (203). Of the two H2A.Z proteins, H2A.Z.1 and H2A.Z.2, H2A.Z.1 is regulated by ERa through an ERE site in its gene h2afz. Furthermore, H2A.Z is recruited to hypomethylated DNA at ERa-active enhancers (205) and is important for estrogendependent ERa activity at FoxA1/ERa binding sites (206). Interestingly, ectoptic expression of H2A.Z was shown to increase MCF-7 cell proliferation in the absence of estrogen or in the presence of TAM suggesting a potential role of this protein in ENDO-R (204). Overexpression of H2A.Z is associated with poor outcome in BC (95).

DNA Methylation
Compared to the MCF-7 parental cell line the DNA methylation pattern is different in FULV-R MCF-7 sublines (33,189,207,208). Both altered hyper-and hypomethylation of promoters and enhancers were found to coincide with FULV resistance. Hypermethylation of promoters in the anti-estrogen-resistant sublines was linked to either a higher expression of DNA methyltransferase (DNMT) 3B or DNMT1 (207,209). In FULV-R MCF-7 cells, promoter A of the esr1 gene is one of the hypermethylated promoters giving rise to strongly reduced ERa expression (208). In contrast, in FULV-R T47D cells, loss in promoter A activity did not coincide with hypermethylation. PTEN is another gene whose promoter can be highly methylated in anti-estrogen resistant MCF-7 cells (209).
In TAM-R MCF-7 cells, hypermethylation was predominantly found in enhancers (189). Of these enhancers~20% were ERaresponsive, of which approximately half contained FoxA1 binding sites. Importantly, methylation in the ERa-responsive enhancers significantly reduced ERa binding and the expression of the enhancer-driven genes. A higher methylation status in these enhancers was found to be linked to a higher risk of relapse on TAM treatment. Methylation of the ERa-responsive enhancers seems also to play a role in regulating ERa transcriptional activity in the different BC subtypes. The highest median methylation of ERa-responsive enhancers was found in the ERa-negative subtype, whereas it was lowest in luminal A tumors (189).

CANCER STEM CELLS (CSC)
There is a great body of evidence that a minor population of cells with stem-like activities, CSCs, are responsible for BC growth initiation and progression (210,211). To identify CSCs in BC, several markers have been established, among them CD44, CD24, CD133 and aldehyde dehydrogenase 1 (ALDH1) (212,213). By being multidrug resistant (214) and by showing low expression of ERa (98,215) CSCs are highly likely to escape endocrine treatment. If so, anti-estrogens, by eradicating non-CSCs while leaving CSCs alive, would increase the proportion of the CSC population. Indeed, treatment with FULV or TAM has been found to enrich the CSC fraction in the MCF-7 cell line (98,216). Also, high expression of ALDH1 is associated with failure of ERa-positive BCs to respond to TAM (98). As shown with MCF-7 cells in mouse xenografts, one subpopulation of CSCs (CD133 hi /CD44 low ) may be of particular importance in FULV-R (217). In BC, higher expression of CD133 correlates with lower response rates to chemotherapy (218) suggesting a general role of CD133 in drug response. CSC activity is maintained by a number of transcriptionregulating factors, such as cleaved fragments of the Notch pathway, b-catenin activated by the Wnt pathway and epithelialto-mesenchymal transition (EMT)-inducing-TFs (211).

The Notch Pathway
The Notch signaling pathway, important for the maintenance of CSC activity in BC (211), has been linked to ENDO-R (219, 220).
To stimulate signaling through the Notch pathway, a Notch receptor interacts with a ligand of the Delta-Serrate-Lag2 (DSL) family, such as JAG1, presented by a neighboring cell. This leads to a g-secretase-dependent cleavage of the Notch protein resulting in the Notch fragment Notch intracellular domain (NICD) (221,222). Imported into the nucleus, NICD induces transcription of genes, such as hairy and E(spl (Hes), engaged in regulation of cell fate decisions.
In MCF-7 and T47D cells, Notch1 and Notch4 activities are negatively regulated by estrogen-activated ERa (91,223). The ERa-dependent repression of the notch4 gene involves the transcriptional repressor death domain associated factor 6 (DAXX), a protein stabilized by ERa. In turn, DAXX recruits DNMT1 to the notch4 promoter leading to DNA methylation. Besides Notch4, DAXX also down-regulates other stemnessrelevant genes, including ALDH1A1, thereby causing the tumor-initiating capacity of BC cells to decline. Importantly, higher expression of DAXX correlates with more favorable outcome of patients who received endocrine treatment (91).
The link between the ERa and Notch pathways may be more complex. One study shows that Notch1 and JAG1 are involved in ERa expression (227) and that silencing of either protein resulted in a loss of luminal marker genes and a gain in basallike marker genes. Another study suggested an ERa-driven cross-talk between non-CSCs and CD44 + /Epcam + /CD24 --CSCs, by which the Notch pathway is activated to increase the CSC population (228).
Inhibitors of the Notch pathways, such as g-secretase inhibitors, are tested in breast cancer trials (220) and may be suitable tools to treat Notch-dependent ENDO-R.

The Wnt Pathway
The Wnt pathway is an important pathway in mammopoiesis, involved in mammary stem cell regulation and cell fate decisions (229). Its deregulation can lead to BC. In the canonical Wnt pathway, a Wnt ligand interacts with the Wnt receptor Frizzled, which in concert with its co-receptor low-density lipoprotein receptor-related (LRP) leads to stabilization of the protein bcatenin in the cytoplasm (230). Translocated to the nucleus, this key effector of the Wnt pathway drives transcription by interacting with the transcription factor T cell factor/lymphoid enhancer-binding factor. Among the target genes are the EMT-TFs Twist and Slug. Through a different pathway Wnt/Frizzled interaction leads to increased ATF-2/c-Jun activity (230), two factors of the AP-1 family discussed to be involved in ENDO-R (AP-1).
In FULV-R and TAM-R MCF-7 sublines, expression of Wnt pathway components, including b-catenin, are increased (33). Also, overexpression of b-catenin in MCF-7 cells decreased their sensitivity to FULV (231). Higher b-catenin cytosolic and/or nuclear abundance have been linked to poor survival in BC (232). Since this was found for all BC subtypes, it may simply reflect a higher degree of CSC activity.

EMT-TFs
EMT is an essential process in embryonal development and wound healing allowing stationary cells to switch to a migrating phenotype (233). This is caused by a set of EMT-TFs, such as Twist, Slug and zinc-finger E-box binding homeobox 1 (ZEB1). EMT can bestow cancer cells stem cell features and convert them to CD44 + /CD24 -CSCs (234). EMT may not lead to a fully formed mesenchymal phenotype, but may give rise to intermediate states, now called quasi-mesenchymal phenotypes (235). Quasi-mesenchymal CSCs may be of particular importance for cancer progression.
Overexpressed in MCF-7 and T47D cells, Twist was shown to bind to the esr1 promoter and to inhibit ERa expression, leading to estrogen-independent proliferation and FULV and TAM resistance (236). Twist-induced suppression of esr1 transcription coincided with DNA methylation, caused by the Twist-recruited DNMT3B. Like Twist, ZEB1 induces esr1 promoter hypermethylation and ENDO-R (237), while ZEB1 downregulation increases FULV sensitivity (238). In FULV-R and TAM-R MCF-7 sublines, primarily Slug was found to be overexpressed (239).
Clinically, higher expression of Twist, Snail or Slug was found to be associated with a higher probability to relapse in ERapositive BCs (99). Twist and Snail combined were even more powerful in predicting the risk of relapse than each protein alone. However, it remains unclear whether the link between EMT-TF expression and poor clinical outcome is based on the downmodulatory effects of EMT-TFs on ERa signaling or on their ability to promote cellular migration.

CONCLUSIONS AND FUTURE PERSPECTIVES
Currently, the PI3K/AKT/mTOR pathway and CDK4/6 are the prime targets to manage endocrine resistance. However, inhibitors against these targets may fail. For instance, ENDO-R resulting from Her2 mutations or FGFR1 amplification are also resistant to the CDK4/6 inhibitor palbociclib (60,240). Hence, there is a need for a list of biomarkers predicting the responses to the currently used inhibitors. Unraveling the nuclear mechanisms involved in ENDO-R may lead to the discovery of additional biomarkers which may help to optimize treatment of endocrine resistant BCs.
Furthermore, by exploring the nuclear mechanisms that allow escape from endocrine treatment new druggable targets may come to light. However, the diversity of nuclear mechanisms leading to ENDO-R requires additional diagnostics to clarify which nuclear changes are responsible for the observed resistance. One such recently identified promising target is the histone PTM reader BRD4 (28), whose activity can be blocked by BET-inhibitors already available for therapy of cancer patients (191). HDACi, also available for treatment of cancers (192), may be an option to overcome HDAC-dependent suppression of estrogen-driven transcription (96).
Transcription factors are more difficult to target, as they usually lack enzymatic activity. However, drugs can interfere with these factors indirectly, for instance, by blocking enzymes responsible for their activation or by inhibiting their interactions with essential co-factors. Transcription factors that are druggable through such an approach include YB-1, NFкB, Notch and ERRa. YB-1 activity can be suppressed by blocking the kinases that catalyze an essential activating phosphorylation event (180). NFкB can be kept in an inactivated state by IKK inhibitors or by the antialcoholismus and anti-cancer drug disulfiram, which seems to interfere with an essential proteolytic step in the NFкB pathway (161). Notch activity can be blocked by g-secretase inhibitors, which are already used in the clinic to treat cancers (241). As the Notch pathway is also important for maintaining the CSC population in BC, g-secretase inhibitors may also counteract the rise of the CSC population during endocrine treatment. ERRa inhibitors, developed to treat diabetes (102), interfere with the interaction of ERRa with its co-factor SRC.
Transcriptional activities could also be controlled by interfering with certain miRNAs. For instance, by blocking miR221/222 b-catenin-dependent transcription FULV resistance can be suppressed (231).
Thus, understanding the nuclear mechanisms involved in ENDO-R may help to dissect those patients who benefit most from treatment with PI3K/AKT/mTOR pathway and CDK4/6 inhibitors and, additionally, may allow the identification of new druggable targets.

AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and has approved it for publication.