Mechanisms of Aromatase Inhibitor Resistance

REVIEWS

Mechanisms of aromatase inhibitor resistance

Cynthia X. Ma1, Tomás Reinert2, Izabela Chmielewska3 and Matthew J. Ellis4 Abstract | Oestrogen receptor-positive (ER+) breast cancer is a major cause of cancer death in women. Although aromatase inhibitors suppress the function of ER and reduce the risk of recurrence, therapeutic resistance is common and essentially inevitable in advanced disease. This Review considers both genomic and cell biological explanations as to why ER+ breast cancer cells persist, progress and cause an incurable, lethal, systemic disease. The design and outcomes of clinical trials are considered with the perspective that resistance mechanisms are heterogeneous, and therefore biomarker and somatic mutation-based stratification and eligibility will be essential for improvements in patient outcomes.

Aromatase inhibitors Ever since Beatson’s historic observation that breast are not precise, however, and the underlying mecha- 1 (AIs). A class of drugs, including tumours can regress following oophorectomy , oestro- nisms between intrinsic and acquired resistance are non-steroidal AIs (for example, gen deprivation has been an important treatment for likely to overlap. In addition, resistance to endocrine anastrozole and letrozole), and oestrogen receptor-positive (ER+) breast cancer. For therapy may be agent-selective. For example, after failure steroidal AIs (for example, postmenopausal women aromatase inhibitors (AIs; such of AI therapy, tumours can respond to other endocrine exemestane), that lower oestrogen levels by inhibiting as anastrozole, letrozole and exemestane) are the cur- therapy approaches such as another AI of a different the enzyme aromatase. rent standard-of-care for preventing relapse (which is class (steroidal versus non-steroidal), oestrogen recep- referred to as adjuvant therapy) (FIG. 1), because several tor modulators (tamoxifen) or SERDs (fulvestrant) and trials over the past two decades have demonstrated the even physiological doses of oestradiol9. superiority of AIs compared with tamoxifen2. Recent Biological insights into the causes of AI resistance studies have also indicated modestly increased efficacy in ER+ breast cancer have developed fairly slowly and

1 for exemestane compared with tamoxifen as adjuvant the aetiological events that drive the prolonged natu- Division of Oncology, + Department of Medicine, treatment for premenopausal women when combined ral history of ER breast cancer, whereby the risk of Siteman Cancer Center, with a gonadotrophin-releasing hormone agonist recurrence persists for several decades, remain unclear. Washington University School (to reduce ovarian oestrogen synthesis)3. In advanced Although multi-gene assays provide an approach of Medicine, 660 South Euclid disease, AI therapy is the standard initial treatment for the identification of low-risk, early-stage AI or Avenue, St Louis, Missouri 63110, USA. but recent data on treatment with higher doses of the tamoxifen-sensitive disease that is treatable with these 2Department of Medical selective oestrogen receptor downregulator (SERD) agents alone (or in sequence), current expression pro- Oncology, Instituto Nacional fulvestrant have indicated that it is a promising alter- filing assays do not provide individualized treatment de Câncer (INCA), native, with evidence for improved disease control and information for high-risk tumours beyond a recom- Praça da Cruz Vermelha, 23, an emerging survival advantage against anastrozole4,5. mendation for generic and only partially effective 20230–130, Rio de Janeiro, 10 Brazil. Unfortunately, despite the potency of AI therapy, over chemotherapy . The use of gene expression profiling to 3Department of 20% of patients with early-stage disease suffer a relapse, assess the risk of developing AI-resistant breast cancer Pneumonology, Oncology which can occur years or decades after diagnosis6. In has been reviewed elsewhere11. Genetically determined and Allergology, Medical patients with advanced or metastatic disease, although differences in endocrine drug metabolism, particularly University of Lublin, Jaczewskiego 8 St., 20–954, the majority derive benefit from initial endocrine of tamoxifen, are not considered in this Review, as Lublin, Poland. approaches of any type, subsequent disease progression these studies have yet to translate into clinical action 4Lester and Sue Smith Breast invariably occurs7. or to improved patient outcomes and are not currently Center, Baylor College of Two general patterns of endocrine therapy resistance a major focus in the use of AI therapy12. Improvements Medicine, Houston 77030, are recognized clinically: intrinsic resistance, whereby in therapeutic approaches for AI-resistant disease Texas, USA. + Correspondence to M.J.E. ER cancers never adequately respond to endocrine are therefore the focus of this Review, as success will e-mail: [email protected] treatment, and acquired resistance, which develops fol- directly stem from the rich database of biological and doi:10.1038/nrc3920 lowing an initial response8. These clinical distinctions genomic insights into the pathogenesis of ER+ breast

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Tamoxifen cancer and the biological consequences of target- fall in Ki67 expression from baseline, although the meas- A selective oestrogen receptor ing oestrogen-dependent transcription by oestrogen urement of Ki67 expression at two time points rather (ER) modulator (SERM), which deprivation. than one increases assay variability, which remains a antagonizes ER function in problem with Ki67 immunohistochemistry (IHC)18,19. breast tissue but also acts as AI resistance in early-stage disease an ER agonist in other tissues, The ACOSOG Z1031 Cohort B trial successfully pio- including the endometrium and Although endocrine therapy resistance in advanced dis- neered a tailored treatment strategy based on real-time blood coagulation system, ease is clinically obvious and is associated with incur- central Ki67 assessment on tumour biopsies taken at resulting in side effects such as ability, the detection (or prediction) of AI resistance 2–4 weeks following neoadjuvant AI therapy to triage endometrial cancer and an in primary tumours before relapse remains a crucial patients to neoadjuvant chemotherapy if the proportion increased risk of thrombosis, + respectively. research endeavour because early-stage disease is still of cells in the biopsy sample that were Ki67 remained within the ‘curability window’. Therapeutically address- above 10%. Preliminary data from this trial indicated that Endocrine therapy ing AI-resistance mechanisms early in the course of dis- about 20% of patients are in this intrinsic AI-resistance Targets the oestrogen receptor ease is inherently more likely to improve the cure rate category and the feasibility of using on-treatment Ki67 to (ER) pathway for the treatment of ER+ breast cancer either by because treatment when the disease burden is lower identify endocrine-resistant disease in the neoadjuvant 10,22 lowering oestrogen level or by has a reduced probability of a pre-existing AI resistance setting for triage to alternative therapy is underway . antagonizing ER function. mutation or subclone that could drive relapse13–15. Ki67 expression at surgery following neoadjuvant AI A direct but investigational approach to the diagnosis therapy, in addition to three other independent prog- Neoadjuvant of endocrine therapy resistance in primary tumours is to nostic factors, pathological tumour size, lymph node (also known as preoperative treatment). Refers to therapy assess the Ki67 proliferation index as early as 2–4 weeks, status and ER level, has also been incorporated into a administered before curative or any time afterwards, following the initiation of neo- prognostic tool: the preoperative endocrine prognostic surgery of the primary cancer. adjuvant endocrine therapy (typically an AI16,17 (BOX 1)). index (PEPI)17 (BOX 1), which further distinguishes endo- This is often used to reduce Continued Ki67 expression despite AI treatment identi- crine therapy-sensitive from therapy-resistant tumours tumour size and render large or locally advanced cancers fies oestrogen-independent proliferation that is clearly to guide systemic adjuvant therapy. In the P024 and operable. associated with an increased risk of disease recurrence IMPACT neoadjuvant endocrine therapy trials the early and death16,18–20. A cut-off point of 10% Ki67 expression relapse rate was very low in patients with a PEPI score of Natural logarithm on-treatment has been used to define sensitive versus 0 (pathological stage 1 or 2A, tumour Ki67<2.7%, ER+)17. The natural logarithm (log ) of e intrinsically resistant patient populations for the pur- The 2.7% cut-off point was derived from the finding that 2.7% is ~1. A natural scale poses of genomic discovery and clinical trial design20,21. each natural logarithm increase in the proportion of cells based on multiples of loge=1 was used in the preoperative Clearly, other definitions could also be valid, such as the in a biopsy sample with Ki67 expression was associated endocrine prognostic index because it creates Ki67 intervals, measured during neoadjuvant aromatase Peripheral tissues (e.g. adipose tissue and skin) inhibitor therapy, associated Androstenedione Oestrone (E1) with significant stepwise increased risk of relapse and Aromatase death. Postmenopausal Testosterone Oestradiol (E2) Premenopausal Ovaries and adrenal glands Ovaries Circulation Testosterone Testosterone

Aromatase Androstenedione Breast tumour tissue Androstenedione Oestrone (E1)

Aromatase Oestradiol (E2) Testosterone Oestradiol (E2) FSH

Pituitary ER

Target gene GnRH ER ER CoA Tumour ERE growth Hypothalamus

Figure 1 | Mechanism of action of AIs. In premenopausal women, the ovaries are the major sourceNature of oestrogen, Reviews | andCancer oestrogen biosynthesis is regulated by the hypothalamus and pituitary gland via the actions of gonadotropin-releasing hormone (GnRH) and follicle-stimulating hormone (FSH). In postmenopausal women, oestrogen is synthesized in peripheral tissues such as adipose tissue, breasts and skin through the action of aromatase, which converts androstenedione and testosterone released from ovaries and adrenal glands to oestrone and oestradiol, respectively. In addition, oestrogen receptor-positive (ER+) breast cancer cells could express aromatase, leading to intratumoural oestrogen production. In postmenopausal women, aromatase inhibitors (AIs) effectively reduce oestrogen production. In premenopausal women, however, the reduced oestrogen levels by AIs induce feedback stimulation of the hypothalamus–pituitary–ovary axis; therefore, ovarian function suppression by GnRH agonists or ovarian ablation such as oophorectomy is required for AIs to be used in premenopausal women. CoA, co-activator; ERE, oestrogen response element.

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Pathological complete with a significantly increased risk of relapse, with the patients with intrinsically resistant tumours, defined by response lowest risk associated with a natural logarithm of Ki67 levels of above 10% in tumour biopsy samples at (pCR). Commonly defined as the Ki67 value of 1 (2.7%) or less, or in the rare patient 4 or 12 weeks or by a PEPI score of above 0 at surgery the absence of residual with a pathological complete response (pCR). However, for chemotherapy or an investigational approach. The invasive cancers in the breast and in the axillary lymph PEPI scores above zero (either because of a high dis- genomic analysis of fulvestrant or AI-resistant tumours node following completion of ease burden at surgery, or high Ki67 levels, or both) and targeting them on the basis of their mutational neoadjuvant systemic were associated with an incrementally higher risk of profile is clearly the next step in this therapeutic strategy. therapy, but other definitions relapse. The occurrence rate of PEPI-0 tumours in Ongoing genomic analyses are also underway for exist. neoadjuvant AI therapy trials ranges from 17% to 37%. endocrine therapy-resistant tumours in the advanced Luminal B Patients with PEPI-0 tumours represent a low-risk popu- disease setting to identify progression or resistance One of the five intrinsic lation that could be potentially treated with endocrine mutations and therapeutic targets. Research to produce molecular subtypes of breast monotherapy; that is, no chemotherapy17,23. a cure for advanced disease is much more daunting and cancer characterized by The ongoing Alliance A011106 trial (also known as the acquisition of tumour samples for analysis is more higher expression levels of proliferation genes and lower the ALTERNATE trial) is testing this concept, and this difficult. Nonetheless, survival improvement for patients expression levels of investigation will prospectively validate the hypothesis with advanced disease could translate into better cure oestrogen receptor that PEPI-0 status is associated with a 5-year relapse risk rates in adjuvant trials and new technologies such as (ER)-regulated genes of less than 5% without the administration of chemo- circulating tumour DNA analysis may circumvent the compared with the luminal A therapy (ClinicalTrials.gov identifier: NCT01953588). need for a tissue biopsy for somatic mutation diagnosis24. subtype, and is associated with a poor prognosis. The Alliance A011106 trial will also identify more effective neoadjuvant endocrine therapy regimens by Somatic mutation patterns randomizing patient treatment between fulvestrant, To uncover relationships between somatic mutation pat- anastrozole or a combination of both for 24 weeks before terns and the effectiveness of AI treatment of primary surgery and by assessing the PEPI-0 rate on each arm. luminal breast cancer, massively parallel sequencing of An important aspect of the trial is the identification of 77 pretreatment tumour biopsy samples from patients treated with neoadjuvant AI therapy was conducted21. This study identified 18 significantly mutated genes Box 1 | Diagnosis of AI resistance in early-stage breast cancer (SMGs), including PI3K catalytic subunit-α (PIK3CA), Intrinsic resistance can be diagnosed at 2–4 weeks following the initiation of neoadjuvant TP53 (which encodes p53), GATA3, E-cadherin aromatase inhibitor (AI) therapy if tumour Ki67 levels are over 10%. This strategy (see the (CDH1), retinoblastoma (RB1), mixed lineage leu- figure) is being used in clinical trials of neoadjuvant AI therapies to identify patients with kaemia 3 (MLL3; also known as KMT2C), MAP3K1, resistant tumours for whom treatment with chemotherapy or participation in clinical cyclin-dependent kinase inhibitor 1B (CDKN1B; which trials of novel therapies is recommended. encodes p27), T-box 3 (TBX3), runt-related transcrip- The preoperative endocrine prognostic index (PEPI) score following 4–6 months of tion factor 1 (RUNX1), low-density lipoprotein recep- neoadjuvant AI or other endocrine therapy provides another strategy to identify tor adaptor protein 1 (LDLRAP1), stathmin 2 (STMN2), endocrine-sensitive versus endocrine-resistant tumours in the early-stage setting. A PEPI score of 0 (pT1/2, node-negative (N0), Ki67<2.7%, oestrogen receptor-positive (ER+)) is myosin heavy chain 9 (MYH9), angiotensin II receptor being investigated prospectively as a surrogate of endocrine therapy-sensitive disease type 2 (AGTR2), splicing factor 3b, subunit 1 (SF3B1) 21 that does not need chemotherapy, and a PEPI of >0 identifies patients with an increased and core-binding factor-β subunit (CBFB) . In addi- risk of relapse. The hazard ratio (HR) of each surgical factor for relapse-free survival (RFS) tion, a variety of structural variations including copy and assigned PEPI points based on the data from the P024 trial are shown in the table. number alterations, deletions, translocations and inver- sions were identified. The number of SMGs in ER+

+ breast cancer was further extended to more than 30 ER breast Surgical factors RFS HR PEPI score cancer diagnosis by the work of The Cancer Genome Atlas (TCGA; see 25 2–4 Tumour size Further information) . The complexity of the genomic weeks T1/2 – 0 data presents serious challenges for biostatistical ana lysis. The biological insights into the remarkable func- T3/4 2.8 3 tional diversity of the luminal breast cancer-associated Node status SMGs are only slowly emerging because many of >10%: AI resistant Ki67 Negative – 0 these genes have never previously been linked to the

Neoadjuvant AI Switch therapy Positive 3.2 3 development of breast cancer. An important follow-on approach for unravelling the Ki67 level ≤10%: AI sensitive importance of luminal breast cancer-associated SMGs is 0–2.7% – 0 Continue AI to conduct targeted sequencing studies in a larger num- >2.7–7.3% 1.3 1 ber of samples in which treatment is controlled and long- Surgery >7.3–19.7% 1.7 1 term clinical outcomes are well documented. Although these studies are mostly pending, within the context of PEPI >19.7–53.1% 2.2 2 neoadjuvant AI trials, this approach has produced some >53.1% 2.9 3 preliminary data on the clinical importance of three of the =0: AI sensitive >0: AI resistant ER status highest frequency SMGs: TP53, MAP3K1 and GATA3. Negative 2.8 3 TP53 mutations were correlated with the luminal B AI alone Additional systematic Positive 0 0 subtype and high Ki67 levels before and after treatment, therapy plus AI whereas mutations in MAP3K1 associated with the

Luminal A luminal A subtype and low levels of Ki67 throughout the histone deacetylase 1 (HDAC1), SHC1 and the hypoxia- 21 One of the five intrinsic treatment course . Interestingly, GATA3 mutation cor- inducible factor 1α (HIF1α)–aryl hydrocarbon receptor subtypes of breast cancer related with greater Ki67 suppression after AI treatment nuclear translocator (ARNT; also known as HIF1β) characterized by high but not with baseline Ki67 levels, suggesting that GATA3 complex were associated with high on-treatment Ki67 expression levels of oestrogen 21 receptor (ER) pathway genes mutation is a predictive marker for AI response . expression levels. These findings are consistent with ear- and low expression levels of Further verification of these results will require the ana lier work on the relationship between proliferation and proliferation genes, and lysis of samples with long-term follow-up to confirm the a hypoxia metagene in AI-treated ER+ breast cancer27. associated with a good hypothesis that patients carrying tumours with MAP3K1 In general, these data support the hypothesis that AI prognosis. or GATA3 mutations will have a favourable outcome, resistance is encoded by the mutation patterns present in Patient-derived xenografts whereas those with TP53 mutation will have worse individual tumour genomes, but detailed and validated (PDXs). Xenograft models outcomes. Using an informatics tool to dissect pathway information is yet to emerge. Overall, the mutational generated by engrafting the activation events (PARADIGM), MAP2K4 mutations maps developed from neoadjuvant AI studies empha- cancerous tissue from a patient were also found to be potentially associated with favour- size the genomic heterogeneity that underlies the clinical into an immunodeficient (FIGS 3,4) mouse to generate an able features (such as luminal A and low PEPI scores), heterogeneity of the disease . The next phase of individualized model of their which is logical as MAP2K4 is a substrate for MAP3K1. this research is to screen a much larger number of sam- disease. In addition, TP53, BIRC6, CDKN1B, RUNX1 and the ples to determine the endocrine phenotype of recurrent long non-coding RNA MALAT1 were connected to high somatic mutations in luminal-type breast cancer and to Ki67 values and luminal B status through pathway infor- understand how their interactions promote prognosis, matics26 (FIG. 2). Various pathway analyses were also con- patterns of metastasis and drug response. Drug targets ducted to gain additional insights into the AI-resistance and biological investigations are expected to be priori process. PathScan analysis indicated that these somatic tized in these studies on the basis of somatic events that mutations affected the caspase cascade and apoptosis, lead to poor outcome. It is also possible that clinically ERBB2, AKT–PI3K–mTOR, p53–RB and MAPK–JUN useful AI resistance and sensitivity classifications based N-terminal kinase (JNK) pathways21 (FIGS 3,4). Further on somatic mutation patterns may emerge. PARADIGM analysis for pathway signature identification indicated that p53, MYC, FYN, MAPK, JUN, ESR1 alterations ESR1 point mutation. Oestrogen receptor 1 (ESR1; which encodes ERα) mutation in the region correspond- PAM50 subtype luminal B +1: very correlated ing to the carboxy-terminal ligand-binding domain of Baseline Ki67 the protein (FIG. 5a) is an acquired AI-resistance mecha- Histopathology grade nism. These mutations were identified through the TP53 mutation analysis of ER+ advanced disease samples obtained from BIRC6 mutation CDKN1B mutation patients whose disease had progressed despite long- RUNX1 mutation term endocrine therapy and also from patient-derived MALAT1 mutation xenografts (PDXs) derived from this patient popula- PEPI score 28–31,34–37 End of treatment Ki67 tion . ESR1 mutation has been hypothesized to be a mechanism of AI resistance since the 1990s32. This RB1 mutation 0: uncorrelated PIK3CA mutation hypothesis was initially overlooked because mutations MLL3 mutation in ESR1 are rare in treatment-naive primary breast CDH1 mutation cancer. In a study of eight ESR1 protein-coding exons ATR mutation from 118 ER+ and 70 ER− primary breast cancers, only MAP2K4 mutation two missense mutations (encoding N69K and M396V) Histopathology type were identified, both in the same ER− breast tumour33. PEPI 0 (Ki67<2.7%, Similarly, no mutations in ESR1 were identified from + – ER , N , T1 or T2) the sequencing of 390 ER+ primary breast cancers by MAP3K1 mutation TCGA25. However, a high frequency (ranging from PAM50 subtype luminal A –1: inversely correlated 11% to 55%) of ESR1 mutation was identified in metastatic disease samples, especially in studies of tumours Figure 2 | DiPSC plot illustratingNature correlations Reviews between | Cancer mutations, biomarkers and subtypes. The correlation that have progressed on serial endocrine therapy that of mutations with luminal subtype, Ki67 expression and includes an AI31,34–37. preoperative endocrine prognostic index (PEPI) score is Most patients with tumours harbouring ESR1 muta- shown. ATR, ataxia telangiectasia and Rad3-related; CDH1, tions experienced a protracted clinical course before E-cadherin; CDKN1B, cyclin-dependent kinase inhibitor sample collection for sequencing. Importantly, the 1B; ER, oestrogen receptor; MALAT1, metastasis associated ESR1 mutations were absent in the matched primary lung adenocarcinoma transcript 1; MLL3, mixed lineage tumours at diagnosis, supporting the idea that ESR1 leukaemia 3; N, lymph node; PIK3CA, PI3K catalytic mutation is generally an acquired resistance mechanism subunit-α (encodes p110α); RUNX1, runt-related transcription factor 1. Adapted by permission from the that emerges after long-term treatment with endocrine American Association for Cancer Research: Goldstein, T. C., therapy. The mutations cluster in the ligand-binding Paull, E. O., Ellis, M. J. & Stuart, J. M., Molecular pathways: domain (LBD) with Y537S, C or N and D538G being extracting medical knowledge from high-throughput the most common. These mutations confer ligand genomic data, Clin. Cancer Res., 2013, 19 (12), 3114–3120. (oestrogen)-independent target gene activation and

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Receptor tyrosine kinases ERBB2 PDGFRA EPHA7 CSF1R DDR1 MET KIT

C 7% C 2% C 9% C 9% C 2% C 4% C 2% M 2% M 4% M 2% M 2% M 2% M 2% M 4%

PIK3CA KRAS C 4% C 13% M 52% C 20% FOXO1 PTEN C 7% BRAF M 2% C 15% C 52% C 7% S 4% FOXO3 AKT M 2% C 13% MAP3K4 M 2% M 4% M 9% C 20% FOXO4 S 4% S 2% C 22% MAP2K4 MAP3K1 MAP2K3 S 2%

C 4% C 2% Cell death MAPK8 S 2% M 7% GATA3 MAPK14 C 9% C 28% M 22% C 4% S 4% MDM2 TP53 ATR M 9%

C 17% CDK4 C 4% ATM S 2% CHK1

C 7% C 7% M 4% CDKN1B CDK2 RB1 CHK2 CDC25 M 2% C 7% E2F Cell cycle progression

Figure 3 | Alterations in key cancer pathway components in luminal breast cancers. FrequenciesNature of genetic Reviews alterations | Cancer identified in 46 oestrogen receptor-positive (ER+) breast cancers by whole-genome sequencing are shown. Key pathways affected include receptor tyrosine kinases, PI3K–AKT–mTOR, RAS–RAF–MAPK and p53–RB, leading to cell cycle progression and resistance to cell death, which could potentially subject cells to oestrogen-independent growth. Genes shown in blue are predicted to be functionally activated, and genes shown in yellow are predicted to be functionally inactivated. C refers to copy number alteration, M refers to mutation and S refers to structural variation.ATM , ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; CDC25, cell division cycle 25; CDK, cyclin-dependent kinase; CDKN1B, CDK inhibitor 1B; CHK, checkpoint kinase; CSF1R, colony-stimulating factor 1 receptor; DDR1, epithelial discoidin domain receptor 1; EPHA7, ephrin A7; FOXO, forkhead box protein O; PDGFRA, platelet-derived growth factor receptor-α; PIK3CA, PI3K catalytic subunit-α. Reproduced from REF. 21, Nature Publishing Group.

cell proliferation in preclinical studies31,34–36. Structural and A kinase anchor protein 12 (ESR1e6-AKAP12)31,38. modelling of the mutant ERs demonstrates a constitutive The ESR1e6-YAP1 fusion gene is best documented, agonist conformation through the formation of hydro- as the corresponding fusion gene was identified in an gen bonds between S537 or G538 and N351 in helix 12 endocrine therapy-resistant PDX model derived from (REF. 34). In preclinical studies, treatment with tamoxifen the breast tumour of a patient presenting with primary and fulvestrant was effective, but higher drug concentra- endocrine therapy-resistant stage IV breast cancer. tions were required. This suggests that clinical studies of In transfection studies, ESR1e6-YAP1 induced strong anti-oestrogen dose escalation or potent ER downregula- hormone-independent growth and activation of clas- tors should be considered for patients with ESR1‑mutant sic oestradiol-regulated genes (such as trefoil factor 1 tumours. (TFF1) and progesterone receptor (PGRG))31. Thus, the YAP1 sequences effectively mimic the ligand-activated ESR1 translocation. An additional, recently uncovered transactivation domain in the C terminus of ERα. genetic endocrine therapy resistance mechanism is ESR1 Another class of translocation involving ESR1 involves chromosomal translocation (FIG. 5b). Several in-frame localized gene rearrangements on chromosome 6 fusion genes preserving the first six or seven exons of between ESR1 and coiled-coil domain containing 170 ESR1 (e6 or e7), including the DNA-binding domain and (CCDC170). CCDC170 resides immediately centromeric hinge region, spliced in-frame into the C terminus of to ESR1. These fusions join the 5ʹ untranslated region and another gene, have been identified to date. Examples promoter of ESR1 to the coding region of CCDC170, gen- of these other genes include Yes-associated protein 1 erating the overexpression of amino-terminally truncated (ESR1e6-YAP1), DNA polymerase-η (ESR1e7-POLH), ΔCCDC170 proteins. These gene fusion events were

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ll cycle inhibitor in experimental models with the subsequent d ce 39 e an restoration of oestradiol-induced growth . The hypoth- ag OR am nd mT d KT a esis provoked by this observation is that a SRC inhibitor NA , A s D I3K d GPCR P s an could be used to restore endocrine therapy sensitivity, ase ng TP gnalli and this idea is supported by a Phase II clinical trial of G C si PL 40 (TABLE 1) nd MAPKs the SRC inhibitor dasatinib . 2+ a a ors The prevalence of ESR1 amplification in breast can- C cept Re cer has been the subject of controversy, as it has been NT W reported to occur either rarely or at rates of up to 20% in sis 41–51 pto different studies . The variability of the data could be po A a result of non-standardized detection methodologies, which have inconsistent sensitivity, specificity and cut- off point47,52. Similar to the ESR1 mutation story, ESR1 amplification may be more common in AI-resistant metastatic tumours, and this should be investigated. Overall, ESR1 alteration, whether point mutation, translocation or amplification, is likely to drive acquired resistance to AI treatment, less commonly intrinsic resistance, and represents an opportunity for clinical trials specific to this population. Unlike ERα, the role of ERβ in endocrine responsiveness is much less well understood. Some researchers have proposed that ERβ is associated with responsiveness to endocrine therapy53,54, whereas others have found that ERβ expression led to endocrine treatment resist- ance55. Thus, the role of ERβ in AI resistance remains SMG mutations an unresolved issue.

Figure 4 | Mutations in key cancer pathways in luminal breast cancers. Concentric Crosstalk between GFR signalling and ER circle diagram depicting mutation events in the eight over-representedNature Reviewscanonical | Cancer The aberrant activation of growth factor receptors + networks that occur in oestrogen receptor-positive (ER ) breast cancer (FIG. 3), which (GFRs), including ERBB family receptors56,57, fibroblast could potentially serve as therapeutic targets. There are 77 luminal breast cancers growth factor receptor 1 (FGFR1)58 and insulin-like included, each arranged as a radial spoke and categorized by mutations in each network growth factor 1 receptor (IGF1R)59,60, and their down- and mutation status. GPCRs, G protein-coupled receptors; PLC, phospholipase C; 61,62 SMG, significantly mutated gene. Reproduced from REF. 21, Nature Publishing Group. stream signalling components, including MAPKs and the PI3K–AKT pathway63,64, has been associated with acquired endocrine therapy resistance65 (FIG. 6). found in eight of 200 primary ER+ breast cancers and The dynamic interplay between GFR signalling and ER were enriched in more aggressive luminal B tumours28. expression has long been the subject of study66. Several ΔCCDC170 may engage the GRB2-associated binding investigations have demonstrated that overexpression protein 1 (GAB1) signalosome to potentiate growth fac- of GFR signalling through epidermal growth factor tor signalling and reduce endocrine sensitivity28. Further receptor (EGFR) or HER2 (also known as ERBB2) acti- elucidation of the biology of CCDC170 will be necessary vates MAPK in ER+ breast cancer, leading to the loss to identify a druggable hypothesis. of ERα expression66–68, and inhibition of MAPK can reactivate ER expression and tumour responsiveness ESR1 amplification. Amplification of ESR1 has also been to endocrine therapy69. In addition, activation of GFR reported as an acquired AI-resistance mechanism. For drives downstream kinases to phosphorylate ERα at key example, an ESR1 amplification event was identified in positions, including S118, S167 and T311, to promote a PDX model obtained after the corresponding human interactions with major co-activators, including ampli- ER+ cancer had progressed on AI therapy31. The amplified in breast cancer 1 (AIB1; also known as SRC3 and con in this study extended across both the promoter and NCOA3), and to recruit CREB-binding protein (CBP; the coding regions of ESR1 and was associated with high also known as CREBBP) and p300 to the ER complex levels of ERα expression. Similar to the clinical response to activate transcription70,71. There are several addi- in the patient who provided the tumour specimen, treat- tional ways that these phosphorylation-enhanced co- ment of the PDX model with oestradiol paradoxically activator interactions could drive hormone-independent induced tumour regression rather than tumour growth9. and/or AI-resistant growth, including enhancement of ESR1 amplification is probably an adaptation to oestrogen the non-genomic functions of ERα, whereby ERα has deprivation, and high-level ESR1 amplification has also growth-promoting functions in the plasma membrane been detected in MCF7 cells after long-term endocrine in association with EGFR72 and IGF1R73. therapy; however, the mechanism of oestradiol-induced Another emerging field concerns the modulation regression remains under investigation. Interestingly, of the unstructured nature of the activation function 1 oestradiol-induced apoptosis can be blocked using a SRC (AF1) transactivation domain in the N terminus of ERα.

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(REF. 74) Synthetic lethality Phosphorylation of S118 by MAPK and CDK7 is these alterations in the ER-binding programme have been 82 The simultaneous perturbation thought to be crucial for promoting an active AF1 confor- predictive of poor clinical outcome . Furthermore, mem- of two genes or processes that mation, with recent data showing recruitment of peptidyl brane-bound ER can promote the phosphorylation of results in cellular or organism prolyl cis–trans isomerase NIMA-interacting 1 (PIN1) at signal transduction components (for example, SRC), lead- death, whereas loss of either alone does not. this phosphorylation site. PIN1 expression is correlated ing to a debate regarding the relative importance of the with the proliferative status of experimental tumours and genomic, that is, transcriptional, role of ER versus promotes ligand-independent activity. These emerging the non-genomic signalling role of ER in the biology of data suggest that pathogenic AF1 activation mechanisms endocrine therapy resistance83. Clinical investigations continue to be an important area of investigation75. Indeed, of GFR pathway inhibitors in combination with endocrine dual inhibition of both the ligand-dependent (AF2) therapy in ER+ breast cancer are ongoing, with evidence of domain of ERα and the AF1 domain, is thought to be a key a positive benefit when HER2-targeted agents and endo- advantage of the use of SERDs, such as fulvestrant76. This crine therapies are combined in ER+ HER2+ breast can- could explain why fulvestrant can have therapeutic activity cer, as shown in several clinical trials65,84–87. Unfortunately, after the development of AI-resistant disease. these HER2‑targeted agents have not been as successful In preclinical studies, combined targeting of ER and when HER2 has not been overexpressed or amplified88, the aberrantly activated GFR signalling pathway is often and inhibitors against other GFRs, including EGFR and required to maximize treatment efficacy63,77. Interestingly, IGF1R, have been disappointing in early phase clini- the activation of GFR signalling influences genome-wide cal trials65,89,90. The limited ability to dissect the complex ER DNA-binding patterns — the ‘ER cistrome’ (REF. 78) mechanisms that lead to endocrine resistance in clinical — which is distinct from the classical oestrogen-induced samples and the lack of predictors of response are among ER-binding sites79–81. This is likely to be important because the barriers to progress. From the pharmacological stand- point, feedback mechanisms that are activated in the pres- 91 a ence of tyrosine kinase inhibitors may reduce efficacy . An alternative strategy is to target HER2 mutations that, although rare, occur more commonly in ER+ breast cancer than in ER− breast cancer. A trial with neratinib in HER2- AF1 domain mutant disease is ongoing (ClinicalTrials.gov identifier: DBD Y537C/S/N (43) D538G (32) NCT01670877), and a combination study of an endocrine Hinge domain agent with neratinib is a logical proposition92. LBD and AF2 WW protein–protein interaction domains PI3K pathway in ER+ breast cancer Missense mutation Components of the PI3K–AKT–mTOR pathway are fre- S463P quently altered in breast cancer93 and mutation in the P535H L536R α-catalytic subunit of PI3K (PIK3CA; which encodes

E380Q V392I V534E R555S p110α) is the most frequent genetic abnormality in luminal-type breast cancer (occurring at a frequency of 21,25 ERα 30%–40%) . There is a relationship between favour- 0 100 200 300 400 500 595 able prognosis and PIK3CA mutation, but the correla- Scale (amino acids) tion is modest and does not diminish the importance of this eminently druggable target94,95. ER+ breast cancer b Fusion with YAP1 after exon 6 of ESR1 cells with PIK3CA mutation are highly dependent on the PIK3CA‑encoded protein p110α for cell survival in vitro, ERα-YAP1 as genetically ablating p110α expression or the use of 0 100 200 300 400 500 600 Scale (amino acids) p110α inhibitors can induce apoptosis, particularly when combined with an ER-targeting approach63,77. Activation Figure 5 | ESR1 mutation and ESR1‑YAP1 translocation. A schematicNature Reviews diagram | Cancer (part a) of oestrogen receptor- (ER ) mutations and their frequencies in ER+ of the PI3K pathway has been shown to regulate ER α α 96 metastatic breast cancer after therapy with aromatase inhibitors (AIs) and other expression . Activated PI3K phosphorylates the trans endocrine agents (combined data from five studies34,35,37,179,180). The structural domains cription factor forkhead box O3A (FOXO3A), prevent- of ERα are shown, including the transcription activation function 1 (AF1) domain, the ing the nuclear localization of FOXO3A and binding to DNA-binding domain (DBD), the receptor dimerization and nuclear localization the ESR1 promoter. Thus, inhibition of PI3K induces ER (hinge) domain, and the ligand-binding domain (LBD) and AF2 domain. The WW expression through FOXO3A97–99. This might explain the protein–protein interaction domains in Yes-associated protein 1 (YAP1) are also shown synthetic lethality of combined PI3K and ER inhibition77. in part b. Mutations in the LBD cause oestrogen-independent activation of ERα and In addition, studies of long-term oestrogen-deprived therefore resistance to oestrogen deprivation therapy such as aromatase inhibition. (LTED) breast cancer cell lines have documented the Mutations at Y537 were not always enumerated as to whether they were S, Y or N and activation of the PI3K pathway in the development of therefore the data were compiled together for the figure. A balanced translocation 63,64,100 (part b) between 6q and 11q was observed in a patient-derived xenograft model of an acquired endocrine resistance . AI- and a fulvestrant-resistant tumour that created a transcript encoding the 5ʹ six Targeting mTOR has been one of the few clinical suc- exons of ESR1 (which encodes amino acids 1–365 of ERα) that preserved the AF1, DBD cess stories in the treatment of AI resistance (TABLE 1). For and hinge domain, fused to the carboxyl terminus of YAP1 (amino acids 230–504). patients with resistance to non-steroidal AIs (anastrozole or The substitution of the LBD of ERα by YAP1 sequences leads to oestrogen-independent letrozole), the addition of the rapamycin analogue everoli- signalling and AI and anti-oestrogen resistance. mus to the steroidal AI exemestane (BOLERO-2 trial)101

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Table 1 | Positive trials in endocrine therapy resistance Resistance target Agent (stage of ER agent Median PFS (months) Refs or pathway development) HER2 Trastuzumab (Phase III) Anastrozole 4.8 versus 2.4 85 mTOR Everolimus (Phase III)* Exemestane 10.6 versus 4.1 101 HER2 Lapatinib (Phase III)* Letrozole 8.3 versus 3.0 87 CDK4 and CDK6 Palbociclib (Phase II)‡ Letrozole 20.2 versus 10.2 111 Proteasome Bortezomib (Phase II) Fulvestrant 12‑month PFS 14% versus 28%§ 181 HDAC Entinostat (Phase II) Exemestane 4.3 versus 2.3 126 SRC Dasatanib (Phase II) Letrozole 20.1 versus 9.9 40 CDK, cyclin-dependent kinase; ER, oestrogen receptor; HDAC, histone deacetylase; PFS, progression-free survival. *Phase III studies of lapatinib in ER+ HER2+ breast cancer and everolimus in ER+ HER2– breast cancer in combination with an aromatase inhibitor led to US Food and Drug Administration (FDA) approval for each combination. ‡The Phase II studies listed require Phase III studies for validation, including palbociclib, which has conditional FDA approval pending Phase III data. §No difference in median PFS.

or to tamoxifen (TAMRAD)102 demonstrated improve- so these agents could be delaying the onset of resistance ments in progression-free survival. However, feed- rather than reversing established resistance. The letrozole back activation of AKT as a result of mTOR complex 1 palbociclib combination recently received conditional (mTORC1) inhibition probably limits the efficacy of approval by the US Food and Drug Administration everolimus, and patients in the BOLERO‑2 trial did (FDA). not experience improved overall survival103–105. More Another potential approach to achieve cell cycle con- potent approaches to PI3K–AKT–mTOR pathway inhi- trol in endocrine-resistant ER+ breast cancer is to thera- bition are available, and several other inhibitors of AKT peutically increase the function of p53. This approach or PI3K, including the pan-PI3K isoform inhibitors, dual takes advantage of the fact that mutations in TP53 are PI3K and mTOR inhibitors, and PI3K-isoform‑specific relatively infrequent in ER+ breast cancer (TP53 muta- inhibitors are under investigation106. The identification tions occur in 30% of luminal B and 12% of luminal A of predictive biomarkers for mTOR inhibition remains tumours) compared with basal-like breast cancer (at least an unmet need, as targeted sequencing efforts in the 80% of which have TP53 mutations). However, the expres- BOLERO‑2 trial failed to show a relationship between sion of p53 may be reduced at the protein level owing to somatic mutation patterns and clinical outcomes107. The amplification of E3 ligases that degrade the p53 protein. relationship between PIK3CA and other mutations and An excellent example is MDM2, for which gene copy gain the efficacy of the next generation of PI3K pathway- was shown to occur in 30% of luminal B tumours and in targeting agents will probably have to be addressed pro- 14% of luminal A tumours in TCGA analyses25. Inhibitors spectively. Trials in which PIK3CA mutation status is an of MDM2 increase the expression of the CDK inhibitor eligibility criterion are currently underway. p21 and induce cell cycle arrest in experimental systems. At least one example of an MDM2 inhibitor (MI-773) is Targeting cell cycle regulation currently in early-stage clinical trials112. Analyses by TCGA demonstrated a significant association between deregulated cyclin D–CDK4 or CDK6–RB Epigenetic regulators of ESR1 pathway and luminal B breast cancer25. These higher risk Sequencing analysis, functional studies and clinical trial breast cancers are enriched for cyclin D1 (CCND1) ampli- results all point to a role for aberrant histone and DNA fication (58% in luminal B versus 29% in luminal A), modifications in endocrine therapy resistance and ER+ gain of CDK4 (25% in luminal B versus 14% in lumi- breast cancer pathogenesis. Mutations in MLL3, a gene nal A), and loss of negative regulators such as CDKN2A encoding a DNA-binding protein that methylates his- (which encodes p16) and CDKN2C (which encodes tone H3 Lys4 (H3K4), occur at a frequency of 7% in p18)25, all of which could lead to oestrogen-independent luminal breast cancer25. In addition, an array of cod- cell cycle progression. In addition, various mitogenic or ing mutations and structural variations occur in other growth factor receptor signalling pathways converge on methyltransferase-encoding genes, MLL2 (also known the cyclin D–CDK4 or CDK6–RB axis, and the activation as KMT2B), MLL4 (also known as KMT2D) and MLL5 of CDK4 or CDK6, which leads to RB phosphorylation, (also known as KMT2E), as well as demethylases was associated with resistance to endocrine therapy108. (KDM6A, KDM4A, KDM5B and KDM5C), acetyltrans- As a result, CDK4–CDK6 inhibitors have the poten- ferases (MYST1 (also known as KAT8), MYST3 (also tial to improve the efficacy of endocrine therapy109. known as KAT6A) and MYST4 (also known as KAT6B)) Improvement in progression-free survival has been and several AT-rich interactive domain-containing pro- observed in a clinical trial of palbociclib, a selective inhib- tein genes: ARID1A, ARID2, ARID3B and ARID4B25. itor of the CDK4–CDK6 kinases, in combination with The accumulation of these mutations in luminal-type letrozole versus letrozole alone110,111. As this was a first- breast cancer implies that they may alter the function of line trial, many of the tumours in this study would have ER in a way that promotes carcinogenesis. It also seems had relatively endocrine therapy-sensitive disease, and likely that the pattern of these mutations could be an

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GFRs: • Cytokines Oestrogen Jagged IGF1R, EGFR, HER2, • Hypoxia or Delta FGFR and MET • Stress

PIP2 PIP3 PIP2 PI(3)P SOS SHC ER Notch GRB2 PTEN SHIP1 or INPP4B P P SHIP2 ER ER p38 JNK P P PDK1 Cytosol IRS1 p85 p110 RAS-GDP RAS-GTP PI3K P P mTORC2 AKT RAF LKB1 • GSK3 • FOXO Tuberin MEK • BAD AMPK • ASK1 RHEB

4EBP1 MAPK S6K mTORC1 eIF4B S6 eIF4E

Nucleus P P P P Transcription ER ER FOS, JUN a CoA ER, CoA, FOS, and MYC ERE JUN and MYC

P P Transcription P P P P P P Transcription ER ER b CoR c ER ER CoA ERE AP1 or SP1

Cyclin D1 CDK4 RB E2F S Cell cycle p16 progression MDM2 p53 p18 • Apoptosis • Senescence p21 p27 G1

Figure 6 | Growth factor receptor signalling, PI3K, MAPK, ER and the p53–RB pathway in ER+ breast cancer. Oestrogen-bound oestrogen receptor (ER), in association with a variety of co-activators (CoAs) (part a) and co-repressors (CoRs) (part b), exerts its classical genomic action as a transcription factor through the oestrogen response element (ERE) of target genes. ER can also mediate an ERE-independent genomic effect via interaction with other transcriptionNature Reviews factors, | Cancer such as AP1, through AP1‑binding sites of target genes (part c). In addition, ER can be activated via plasma membrane crosstalk with other growth factor receptor (GFR) pathways that phosphorylate (P) ER or its co-regulators. GFRs — for example, HER2, epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R) and fibroblast growth factor receptor (FGFR) — undergo phosphorylation and dimerization upon ligand activation, which triggers their intracellular association with the regulatory subunit (p85) of PI3K, either directly or indirectly via an intermediate adaptor molecule such as insulin receptor substrate 1 (IRS1). This releases the inhibitory effect of p85 on the catalytic subunit p110 of PI3K. PI3K converts phosphatidylinositol bisphosphate (PIP2) to phosphatidylinositol trisphosphate (PIP3), which allows the activation of AKT and downstream signalling components of the PI3K pathway to promote cell growth, proliferation and survival. PTEN and inositol polyphosphate 4-phosphatase type II (INPP4B) are two phosphatases that negatively regulate the PI3K pathway by dephosphorylating PIP3 and PIP2, respectively. In addition, activation of receptor tyrosine kinases (RTKs) also activates the MAPK pathway. PI3K and MAPK pathway activation leads to phosphorylation of ER and ER coregulators. Cyclin D1 is a direct transcription target of ER or other GFR signalling pathways. Cyclin D1 activates cyclin-dependent kinase 4 (CDK4) and CDK6, which phosphorylates RB, which releases the E2F transcription factors and activates the expression of genes required for the G1 to S phase transition of the cell cycle. CDK4 and CDK6 are negatively regulated by specific and universal inhibitors including p16, p18, p21 and p27. p53 negatively regulates the cell cycle through the activation of p21. MDM2 inhibits p53 function. RB and p53 are important mediators of apoptosis and/or senescence with intrinsic or extrinsic signals such as genotoxic stress. The crosstalk between ER and GFR signalling, as well as constitutive activation of their downstream effectors, are potential mechanisms of oestrogen-independent cell growth of ER+ breast cancer, leading to aromatase inhibitor (AI) resistance. 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; AMPK, AMP-activated protein kinase; ASK1, apoptosis signal-regulating kinase 1; BAD, BCL-2-associated agonist of cell death; eIF4, eukaryotic translation initiation factor 4; FOXO, forkhead box protein O; GRB2, growth factor receptor-bound protein 2; GSK3, glycogen synthase kinase 3; JNK, JUN N-terminal kinase; LKB1, liver kinase B1; mTORC, mTOR complex; PDK1, 3-phosphoinositide‑dependent protein kinase 1; RHEB, RAS homologue enriched in brain; SHIP, SH2 domain-containing inositol 5ʹ phosphatase.

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important determinant in endocrine therapy responsive- this pathway to be involved in adaptive or acquired ness, as well as targets for epigenetic therapy (as discussed resistance. Cancer stem cells (CSCs) are intrinsically below). However, this remains a mostly unexplored resistant to treatment because they are characterized area of research. by certain biological features, such as induction of a At the level of DNA methylation, it is well established G0 state133; overexpression of proteins associated with that epigenetic transcriptional silencing of ESR1 occurs drug efflux and metabolism134,135; high levels of anti- as a result of closed or compact nucleosome structure apoptotic proteins such as survivin and the BCL-2 fam- due to the methylation of CpG islands in the ESR1 pro- ily136; deregulation of signalling pathways including moter113,114. However, histone deacetylation can also con- Notch137, Hedgehog (HH)138, WNT, PI3K–AKT and tribute to the loss of ESR1 expression113–117. Treatment of others129,139; and enhanced DNA damage responses140,141. ER− human breast cancer cell lines with an HDAC inhibi- The existence of CSCs in breast cancer is supported tor or a DNA methyltransferase 1 (DNMT1) inhibitor by the identification of a CD44+CD24− subpopulation leads to the re-expression of ESR1 mRNA114,116,118–121, with of tumour cells that are capable of self-renewal and synergism observed for these two classes of agents when tumour initiation142,143. In ER+ breast cancers, these combined in cell culture studies116,119. HDAC inhibitors CD44+CD24− CSCs show low or absent ER expres- were also capable of restoring endocrine sensitivity in sion and increased PI3K pathway signalling144,145, resistant ER+ breast cancer cells122. However, the mecha- both of which are associated with AI resistance. Gene nisms of action of HDAC inhibition are complex123. For expression analysis of residual tumours following neo- example, HDAC inhibitors have been shown to deplete adjuvant letrozole treatment indicated a significant EGFR expression and downstream signalling121, to induce increase in the CD44+CD22−/low mammosphere sig- the expression of p21 (also known as WAF), an important nature and an elevated expression of mesenchymal- inhibitor of CDKs, and to inhibit p21-activated kinase 1 associated genes146. Similarly, prolonged oestrogen (PAK1), a Ser/Thr kinase that functions downstream of deprivation, which mimicked AI treatment, expanded RAS122,124. Initial success of HDAC inhibitors has been the subpopulation of ER− progesterone receptor (PR)− reported in Phase II clinical trials in patients with meta- cytokeratin 5 (CK5)+ luminobasal cells with basal-like static ER+ breast cancers progressing despite endocrine and claudin-low subtype signatures in an ER+PR+ lumi- therapies, and larger studies are ongoing125,126. nal breast cancer xenograft147. These cells were sensitive to Notch inhibition with γ-secretase inhibitors147. Resistance to cell death Pharmacological interventions to activate cell death to Heterotypic cellular interactions enhance the efficacy of endocrine therapy are of great In addition to cancer cell-autonomous factors, the host interest, as acquired resistance to AIs would be circum- microenvironment, including the extracellular matrix vented if ER+ cells could be induced to die rather than (ECM) and various stromal and immune cells, can con- simply to stop proliferating pending a reactivation event tribute to drug resistance148 (FIG. 7). These are complex (such as a rise in oestradiol owing to the termination of interactions to dissect and can be considered on the basis AI therapy or a mutation in the ERα LBD). Increased of each cell type that is potentially involved148. expression of BCL-2 and/or BCL-W (also known as BCL2L2) has been shown to correlate with endocrine Cancer-associated fibroblasts. Cancer-associated fibro- therapy resistance127. Inhibitors of BCL-2 induced both blasts (CAFs) express high levels of α-smooth muscle apoptosis and autophagy in endocrine therapy-resistant actin (αSMA), which is a marker of myofibroblasts, and breast cancer cells in preclinical models128. they secrete a number of soluble factors that promote Other small-molecule inhibitors targeting nuclear tumour growth and angiogenesis, including transforming factor-κB (NF-κB), which have been implicated in growth factor-β (TGFβ), platelet-derived growth factor apoptosis evasion, and inhibitor of apoptosis proteins receptor (PDGFR), vascular endothelial growth factor A (IAPs), which are key negative regulators of the terminal (VEGFA), hepatocyte growth factor (HGF) and stromal effectors caspase 3 and caspase 7, are also being devel- cell-derived factor 1 (SDF1; also known as CXCL12)149–151. oped129,130. Interestingly, cIAP1 (also known as BIRC2) CAF-secreted HGF was associated with fulvestrant resist- may be pharmacologically engaged for the degradation ance in vitro owing to activation of MET (the HGF recep- of ER therapeutically131. In addition, the activation of tor)152, whereas SDF1 caused ER+ breast cancers to become cIAP2 (also known as BIRC3) by inflammatory cytokines resistant to fulvestrant through binding to CXCR4 and from immune cells in the stroma could help to explain subsequent activation of ERK1, ERK2 and p38 MAPK153. the resistance of ER+ breast cancer cells to apoptosis, and In addition, CAF-derived TGFβ induced epithelial-to- suggests a possible therapeutic use for IAP inhibition132. mesenchymal transition (EMT) of ER+ breast cancer cells148, Bortezomib is a nonspecific proteasome inhibitor that a phenotype that was shown to drive resistance to tamox- blocks the NF-κB pathway, among others, and its com- ifen154. Additionally, endocrine-resistant ER+ breast cancer Epithelial-to-mesenchymal bination with fulvestrant in AI-resistant disease is under cells are reported to gain a more basal phenotype, which transition investigation104 (TABLE 1). is associated with a reduction in E-cadherin expression155, (EMT). A process by which as well as enhanced motility and invasion, through the epithelial cells lose their cell polarity and cell–cell adhesion, Induction of stem cell-like features upregulation of SRC kinase, NF-κB, CD4, TGFβ and and gain migratory and Cancer stem cell-like properties have also been reported receptor tyrosine kinase ligands that trigger changes in invasive properties. in ER+ disease and there is clearly a potential role for gene expression via complex signalling networks156,157.

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CAF Hypoxia shown to promote oestrogen-independent growth of ER+ breast cancer in mouse xenograft models160. The VEGFA • CAV1 loss EMT protective effect of MSCs on cancer cells is thought to • Autophagy be mediated both by direct cell–cell interaction and TGFβ by secreted proteins that include HGF, TGFβ, SDF1, Soluble factors hyaluronan, interleukin-6 (IL-6), IL-8, CCL5 and • Lactate (REF. 148) • Ketone CCL10 . EGFR MET CXCR4 Inflammatory and immune cells. Another aspect of P P P P P P tumour stromal biology is the chronic inflammation Fibronectin MAPK TIGAR that is often present in breast cancer. In the setting of Integrin α persistent chronic inflammation, dynamic interactions Integrin 1 β • Apoptosis P P between the host and breast cancer cells can promote Laminin 5 PI3K–AKT P P • Autophagy 161 Integrin α6 ER ER disease progression and metastasis . In preclinical • Glycolysis + Integrin β4 breast cancer models, IL-4‑expressing CD4 T cells indirectly promote invasion and metastasis by regulat- 162 • CCL2 HER3 ing tumour-associated macrophages (TAMs) . These • CSF1 TAMs are capable of enhancing metastasis through the P HGF, TGFβ, SDF1, Breast hyaluronan, IL-6, activation of EGFR signalling. In addition, TAMs stim- cancer cell P IL-8, CCL5 ulate breast cancer metastasis by activating NF-κB163. and CCL10 Furthermore, intratumoural inflammation results in high levels of prostaglandin E2 (PGE2) and an TAM (M2) B cell inhibitory environment for dendritic cell (DC) and NK TH1 TAM T cell function. PGE2 can induce FOXP3 expression in cell cell (M1) MSC T regulatory (TReg) cells within the tumour microenvironment, thereby altering the phenotype of incoming TReg cell effector T cells to that of TReg cells that suppress intra- Dendritic cell tumoural immune responses164. Consistent with a role in inducing an immune-suppressive effect, tumour MDSC infiltration by TReg cells has been associated with an Figure 7 | The tumour microenvironment and AI resistance. The extracellular matrix increased risk of relapse in patients with ER+ breast can- 165 molecules fibronectin and laminin 5 activate cancer cell growthNature and survival Reviews signalling | Cancer cer . In addition, in the neoadjuvant setting, treatment via integrin receptors. Cancer-associated fibroblasts (CAFs) secrete a number of soluble with letrozole has been shown to reduce intratumoural factors that promote tumour growth and angiogenesis, including transforming growth + + FOXP3 TReg cells, with a greater decrease in FOXP3 factor-β (TGFβ), which promotes epithelial-to-mesenchymal transition (EMT), vascular T cells observed in responding patients after 6 months endothelial growth factor A (VEGFA), which promotes angiogenesis, and factors that of neoadjuvant letrozole therapy166 activate epidermal growth factor receptor (EGFR), MET and CXC chemokine receptor type 4 (CXCR4), leading to activation of PI3K–AKT and MAPK pathways and endocrine In clinical studies, the presence of tumour-infiltrating resistance. In addition, hypoxia-induced lactate and ketone in the tumour T lymphocytes (TILs) and mature DCs correlates with microenvironment upregulates TP53-induced glycolysis and apoptosis regulator (TIGAR) lymph node involvement and tumour grade, and the to inhibit apoptosis. Mesenchymal stem cells (MSCs) protect cancer cells through direct presence of plasmacytoid DCs in primary breast tumours cell–cell interaction and by secreted proteins. In addition, tumour-associated has been correlated with an adverse outcome, suggesting macrophages (TAMs) and immune cells contribute to local inflammation and immune their contribution to the progression of the disease and suppression. There is emerging evidence implicating the contribution of these tumour possibly to endocrine therapy resistance167. Furthermore, cell-protective mechanisms from the tumour microenvironment in aromatase inhibitor in an analysis of gene expression data from 81 paired (AI) resistance. CAV1, caveolin 1; CCL, CC motif chemokine; CSF1, colony-stimulating tumour samples taken at baseline and after 2 weeks of factor 1; ER, oestrogen receptor; HGF, hepatocyte growth factor; IL, interleukin; MDSC, treatment with neoadjuvant anastrozole, higher expres- myeloid-derived suppressor cell; NK cell, natural killer cell; SDF1, stromal cell-derived factor 1; T 1 cell, T helper 1 cell; T cell, T regulatory cell. sion of immune-associated genes such as SLAM fam- H Reg ily member 8 (SLAMF8) and tumour necrosis factor (TNF), as well as lymphocytic infiltration by histologi- Other mechanisms of CAF-induced endocrine resistance cal examination, was associated with poor response168. include the upregulation of TP53-induced glycolysis and Further analysis of the gene expression data identi- apoptosis regulator (TIGAR) in cancer cells owing to the fied the profile most closely aligned to that of DCs169, release of lactate and ketones158. which have been associated with an adverse outcome 168 Plasmacytoid DCs in AI-treated patients . These data suggest a possible Innate immune cells that Mesenchymal stem cells. These pluripotent stem cells avenue for immunotherapy to overcome endocrine circulate in the blood and that are capable of differentiating into connective tissue, resistance. Studies are ongoing to evaluate vaccines that are found in peripheral osteoblasts, adipocytes and chondrocytes. Although target HER2 or carbohydrate antigens such as mucin 1 lymphoid organs, specialized in mesenchymal stem cells (MSCs) predominantly reside (MUC1), as well as immune checkpoint modulators such rapid and massive secretion of type I interferon (IFNα/β) in in the bone marrow, they are found in many other as those antagonizing cytotoxic T lymphocyte-associated response to foreign nucleic tissues and cancers, including breast cancer, and they antigen 4 (CTLA4) and programmed cell death protein 1 acids. promote breast cancer metastasis159. MSCs have been (PD1; also known as PDCD1)170.

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Tumour microenvironment growth could be due to cancer cell-intrinsic mechanisms • ECM: fibronectin, collagen such as the acquisition of resistance mutations; however, • Cellular components: TAM the cell biological mechanisms that are responsible for the (M1, M2), CAF, MSC, MDSC, loss of tumour dormancy and cancer reactivation are TH1 cell, TH2 cell, B cell, ER pathway dendritic cell, NK cell, poorly understood. Evidence suggests that the bone mar- • Loss of ER expression cytotoxic T cell, osteoclast, Growth factor row is a potential sanctuary site where cancer cells reside in • ESR1 mutation, osteoblast and platelet receptor pathway amplification HER2, IGF1R and the haematopoietic stem cell niche, protected from treat- or translocation EGFR overexpression, ment by the biologically active molecules that are produced • Aberrant expression mutation or there173. It has long been recognized that disseminated or mutation of ER amplification co-regulators tumour cells (DTCs) are present in the bone marrow in 30% of patients with localized or early-stage breast can- AI resistance 174 Apoptosis and senescence Secondary messengers cer , and these patients are at a higher risk of both skel- • Mutation of TP53 • PI3K pathway etal and extraskeletal metastasis175. These DTCs can persist • Amplification of MDM2 (e.g. mutations in in the bone marrow for years in a quiescent state and are • BCL-2 and survivin PIK3CA, PTEN and AKT1) ↑ resistant to cancer therapies176. Some DTCs have CSC fea- • ↑ Telomerase • MAPK pathway tures with high expression of SNAIL, a transcription factor involved in EMT induction177. Interventions that alter Cell cycle machinery EMT and CSC • Loss of RB, p16 and p18 • Notch, Hedgehog, bone physiology, such as bisphosphonates, clearly have the • Amplification WNT and TWIST1 potential to favourably alter the natural history of DTCs in of CCND1 • Tumour dormancy the bone marrow, at least in postmenopausal women178.

Figure 8 | The hallmarks of AI resistance. Several cell-autonomous and non-cell- Conclusions + Nature Reviews | Cancer autonomous mechanisms in oestrogen receptor-positive (ER ) breast cancer and the The FDA approvals for the delay in tumour progression tumour microenvironment could lead to aromatase inhibitor (AI) resistance. These include achieved by everolimus and the CDK4 and CDK6 inhibi- deregulation of the ER pathway, growth factor receptor signalling, secondary messengers, tor palbociclib in combination with an AI promote opti- the cell cycle machinery, apoptosis and senescence, epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSCs), tumour dormancy, and the tumour mism that considerable improvements in the treatment + microenvironment. CAF, cancer-associated fibroblast; CCND1, cyclin D1; ECM, of AI therapy-resistant ER breast cancer are a near-term extracellular matrix; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth possibility. However, there is currently no evidence to factor 1 receptor; MDSC, myeloid-derived suppressor cell; MSC, mesenchymal stem cell; date that these dual-targeting approaches improve overall

NK cell, natural killer cell; TAM, tumour-associated macrophage; TH, T helper. survival or are active adjuvant treatments. Improvements in the fidelity of preclinical models are therefore essential, and PDX models clearly have a role in moving the Tumour dormancy and disseminated tumour cells endocrine resistance field beyond the restricted discov- Tumour dormancy is defined as the state of microscopic ery space afforded by the few ER+ cell lines available31. non-progressing metastases that remain capable of pro- However, the value of PDX models for the study of gression to overt advanced disease after a long period of tumour dormancy, stem cell-like behaviour and stro- time. The natural history of ER+ breast cancer indicates mal–epithelial interactions is unclear. Nonetheless, PDX that micro-metastatic ER+ cancer cells are in a reversible studies, paired with data from the omics analysis of clini- dormant state and the long-term efficacy of endocrine cal samples, should instruct the design of trials of more therapy, including AI therapy, involves the maintenance of effective multi-agent combinations, individually tailored the dormant state rather than the induction of cell death or to resistance mechanisms diagnosed through sequencing irreversible cell cycle arrest171,172. Reactivation of cancer cell or other omics techniques (FIG. 8).

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