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Estrogen receptor mutations and functional consequences for breast cancer Christoforos Thomas and Jan-A˚ke Gustafsson Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, 3605 Cullen Boulevard, Houston, TX 77204, USA

A significant number of estrogen receptor a (ERa)positive breast tumors develop resistance to endocrine therapy and recur with metastatic disease. Several mechanisms of endocrine resistance have been proposed, including genetic alterations that lead to ERs with altered protein sequence. By altering the conformation of the protein and increasing the interaction with coactivators, point mutations in ESR1, the gene encoding ERa, promote an active form of the receptor in the absence of hormone that assists tumor cells to evade hormonal treatments. Recent studies have confirmed that ESR1 point mutations frequently occur in metastatic breast tumors that are refractory to endocrine therapy, and suggest the development of novel strategies that may be more effective in controlling ER signaling and benefit patients with recurrent and metastatic disease. Introduction Estrogen receptors a (ERa) and ERb are two members of the nuclear receptor superfamily that mediate the effects of estrogens in target tissues [1–3]. Similarly to most nuclear receptors, ERs contain a domain with ligand-independent activation function (AF-1) at the N-terminus (A/B domain), a DNA-binding domain (DBD, C domain), followed by a hinge domain (D domain), and a ligand-binding/ dimerization domain (LBD) at the C-terminus (E/F domain) that contains a ligand-dependent transcription activation function (AF-2) (Figure 1). In mammary gland, ERs regulate cell growth and differentiation by controlling gene expression in response to hormone binding. Upon binding to a ligand, such as 17b-estradiol (E2), they recruit coactivators or corepressors (see Glossary) and initiate or repress transcription by acting as transcription factors or by interacting with and altering the activity of other transcription factors. They bind to DNA either directly at estrogen response elements (ERE) at transcriptional regulatory sites of target genes, or through other DNA-bound transcription factors [4]. In addition to agonist-dependent Corresponding authors: Thomas, C. ([email protected]); ˚ . ([email protected]). Gustafsson, JA Keywords: estrogen receptor mutations; breast cancer; metastasis; endocrine therapy; resistance. 1043-2760/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2015.06.007

activation, ERs elicit transcriptional responses in the absence of ligands. This activation usually occurs when aberrant growth factor signaling alters the phosphorylation and activity of the receptor [4–6]. To date, sustained exposure to estrogens is considered a risk factor for breast cancer [7]. In addition to altered hormone levels, changes in the expression of ERs correlate with clinical outcome. ERa is expressed in nearly 70% of breast tumors at levels that are significantly higher than those of ERb. ERa drives proliferation and promotes the survival of breast cancer cells [8]. Conversely, anti-proliferative and anti-invasive responses have been observed following expression of wild-type ERb in breast cancer cell lines [9–11]. ERa is the principal biomarker for directing

Glossary Array comparative genomic hybridization (aCGH): a molecular cytogenetic technique that identifies regions of DNA duplication, amplification, or deletion in a DNA sample. Test and control DNA are labeled with fluorophores and the probes that are generated are co-hybridized with cloned known DNA fragments in an array. The relative fluorescence intensities of the hybridized fluorophores are quantified following digital imaging. Coactivators: proteins that activate transcription of specific genes by interacting with transcription factors and by regulating the activity of the transcriptional machinery and the DNA structure at the transcriptional regulatory sites. Corepressors: proteins that decrease gene expression by regulating the activity of the transcriptional apparatus and chromatin structure. Deep sequencing: a method of sequencing the genome where each base within the genome is sequenced (read) multiple times. Fluorescence in situ hybridization (FISH): a cytogenetic method that detects specific DNA sequences including deletions, amplifications and other DNA rearrangements as well as specific RNA targets. Fluorescent probes are hybridized to the target nucleic acid in the tissue sample and are visualized by fluorescence microscopy. Gene amplification: a selective increase in the number of copies of a gene. Missense mutation: a single-nucleotide substitution that results in a codon that specifies a different amino acid. Multiplex ligation-dependent probe amplification (MLPA): a high-resolution method to detect copy-number variations in genomic sequences. Pairs of fluorescent probe oligonucleotides are only amplified by PCR when they are hybridized to their DNA targets and ligated into a complete probe. The amplification product that generates fluorescence is separated on a capillary sequencer and reflects the number of target sites in the sample DNA. Non-genomic mechanisms: non-transcriptional mechanisms of signal transduction. Nonsense mutation: a single base modification that generates a stop codon. Single-strand confirmation polymorphism (SSCP) analysis: a technique to discover DNA polymorphisms. Genomic DNA is radiolabeled during PCR amplification, denatured, and the single-stranded nucleic acids with different sequences (even a single base pair) are separated by electrophoresis. DNA sequencing can be performed from the shifted bands from the SSCP gels.

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AF-1

DBD

HD

AF-2 and LBD

K303R

A/B 1

C 180

S463P

344insC

D 263

L536P L536R V534E L536Q D538G

E/F 302

595 E380Q R503W

P535H

ERα-66 (wild type or with point mutaons)

Y537C Y537N Y537S

ERα-Δ3 ERα-46 411

ERα-36 273

ERβ1 (wild type) 1

149

214

248

530

ERβ2 469 495

ERβ5 469 472 TRENDS in Endocrinology & Metabolism

Figure 1. Schematic representation of genomic and functional structure of estrogen receptors (ERs) and the locations of alterations. The structural domains are labeled A–F and the numbers of amino acids are indicated in black below. The locations of the functional domains are represented by solid blue bars on top. The regions A/B contain the activation function 1 (AF-1) domain with ligand-independent activity, and a co-regulatory domain that provides binding sites for the recruitment of coactivators and corepressors. The C and D regions correspond to the DNA-binding domain (DBD) and hinge domain (HD), respectively. The hinge domain is a multifunctional domain that binds co-regulatory proteins and regulates conformation, DNA binding, stability, and intracellular localization of the receptor. The region E/F contains the activation function 2 (AF-2) domain with hormone-dependent activity and the ligand-binding domain (LBD). This region binds co-regulatory proteins and controls receptor dimerization and nuclear translocation. Locations of ERa point mutations with altered amino acid residues are indicated in red. Solid and dashed lines indicate mutations found in metastatic and primary tumors, respectively. ERs can also be modified by alternative splicing, resulting in truncated proteins. Truncated receptors can also be generated due to nonsense point mutations, such as single-nucleotide deletion, that generate frame-shifts and translation termination codons. Human ERa and ERb truncated isoforms that are expressed in breast tumors are shown below the wild-type forms. ERb isoforms are formed from alternative splicing of the last coding exon (shown by the striped bars).

endocrine therapies and is the primary therapeutic target in breast cancer [12]. Endocrine therapies include selective ER modulators (SERMs) such as tamoxifen, that acts as an ER antagonist in the breast, and fulvestrant, an antagonist that induces proteasomal degradation of ERa, as well as aromatase inhibitors such as letrozole and exemestane that block the synthesis of estrogen and deprive the receptor of hormone [13]. Approximately 50% of ERa-positive breast cancers initially respond to adjuvant endocrine treatment. However, a significant number of these tumors eventually develop resistance and recur as metastatic disease. The majority of ERa-positive tumors with acquired endocrine resistance retain ERa expression, suggesting that ERa is involved in therapeutic resistance [14]. In endocrine-resistant tumors, ERa is unresponsive to ligands and can be activated either by aberrantly active coactivators and growth factor signaling, or by mutations that alter its structure and function [5,15–19]. Several variant forms of ERs have been identified in breast cancer. These include truncated isoforms and ER proteins with single amino acid alterations [15]. ERa and ERb truncated variants affect cancer cellular responses by modulating the function of wild-type receptors and other tumor-associated factors [4,15]. In clinical studies, the 2

truncated ER isoforms have been associated with disease outcome including response to endocrine therapy [20– 22]. In addition to truncated proteins, point mutations in the ESR1 gene were first detected at a low frequency in breast cancer [23–25]. Some of the mutations identified in metastatic cancers were found to lead to receptors with hormone-independent transcriptional activity [25–27]. More recent studies analyzing large numbers of primary tumors with sensitive sequencing methodology detected ESR1 mutations at a very low frequency, strengthening the notion that the presence of ESR1 mutations is a rare event in breast cancer [28–31]. This idea has been prevalent until very recently, when several independent groups reported a substantial frequency of ESR1 mutations in metastatic cancers that progressed during hormonal therapy [14,32–34]. In contrast to point mutations, ESR1 gene amplification has been described as an uncommon event in breast cancer, but its clinical significance remains to be determined [14,28,34–37]. Unlike ESR1, ESR2, which codes for ERb, is not among the significant mutated genes in breast cancer [28]. In this review we focus on frequentlyoccurring ESR1 point mutations that have recently been reported to mediate clinical resistance to hormonal therapy. Finally, we discuss how ER mutations could lead to

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Review development of novel strategies to effectively regulate ER signaling and benefit patients with recurrent and metastatic disease. Truncated ER variant isoforms in breast cancer Exon-deleted mRNA variants of both ER subtypes have been found in normal breast as well as breast cancer tissues. What remains unclear today is whether all of these variants produce proteins, and then whether the truncated proteins have any effects on the development and progression of breast cancer [15]. Truncated ER proteins possess unique structures that determine their interactions with components of intracellular signaling pathways and also their subcellular localization and function [4]. Owing to the lack of antibodies and sensitivity of other methods of detection, only a few truncated ER proteins have been analyzed in tumor samples [20,21,38]. Among the truncated ERa isoforms, ERa-36 has been found in both ERapositive and -negative breast cancer cells [39]. This ERa isoform lacks the AF-1 and a large portion of the LBD, and is localized in both plasma membrane and cytoplasm where it mediates non-genomic oncogenic signaling in the presence and absence of ligand (Figure 1) [40]. Induction of ERa-36 expression increases the survival, invasiveness, and anti-estrogen resistance of breast cancer cells [41–45]. Consistent with its cellular effects, increased expression of this variant in tumors from tamoxifen-treated patients correlates with poor survival [22]. Another ERa isoform, ERa-46, that is expressed in breast cancer cells, lacks only the AF-1 domain (Figure 1) [46]. ERa-46 has been shown to mediate membrane-associated estrogen signaling and is implicated in estrogen-dependent growth of breast cancer cells [47]. Lower expression of the variant was observed in endocrine-resistant breast cancer cells, and its upregulation in these cells restored tamoxifen sensitivity [48]. Some ERa-46 actions in ERa-positive breast cancer cells are attributed to its interaction with the wild-type isoform that results in altered wild-type ERa transcriptional responses [15]. A similar mechanism is employed by another isoform that lacks part of the DBD. ERa exon D3 was found to decrease proliferation and invasion in breast cancer cells (Figure 1) [49]. Five ERb isoforms exist as a result of alternative splicing of the last coding exon (Figure 1) [4]. Of all five variants, only protein forms of wild-type ERb (ERb1), ERb2 (also known as ERbcx), and ERb5 have been detected in clinical breast cancer specimens [20,21,50,51]. Owing to differential splicing within the LBD, ERb2 and ERb5 either have disoriented or completely lack helix 12 that is required for ligand-dependent activation function (AF-2), and thus determines receptor activity in response to ligand binding [52]. Although these structural modifications prevent ERb2 and ERb5 from binding ligand, both variants can heterodimerize with wild-type ERs. As a result of this interaction, ERb2 was found to inhibit ERa-mediated transcription [53]. Although in a few preclinical studies expression of ERb2 was not found to alter the phenotype of breast cancer cells [54], in clinical studies ERb2 and ERb5 have been reported to be associated with disease outcome. High nuclear expression of ERb2 and ERb5 was associated with better survival and cytoplasmic or combined nuclear

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and cytoplasmic ERb2 expression with worse outcome [21]. More recently, ERb2 was found to predict poor prognosis [55,56], whereas ERb5 appeared to be marker of worse outcome in HER-2-positive and triple-negative patients [57]. A growing body of evidence points to different roles of ERb isoforms in breast cancer biology and suggests their potential prognostic role for patient management. ESR1 point mutations in breast tumors Alterations in the protein sequence of ERa are among the mechanisms that were proposed to contribute to acquired resistance to hormonal therapy in patients with ERapositive breast cancer [16]. Several groups initially attempted to test this hypothesis by analyzing the sequence of ESR1 gene in endocrine-resistant and -sensitive tumors using conventional sequencing methods. In the late 1980s, sequence analysis of mRNA and genomic DNA of breast tumors identified ESR1 point mutations that were correlated with decreased hormone–receptor binding [58,59]. The presence of a single-nucleotide deletion in endocrine-resistant recurrent metastatic tumor, but not in its primary counterpart, was first reported during single-strand conformation polymorphism (SSCP) analysis of a relatively small number of tumors that relapsed with lymphatic metastasis after adjuvant tamoxifen treatment [24]. Point mutations were also detected at low frequency in metastatic breast tumors in a contemporary study. Among these mutations, a Tyr537Asn alteration that is located in the LBD was present in recurrent bone metastasis of a patient that received hormonal therapy [25]. Mutations in Tyr537 residue were found to alter the conformation of the receptor, increasing its interaction with coactivators and its transcriptional activity in the absence of hormone. It was also shown that the constitutively-active mutant ERa was partially or fully inhibited by tamoxifen [25,60,61]. Further functional analysis led to a proposed mechanism of the mutation on receptor action. By regulating the conformation of helix 12, Tyr537 maintains the receptor in an inactive state in the absence of hormone. Replacement of this amino acid as a result of mutation alters the conformation of helix 12 to form an interacting surface that allows recruitment of coactivators independent of hormone binding [26,27,62]. By sequencing a larger number (188) of unselected primary breast tumors, Roodi and colleagues detected ESR1 point mutations of unknown significance in one ERa-negative tumor [23]. The low frequency of ESR1 point mutations in primary breast cancer was further supported by more recent studies that analyzed a large number of tumors with next-generation sequencing. Although they identified several significantly mutated breast cancer-associated genes, ESR1 was not included in this these genes [28,30,31]. However, this analysis was limited to samples from untreated patients, and it did not rule out that ESR1 mutations are common in recurrent cancers. Three independent groups have recently tested this hypothesis and they found activating ESR1 point mutations in a relatively high number of metastatic tumors from patients that relapsed after hormonal therapy [14,32–34]. The results of these studies will be presented in more detail in the next sections of the article. 3

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Review Sequence of breast cancers and ESR1 mutations Toy and colleagues analyzed genetic alterations in two independent cohorts of patients with ERa-positive metastatic breast cancer using deep-sequencing approaches, and matched normal DNA for comparison. Both cohorts included tumors that had progressed during adjuvant treatment with hormonal therapy, including aromatase inhibitors. In the first cohort, the authors analyzed 36 samples from patients that had been enrolled in the metastatic breast tumor procurement protocol (NCT00897702). TP53 (tumor protein 53), PIK3CA (phosphatidylinositol-4,5bisphosphate 3-kinase, catalytic subunit a), and GATA3 (GATA binding protein 3) mutations were identified at comparable prevalence with that reported in the untreated primary ERa-positive breast cancers by The Cancer Genome Atlas (TCGA) network [28]. By contrast, ESR1 mutations were detected at much higher frequency (9/36 cases, 25%) in the set with relapsed tumors compared with the TCGA cohort. Similar to the first cohort of patients, analysis of tumors from the BOLERO-2 clinical trial (NCT00863655) revealed that the LBD of ESR1 was frequently mutated (5/44 cases, 11%) in metastatic tumors that were refractory to aromatase inhibitors, but not in the untreated primary tumors (6/183 cases, 3%). In both cohorts, the majority of ESR1 mutations affected amino acids Tyr537 or Asp538 in helix 12 of the LBD (Table 1) [34]. In the second study, Robinson and colleagues described a prospective clinical study that included 11 patients with metastatic ERa-positive breast cancer that had received hormonal therapy. Six of the 11 patients (54.5%) treated with anti-estrogens and aromatase inhibitors had missense mutations within the LBD of ESR1 [33]. As in the first study, the most frequently observed mutations affected amino acids Tyr537 or Asp538 (Table 1). The third group of investigators retrospectively analyzed ERa-positive primary and endocrine-treated metastatic tumors, as well as ERa-negative tumors. Primary and metastatic tumors had similar mutation frequency in commonly altered genes in cancer, but differed in ESR1 gene. Twelve somatic point mutations were identified in the LBD of ESR1, one (residue 503) in an untreated primary tumor (1/58, 1.72%), and 11 in metastatic tumors (11/76, 14%). Hotspot mutations (Tyr537, Asp538) were detected in 12% (9/76) of the metastatic specimens (Table 1). Assessment of the mutation frequency in a subgroup of heavily endocrine-treated metastatic tumors showed even higher rates (5/25, 20%) [14]. In a contemporary study, the same group of investigators detected the Asp538Gly mutation in liver metastases in five patients (5/13, 38%) with endocrine-resistant ERa-positive cancer [32]. Collectively, these studies reported the presence of ESR1 point mutations in 25%, 11%, 54.5%, 38%, and 14% of the metastatic tumors. 75% of the mutations affected amino acids Tyr537 and Asp538. Importantly, in the matched primary and metastatic specimens that were analyzed, mutations were detected only in the metastatic samples. When large cohorts of untreated primary tumors were examined, mutations in the LBD of ESR1 were detected at very low rates (1.72 and 3%) that are still higher compared with that reported by the TCGA network (0.6%) [28]. 4

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Point mutations in the LBD of ESR1 were also identified in three of the six circulating tumor cell (CTC) lines derived from patients with metastatic ERa-positive breast cancer. The patients received hormonal therapy including aromatase inhibitors, and the mutations were not present in the primary tumors or metastatic lesions before treatment [63]. In addition, ESR1 point mutations were detected in patient-derived xenografted (PDX) samples. The PDXs were derived from ERa-positive tumors that recurred after hormonal therapy and which were transplanted into oophorectomized mice. Tyr537Ser was identified in both the originating tumor that was aromatase inhibitor-resistant, and in the PDX counterpart that exhibited estrogenindependent growth. By contrast, Glu380Gln was detected only in the low estradiol-grown PDX, suggesting that hormone deprivation during xenografting favors clonal selection of cells with ESR1 mutations [64]. Functional roles of ESR1 mutations Based on previous findings demonstrating the increased hormone-independent transcriptional activity of ERaTyr537 variants [25–27,60–62], the three groups of investigators performed functional studies to determine the activity of the ESR1 mutants that were detected in breast tumors. ER expression constructs containing different point mutations were co-transfected with ERE reporter plasmids into cells. ERE-dependent transcription and expression of ER target genes, cofactor recruitment, and other indicators of ER activity were measured in cells in the presence or absence of the ER ligand, E2, and compared with the activity of wild-type receptor. Toy and colleagues reported a dramatic increase in the ERE-dependent transcriptional activity following transfection of breast cancer cells with the single Tyr537Ser and Asp538Gly mutants, or with the Ser463Pro/Asp538Gly double mutant, in the absence of hormone. A modest further increase in the activity of these mutants was observed by the addition of E2. By contrast, the weakly-active under basal conditions Ser463Pro mutant, and the wild-type receptor, demonstrated elevated activity in the presence of E2. Similarly to the ERE-dependent transcription, Tyr537Ser, Asp538Gly, and Ser463Pro/ Asp538Gly mutants significantly induced the expression of ER target genes in the absence of hormone. Gene expression microarray analysis revealed a distinct expression pattern in MCF-7 cells transfected with Tyr537Ser, Asp538Gly, or Ser463Pro/Asp538Gly mutants, compared with that of the wild-type receptor. In addition to the ER-dependent gene expression, mutations in Tyr537 and Asp538 residues increased ERa phosphorylation on Ser118, recruitment of the coactivator AIB1, and stability of the receptor, further supporting the increased ligand-independent activity of the mutants (Table 2). Unlike the hotspot mutations, the Ser463Pro alteration that is located in the dimerization region (E domain) was found to induce dimerization of mutant with wild-type receptor, and this may account for the ligand-dependent activation of this mutant (Figure 2E). Finally, expression of Tyr537Ser and Asp538Gly ERa proteins in tumors grown in estrogen-deprived mice dramatically increased tumor growth, strengthening the association of the mutations with the stimulation of hormone-independent oncogenic ERa function (Table 2) [34].

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Table 1. Description of ESR1 point mutations in metastatic breast cancer and their relationship with hormonal therapya ERa protein alteration Truncated Tyr537Asn Asp538Gly

ERa status in primary tumor WT N/A WT

Metastatic site Lymph node Bone N/A

Ser463Pro/Asp538Gly

N/A

N/A

Tyr537Ser/Asp538Gly

N/A

N/A

Leu536Arg

N/A

N/A

Val534Glu

N/A

N/A

Tyr537Asn

N/A

N/A

Tyr537Ser

N/A

N/A

Tyr537Ser

N/A

N/A

Tyr537Ser

WT

N/A

Ser463Pro/Tyr537Asn

N/A

N/A

Pro535His

N/A

N/A

Tyr537Cys

N/A

N/A

Tyr537Ser

N/A

N/A

Asp538Gly

N/A

N/A

Leu536Gln

N/A

Lung, liver

Tyr537Ser

WT

N/A

Asp538Gly

WT

Liver

Tyr537Ser

WT

Skin, bone, liver

Asp538Gly

N/A

Brain

Tyr537Ser

N/A

Metastatic lobular

344insCys Glu380Gln Tyr537Asn Tyr537Asn Tyr537Cys Tyr537Cys Tyr537Ser Tyr537Ser Asp358Gly Asp358Gly Asp358Gly Asp538Gly Asp538Gly Asp538Gly Asp538Gly Asp538Gly Tyr537Ser

N/A N/A N/A WT b N/A WT N/A N/A N/A N/A WT WT WT WT WT WT WT

Liver Bone Lymph node Lymph node Bone Lung Liver Liver Liver Bone Skin Liver Liver Liver Liver Liver CTC

Asp538Gly

WT

CTC

Hormone treatment (duration) SERM DES AI, SERM (6.5 years) AI, SERM (7.5 years) AI, SERD, SERM (3.3 years) AI, SERM (10 years) AI, SERD (5.5 years) AI, SERD (5.5 years) AI, SERD (1.5 years) AI, SERD (2 years) AI, SERM (2.5 years) AI (2 years) AI (5 years) AI (3 years) AI (5 years) AI (6 years) AI, SERM, SERD (>5 years) Oophorectomy, AI, SERD (1 year) AI, SERM, SERD (>5 years) Oophorectomy, AI, SERD, SERM (>6 years) AI, SERM (>5 years) AI, SERM, SERD (>5 years) AI, SERM, SERD AI AI, SERM, SERD SERM AI, SERM, SERD AI AI, SERM, SERD SERM AI, SERM, SERD AI, SERD AI AI, SERD AI, SERM AI, SERM, SERD AI, SERM, SERD AI, SERM, SERD AI, SERD (>1 year)

Protocol/program

Ref.

TPC, CCRF N/A Metastatic procurement protocol NCT00897702

[24] [25] [34]

Bolero-2 NCT00863655

MI-ONCOSEQ

[33]

MDACC/ BIDMD/HCUV/ NCT00780676

[14]

N/A

[32]

N/A

[63]

5

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Table 1 (Continued ) ERa protein alteration

ERa status in primary tumor

Metastatic site

Leu538Pro

WT

CTC

Tyr537Ser ESR1/YAP1 fusion

N/A N/A

Skin, nodes Bone, lung, nodes, liver, brain

Hormone treatment (duration) AI, SERD (>1 year) AI, SERM (>1 year) AI, SERM, SERD AI, SERM, SERD

Protocol/program

Ref.

N/A

[64]

a

Abbreviations: AI, aromatase inhibitor; BIDMD, Beth Israel Deaconess Medical Center; CCRF, Cleveland Clinical Research Foundation; CTC, circulating tumor cell; DES, diethylstilbestrol; HCUV, Hospital Clinico Universitario of Valencia; MDACC, MD Anderson Cancer Center; N/A, not available; SERD, selective ER degrader; SERM, selective ER modulator; TPC, tissue procurement core; WT, wild type, YAP1, Yes-associated protein 1.

b

Specimen from the same metastatic site from an earlier time-point.

The two other groups found that all ERa mutants (Leu537Gln, Tyr537Asn, Tyr537Cys, Tyr537Ser, Asp538Gly) possess significantly higher activity than the wild-type receptor when they are expressed in cells in the absence of E2, and that their activity is not further induced by the addition of hormone [14,32,33]. Similarly to Toy and colleagues, Jeselsohn and colleagues reported that, unlike wild-type ERa, the Tyr537Asn and Asp538Gly mutants induced transcription of ER-regulated genes in the absence of hormone, and this was associated with increased proliferation and migration of the mutant ERa-transfected breast cancer cells in estrogen-deprived conditions (Figure 2E) [14,32]. Response of ERa mutants to anti-estrogen treatment The ligand-independent activation of mutant ERa can account for the progression of breast tumors that express mutant receptor during treatment with hormone-deprivation therapies. Several studies assessed whether drugs

that directly target the receptor, such as tamoxifen and fulvestrant, are effective at inhibiting the activity of ERa mutants. While the group that first identified the Tyr537Asn mutant in metastatic breast cancer indicated slight inhibition of its basal activity by tamoxifen [25], contemporary studies reported complete response of the Tyr537 ERa mutants at the same concentration of the drug (100 nM) [26,61]. More recent studies demonstrated a gradual decrease in the transcriptional activity of the Tyr537Ser and Asp538Gly mutants with increasing concentrations of tamoxifen and fulvestrant. Higher concentrations of the drugs (50–100 nM) were required to bring the activity of the mutants close to that observed with the wild-type ERa [32,34]. Similar concentrations of the anti-estrogens were found to potently inhibit the activity of all ERa mutants (Leu536Gln, Tyr537Ser, Asp538Gly, Tyr537Cys, Tyr537Asn) that were identified in metastatic tumors by Robinson and colleagues [33]. Finally, Jeselsohn and colleagues achieved potent inhibition of the activity of

Table 2. Functional roles and biological effects of ESR1 mutationsa ESR1 mutation Tyr537Ser

Lys303Arg

Functional role Induction of basalb ERE activity and ER target genes; increased interaction with coactivators; increased stability; partial or complete inhibition by anti-estrogens Induction of basal ERE activity; partial or complete inhibition by anti-estrogens Increase of basal ERE activity and ER target genes; strong interaction with coactivators; increased stability; partial or complete inhibition by anti-estrogens Induction of basal ERE activity and ER target genes; increased interaction with coactivators; increased stability; relative resistance to anti-estrogens Modest constitutive activity; modest interaction with coactivators Modest induction of basal ERE activity; no induction of ER target genes Induction of basal ERE activity; partial inhibition by anti-estrogens Enhanced binding to coactivators

Leu536Pro

Increased stability

Tyr537Cys Tyr537Asn

Asp538Gly

Glu380Gln Ser463Pro Leu536Gln

a

Biological activity Increased growth in cell culture and mice; decreased CTC survival with HSP90 inhibitors or anti-HSP90/anti-estrogen treatment

Refs [33,34,60–64]

N/A

[14,33]

Increased cell proliferation with or without tamoxifen

[14,25,26,33,64]

Increased cell proliferation and migration; increased tumor growth in mice; E2independent PDX growth

[14,32–34]

N/A

[14,60]

N/A

[34]

N/A

[33]

Increased cell growth in response to estrogen; confers resistance to AI and tamoxifen Decreased CTC survival with HSP90 inhibitors or anti-HSP90/anti-estrogen treatment

[80] [63]

Abbreviations: AI, aromatase inhibitors; CTC, circulating tumor cell; ERE, estrogen response element; HSP90, heat shock protein 90 kDa; N/A, not available; PDX, patientderived xenograft.

b

Basal (in the absence of 17b-estradiol).

6

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(A)

(B)

Helix 3

Helix 3

Helix 12

Helix 12 N348 Y537

D538

(C)

(D)

Helix 3

Helix 3 Helix 12 S537

(E)

(F)

wtERα

D538

CoA E2 wtERα

Helix 12

D351 G538 Y537

D351

CoA ERα S537

Treatment

Cell survival genes E2

CoA ERα wtERα P463

Progression

Clonal expansion of cells with ESR1 mutaons therapy resistance

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Figure 2. Representation of the structures of wild-type (WT) and mutant forms of estrogen receptor a (ERa). (A) WT ERa in an agonist-bound conformation. Hydrogen bond (dashed red line) is formed between Tyr(Y)537 and Asn(N)348 and helix 12 is shifted towards helix 3 forming a surface for the recruitment of a coactivator peptide (colored red). (B) WT ERa in an antagonist-bound conformation. No hydrogen bond is formed between Tyr537 and Asn348, and helix 12 has drifted away from helix 3, thus preventing binding of coactivators. The pattern of hydrogen bond formation is altered in Ser(S)537 (C) and Gly(G)538 (D) mutants of ERa. In the Ser537 mutant, Ser537 hydrogen bonds with Asp(D)351. This bond was observed in X-ray crystal structures and after structural modeling analysis. This mutant is predicted to lack the Tyr537– Asn348 hydrogen bond. In the Gly538 mutant, Gly538 may form hydrogen bond with Asp351. Similarly, this mutant is predicted to lack the Tyr537–Asn348 hydrogen bond and Tyr537 is too far from Asp351 to interact. In both mutants the position of helix 12 is similar to that of the agonist-bound receptor; this allows binding of coactivators and suggests ligand-independent constitutive activity of mutant receptor. (E) Schematic illustration of the mechanism of mutant ERa action. Estrogen enables WT ERa to recruit coactivators (CoA) and initiate transcription. By contrast, ERa mutants (Ser537, Gly538) can bind to coactivators and induce gene expression in the absence of ligands. The Pro463 mutant induces transcription through its increased dimerization with the WT receptor. (F) Clonal evolution of ESR1 mutations in breast tumors. Endocrine therapy induces death of cells with WT ERa (gray cells). This provides a selective pressure that favors the proliferation of cells with mutant ERa (red cells) which resist treatment. These cells eventually dominate the tumor and result in cancer progression and therapy resistance. Panels A–D reproduced from Nettles, K.W. et al. [65] with permission from Macmillan Publishers, copyright 2008.

the Tyr537Asn mutant by using doses of tamoxifen and fulvestrant higher than those reported in other studies. Higher doses of fulvestrant were also required to induce degradation of mutant ERa at similar levels to those observed with the wild-type receptor [14]. These data suggest that tumors expressing mutant ERa may be relatively resistant to the currently used clinical doses of antiestrogens, and higher doses or more-potent ERa antagonists, will be required to effectively block oncogenic mutant ERa signaling.

Structural modeling of ERa mutants Functional studies demonstrated that ERa with mutations in residues Tyr537 and Asp538 mimics the ligand-bound wild-type ERa in the association with coactivators and protein stability [14,33,34]. Comparison of the structure of an agonist-bound wild-type ERa with that of a Tyr537Ser mutant revealed a high degree of similarity. Similarities were also observed in the conformation of helix 12 where the mutation is located [65]. The position of helix 12 determines the interaction with coactivators, and, thus, 7

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the activity of the receptor in response to ligand binding. Upon estrogen binding, helix 12 is folded across helix 3, thereby creating a surface for the binding of coactivators. By contrast, in response to tamoxifen binding, helix 12 has drifted away from helix 3, thus preventing the recruitment of coactivators [32]. Analysis of the crystal structure as well as molecular modeling approaches demonstrated that the position of helix 12 of the mutant receptor is similar to that of estrogen-bound wild-type ERa, and this can explain the ligand-independent activity of mutant ERa (Figure 2). The active conformation of mutant ERa is stabilized by alterations in the pattern of hydrogen bonds that are formed between amino acids in helix 12 and helices 3–5. The hydrogen bond that exists between Tyr537 and Asn348 in the structure of agonist-bound wild-type ERa is lost in mutant receptors. In Tyr537Ser and Asp538Gly mutants this interaction may be replaced by hydrogen bonds that are formed between Ser537 and Gly538 residues in helix 12 and that of Asp351 in helix 3. These amino acid interactions seem to promote shifting of helix 12 towards helix 3 and the rest of the structure, which favors coactivator binding and the active conformation of mutant receptor (Figure 2) [32,34,65]. Modeling the structures of the ligand-bound wild-type ERa and Asp538Gly mutant predicted conformational changes that alter the docking of ligand in the mutant receptor [32]. However, analysis of the structure of the Tyr537Ser mutant revealed no impact on the ligand-binding pocket, indicating its ability to bind ligands including anti-estrogens [65]. These results suggest differences in the ligand-binding affinity of different ERa mutants and wild-type receptor, which may account for the higher concentrations of anti-estrogens that are required to inhibit the activity of mutant receptors compared with those that block the agonist-bound wild-type ERa. Structural modeling seems to provide a mechanistic explanation for how the mutations identified in endocrine-resistant tumors induce conformational changes that promote an active form of the receptor in the absence of hormone.

standard FISH protocol, two groups recently performed FISH after treating the tissue samples with RNase to exclude primary mRNA transcripts from the DNA copynumber evaluation. They also analyzed tumors with multiplex ligation-dependent probe amplification (MLPA). Ooi and colleagues detected increased ESR1 copy number in 5.9% of the specimens. However, when they used as amplification criterion an ESR1/centromere 6 copy number ratio 2, only one in three cases with increased copy number were identified as ESR1-amplified tumors [72]. Moelans and colleagues, although they detected ESR1 amplification in 16% of the tumors using FISH-RNase assay, they did not confirm this increased copy number in half of the cases when MLPA was performed [73]. Consistent with the studies that previously reported a low frequency of ESR1 amplification in breast cancers, ESR1 was not among the significantly amplified genes in the analysis of more than 700 tumors by the TCGA network [28]. However, the majority of these studies analyzed untreated primary tumors and, as in the case of ESR1 point mutations, ESR1 amplification might only occur at a substantial frequency in endocrine-resistant metastatic cancers. ESR1 amplification was detected in a xenograft sample from a patient with metastatic breast cancer that was resistant to aggressive treatment [64]. The groups that identified ESR1 point mutations in metastatic breast cancer used the deep-sequencing data to compute gene copy-number alterations in the same samples. Toy and colleagues reported frequent amplifications in genes that are commonly amplified in breast cancer (i.e., ERBB2) but not in the ESR1 gene [34]. Robinson and colleagues identified ESR1 point mutations in six of the 11 patients, but did not detect ESR1 amplifications and gene fusions [33]. Finally, Jeselsohn and colleagues found ESR1 amplification in 1.7% of primary breast tumors and 1.3% of the metastatic cases [14]. Despite most of the evidence pointing to a low frequency of ESR1 amplification in breast cancer, studies to identify correlations between this type of aberration and response to therapy are warranted.

ESR1 amplification in breast tumors There has been considerable controversy regarding the frequency of ESR1 gene amplification in breast cancer as a result of publication of contradictory data from studies that applied diverse methodologies to measure DNA copy numbers and different criteria to define amplification [4]. By using fluorescence in situ hybridization (FISH) and quantitative PCR (q-PCR), Holst and colleagues initially reported ESR1 amplification in a substantial proportion of breast cancers (20.6%) [66]. These results have been challenged by several groups who measured ESR1 copy number in different cohorts of breast tumors with various methods including comparative genomic hybridization (CGH), FISH, and q-PCR. These groups reported amplification in less than 1% [67,68], 1% [35] and 1.35%, 2.8%, 2.6%, 3.7% and 4.2% of the tumors [36,37,69–71]. In most of these studies ESR1 copy number was lower than that of ERBB2, which codes for HER2 (v-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2), and no significant correlation between amplification and protein expression was observed [37,67,68]. In addition to the

Concluding remarks and future perspectives A substantial frequency of ESR1 point mutations has recently been demonstrated in ERa-positive metastatic breast tumors [74]. These mutations were detected in patients who relapsed after hormonal therapy, suggesting that they play a role in development of acquired endocrine resistance. The absence of detectable mutations in the untreated primary tumors indicates that clonal selection of tumor cells with ESR1 mutations occurs during cancer progression, and that the mutations assist tumor cells to evade hormonal treatments (Figure 2F) [75]. Given that the majority of these patients were treated with various hormonal therapies it has not been fully elucidated if the alterations are developed in the context of anti-estrogen or aromatase inhibitor treatment. The ability of direct ERa antagonists, but not of hormonal withdrawal, to effectively inhibit the activity of ERa mutants suggests that the mutations promote resistance to hormone deprivation and that aromatase inhibitor-unresponsive tumors may benefit from anti-estrogens. Patients that develop resistance to aromatase inhibitors were previously reported to

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Review respond to anti-estrogens [76]. However, one of the patients whose ERa mutant tumor had spread while on hormonal therapy did not respond to tamoxifen and fulvestrant that were given after detection of the mutation [34]. The residual activity of ERa mutants that was observed at lower doses of these drugs may be sufficient to confer clinical resistance. Future prospective clinical sequencing studies with large cohorts of tumors that become refractory to different hormonal therapies will clarify the association of the mutations with mechanisms of endocrine resistance. Implementation of single cell DNA sequencing in tissue and in serial plasma samples to monitor genomic changes in real-time will lead to more precise evaluation of the mutation frequency not only in metastatic tumors but also in primary disease, where discrepancy between studies still exists. It will also help to better understand the evolution of the mutations during cancer progression. Early detection of the mutations in sub-clonal cell populations of the primary tumor can appropriately guide the selection of adjuvant therapy. In addition, given that high doses of anti-estrogens were able to inhibit the activity of ERa mutants, more potent or specific antagonists may benefit patients whose tumors are unresponsive to hormonal therapy. As such, next-generation anti-estrogens are currently being tested in preclinical and clinical settings with promising results. Among these are the orally-bioavailable compounds AZD9496 [77] and GDC-0810 that act as selective ER degraders. GDC-0810 has recently been shown to potently inhibit the growth of anti-estrogen-resistant wildtype and mutant (Tyr537S) MCF-7 tumor xenografts by reducing ERa expression. In Phase I and ongoing Phase II clinical trials including postmenopausal women with locally advanced or metastatic breast cancer that had progressed after endocrine therapy, GDC-0810 showed reversible and manageable toxicity. Two patients with ESR1 mutant liver metastatic tumors showed partial response, with decreased ERa levels in tumors [78,79]. In addition to anti-estrogens, further structure modeling studies will contribute to a better understanding of the conformation of the mutant receptor and determine whether peptide derivatives can be tested as alternative targeted therapies. Finally, given the crucial role of coactivators in the ligand-independent activation of the mutant receptor, compounds that target coactivators may prove effective in reversing ER mutant-driven endocrine resistance in breast cancer. Acknowledgments We thank Margaret Warner for critically reading the manuscript.

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