Differences in the Transcriptional Response to Fulvestrant and Estrogen Deprivation in ER-Positive Breast Cancer

Published OnlineFirst June 10, 2014; DOI: 10.1158/1078-0432.CCR-13-1378

Clinical Cancer Personalized Medicine and Imaging Research

Neill Patani1,2, Anita K. Dunbier1,5, Helen Anderson2, Zara Ghazoui1, Ricardo Ribas2, Elizabeth Anderson3,6, Qiong Gao2, Roger A'hern4, Alan Mackay2, Justin Lindemann3, Robert Wellings3, Jill Walker3, Irene Kuter7, Lesley-Ann Martin2, and Mitch Dowsett1,2

Abstract Purpose: Endocrine therapies include aromatase inhibitors and the selective estrogen receptor (ER) downregulator fulvestrant. This study aimed to determine whether the reported efficacy of fulvestrant over anastrozole, and high- over low-dose fulvestrant, reflect distinct transcriptional responses. Experimental Design: Global gene expression profiles from ERa-positive breast carcinomas before and during presurgical treatment with fulvestrant (n ¼ 22) or anastrozole (n ¼ 81), and corresponding in vitro models, were compared. Transcripts responding differently to fulvestrant and estrogen deprivation were identified and integrated using Gene Ontology, pathway and network analyses to evaluate their potential significance. Results: The overall transcriptional response to fulvestrant and estrogen deprivation was correlated (r ¼ 0.61 in presurgical studies, r ¼ 0.87 in vitro), involving downregulation of estrogen-regulated and proliferation-associated genes. The transcriptional response to fulvestrant was of greater magnitude than estrogen deprivation (slope ¼ 0.62 in presurgical studies, slope ¼ 0.63 in vitro). Comparative analyses identified 28 genes and 40 Gene Ontology categories affected differentially by fulvestrant. Seventeen fulvestrant-specific genes, including CAV1/2, SNAI2, and NRP1, associated with ERa, androgen receptor (AR), and TP53, in a network regulating cell cycle, death, survival, and tumor morphology. Eighteen genes responding differently to fulvestrant specifically predicted antiproliferative response to fulvestrant, but not anastrozole. Transcriptional effects of low-dose fulvestrant correlated with high-dose treatment, but were of lower magnitude (ratio ¼ 0.29). Conclusions: The transcriptional response to fulvestrant has much in common with estrogen deprivation, but is stronger with distinctions potentially attributable to arrest of estrogen-independent ERa activity and involvement of AR signaling. Genes responding differently to fulvestrant may have predictive utility. These data are consistent with the clinical efficacy of fulvestrant versus anastrozole and higher dosing regimens. Clin Cancer Res; 1–12. 2014 AACR.

Introduction Aromatase inhibitors, for example, anastrozole, cause pro- Endocrine therapies abrogate estrogenic signaling found postmenopausal estrogen suppression and are used through distinct mechanisms, impeding estrogen synthesis as first-line neoadjuvant, adjuvant, and metastatic thera- or transcriptional activity of estrogen receptor alpha (ERa). pies. Selective estrogen receptor modulators (SERM), for example, tamoxifen, exert partial agonist activity. In contrast, the selective ER downregulator (SERD) fulvestrant Authors' Affiliations: 1Academic Biochemistry, Royal Marsden Founda- (Faslodex, AstraZeneca) is a pure antiestrogen, inhibiting 2 tion Trust; Breakthrough Breast Cancer Research Centre, Institute of receptor dimerization, nuclear uptake, estrogen-response Cancer Research, London; 3AstraZeneca, Macclesfield; 4Clinical Trials and Statistics Unit, Institute of Cancer Research, Sutton, United Kingdom; element binding, and accelerating ERa degradation (1–5). 5Department of Biochemistry, University of Otago, Dunedin, New Zealand; Fulvestrant is licensed for postmenopausal progression/ 6Boehringer Ingelheim, Vienna, Austria; and 7Massachusetts General Hos- pital, Boston, Massachusetts relapse on first-line endocrine therapy (6–8). Low-dose fulvestrant (250 mg/28 days) provides comparable disease Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). outcome to anastrozole following first-line tamoxifen in metastatic disease (6–8), with utility after progression on Corresponding Author: Mitch Dowsett, Academic Department of Bio- chemistry, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, aromatase inhibitors (9–11). As first-line therapy in metasta- United Kingdom. Phone: 44-207-808-2885; Fax: 44-207-376-3918; tic or locally advanced disease, low-dose fulvestrant provides E-mail: [email protected] comparable disease outcome to tamoxifen (12). High-dose doi: 10.1158/1078-0432.CCR-13-1378 treatment (500 mg on day 0, 14, 28, monthly thereafter) 2014 American Association for Cancer Research. further improved progression-free (13) and overall survival

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ceptible to both aromatase inhibitors and fulvestrant. Con- Translational Relevance temporary models also include activities which do not Aromatase inhibitors are first-line postmenopausal require interaction and may involve either estrogen or ERa agents for estrogen receptor alpha (ERa)-positive breast independently (refs. 21–25; Fig. 1A). Such nonclassical cancer. However, there is considerable response hetero- activities might be affected selectively by aromatase inhibi- geneity and women frequently relapse. Estrogen depri- tors and fulvestrant, respectively (Fig. 1B). In vitro, the vation does not completely arrest ERa activity, and greater antiproliferative effect of fulvestrant (26) has been transactivation of the unliganded receptor may continue attributed to continued ERa activity following estrogen through cross-talk with growth factor pathways. In con- withdrawal, with hypersensitivity to residual estrogen trast with aromatase inhibitors, the selective ER down- and/or estrogen-independent interactions between ERa regulator fulvestrant also abrogates ligand-independent and growth factor pathways. ERa has recently been shown ERa activity. The benefit of fulvestrant as an alternative, to retain genomic binding activity following estrogen with- combination, or sequential therapy to aromatase inhib- drawal and drives a CDK4/E2F-dependent transcriptional itor has been reported, but molecular mechanisms program. Such ligand-independent ERa activity has partic- underpinning its relative efficacy remain unclear and ular relevance to de novo and acquired aromatase inhibitor biomarkers for patient selection are lacking. This study resistance, where ERa is frequently expressed and fulves- demonstrates, for the first time, that the overall tran- trant may remain effective (9, 10, 27). scriptional response to fulvestrant is of greater magni- In this study, global gene expression profiles from tude than estrogen deprivation, consistent with its clin- presurgical studies of fulvestrant or anastrozole, and corre- ical efficacy and more complete blockade of estrogenic sponding in vitro models, were assessed. The primary objec- signaling. Using a robust integrative approach, we iden- tive was to compare and contrast transcriptional responses. tify a subset of genes differentially affected by fulvestrant Secondary objectives included evaluating the biologic that comprises distinct biologic networks, correlates response to low- and high-dose fulvestrant and the extent with antiproliferative response, and has potential utility to which transcriptional consequences were attributable to as predictive biomarkers for fulvestrant. ERa depletion.

Materials and Methods (14) in the COmparisoN of Faslodex In Recurrent or Meta- Presurgical study of fulvestrant static breast cancer (CONFIRM) trial. The Fulvestrant fIRst- Pre- and on-treatment (4-week) core biopsies stored at line Study comparing endocrine Treatments (FIRST) found 20 C in RNA-later (Qiagen) were available from NEWEST an improved time-to-progression with the high-dose com- (ClinicalTrials.gov-NCT00093002; ref. 17; Supplementary pared with anastrozole (1 mg/day) in advanced disease Fig. S1). This phase II study recruited postmenopausal (15, 16). In the Neo-adjuvant Endocrine therapy for Women women with untreated, potentially operable, locally with Estrogen-Sensitive Tumours (NEWEST) trial of locally advanced, ERa-positive, primary invasive cancer 2 cm. advanced disease, high-dose fulvestrant showed greater sup- No data were available for HER2 status. Randomization was pression of ERa, progesterone receptor (PgR), the prolifera- to low- (250 mg/28 days) or high-dose (500 mg on days 0, tion marker Ki-67, and radiological response than low dose 14, 28, monthly thereafter) fulvestrant. The on-treatment (17). These data show signiﬁcant differences between ful- biopsy was taken before the day 28 dose of fulvestrant in vestrant dosing schedules and a mechanism of action which both arms of the study. RNA was extracted with RNeasy, is different to, and may circumvent complete cross-resistance assessed using an Agilent Bioanalyser (Santa Clara) and with, SERMs and aromatase inhibitors. The molecular rejected if RNA integrity number was <5. Following exclu- mechanisms which underpin these clinically important dif- sions, 22 high-dose and 16 low-dose pre-/on-treatment ferences are incompletely understood. pairs were available (Supplementary Fig. S1). The transcriptional response to fulvestrant differs from SERMs, with the latter upregulating particular estro- Presurgical study of anastrozole gen-regulated genes (ERG; ref. 18). In addition to more Pre- and on-treatment (2- and 16-week) core biopsies complete ERG antagonism, fulvestrant exclusively down- were available from postmenopausal women receiving regulates numerous cell cycle, proliferation, and DNA syn- anastrozole monotherapy (1 mg/day) within a randomized thesis genes in vitro (19), and some estrogen-suppressed phase II neoadjuvant trial of anastrozole alone or with genes are upregulated by fulvestrant and not by tamoxifen geﬁtinib in early disease (ClinicalTrials.gov-NCT00255463; (20). The transcriptional response to aromatase inhibitors ref. 28). This subgroup constitutes the Functional and fulvestrant has not previously been compared and may Aromatase Inhibitor Molecular Study (FAIMoS; ref. 29; be pertinent to the clinical utility of fulvestrant as an Supplementary Fig. S1B and S1C). Following exclusions, alternative, sequential, or combination therapy. The poten- 81 two-week and 18 sixteen-week pairs were available tial for difference is supported by their contrasting effects on (Supplementary Fig. S1). Written informed consent was estrogen and ERa. The interaction between estrogen and obtained from each subject and investigations performed ERa underpins classical estrogenic signaling which is sus- after approval by a local Institutional Review Board.

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AB Activity of estrogen

ERα independent estrogen activity Estrogen ERα ERα- Estrogenic Estrogen- levels levels independent ERα activity independent estrogenic ERα activity activity

Estrogenic ERα activity SERD

Estrogen independent ERα activity

Activity of ERα

Figure 1. A, summary of estrogen-dependent ERa signaling (yellow) and the potential independent activities of estrogen (blue) and ERa (red). B, comparison of the impact of aromatase inhibitors and SERDs on classical and nonclassical estrogenic signaling, illustrating activities targeted by both agents (yellow) and those affected speciﬁcally by aromatase inhibitors (blue) or fulvestrant (red). Reduced activity is indicated by downward arrows (solid black) and no change is indicated by horizontal arrows (black outline).

In vitro modeling of fulvestrant or estrogen ing study of ten high-dose pairs from NEWEST and ten two- deprivation week pairs from FAIMoS - HumanHT-12v4, and (iv) in vitro MCF7 cells (ATCC) were cultured in phenol red-free samples - HumanHT-12v4. RPMI-1640, 10% FBS (Gibco Life Technologies), and 1 nmol/L 17b-estradiol (E2). Cells were stripped of steroids Global gene expression analysis for 48 hours in phenol red-free RPMI with 10% dextran- Raw expression data were extracted with BeadStudio, coated charcoal-stripped FBS (DCC). Cells were seeded into transformed by variance-stabilizing transformation and nor- 6-well plates at a density of 3 105 cells/well for 24 hours. malized using Robust Spline Normalization in the Lumi Monolayers were: (i) harvested at this stage, that is, follow- package in Bioconductor. Probes were excluded if they were ing 72 hours of estrogen deprivation (modeling aromatase not present in any samples (detection P>1%). Microarray inhibitor), (ii) treated for 48 hours with 0.1 nmol/L E2 in data are publicly available (29–32). Expression data and DCC (modeling baseline), or (iii) treated for 48 hours with annotation files (HumanHT-12_V4_0_R2_15002873_B, 10 nmol/L fulvestrant and 0.1 nmol/L E2 (modeling ful- HumanWG-6_V3_0_R3_11282955_A, and HumanWG- vestrant). Experiments were conducted in triplicate and 6_V2_0_R4_11223189_A) were imported into Partek Geno- RNA extracted using RNeasy (Qiagen). mics Suite (PGS, 6.6_6.12.0531, Partek Incorporated). Class comparison of pre- and on-treatment clinical sam- Microarray-based global gene expression profiling ples used two-way ANOVA. Treatment status (i.e., pre- or RNA was quantified, amplified, labeled, and hybridized on-treatment) was considered a categorical variable with onto Expression BeadChips (Illumina). Samples were fixed effect (as assignment represents all conditions of processed with the following BeadChips: (i) FAIMoS - interest). Pre- and on-treatment samples were paired HumanWG-6v2, (ii) NEWEST - HumanWG-6v3, (iii) bridg- according to their patient identifier, which was considered

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a random effect variable which encompassed inter-patient (Pearson r ¼ 0.36, P < 0.0001), albeit of lesser magnitude variability (given that patients represent a random sample (slope ¼ 0.29; Fig. 2A; Deming linear regression), support- of all possible patients). Transcripts differentially expressed ing a quantitative difference between the dosing schedules. between fulvestrant-treated, estrogen-deprived, and control None of the alterations in gene expression induced by low- conditions in vitro were identified by ANOVA. FDR of 5% dose treatment were statistically significant after multiple was used to correct for multiple testing. testing corrections (FDR<0.05). However, downregulation of individual proliferation-associated (e.g., AURKA)or Analytical strategy estrogen-regulated (e.g., PGR, PDZK1, and GREB1) genes Matched Illumina probe identifiers were used to enable reached significance (uncorrected P < 0.05; Supplementary valid comparisons between BeadChips. All detected probes Table S1). In contrast, 2,210 transcripts were significantly from HumanWG-6v3 (n ¼ 22550) were used to evaluate affected (977 upregulated and 1,233 downregulated, fulvestrant dosing regimens. Comparisons between NEWEST FDR<0.05) in the high-dose cohort. Further comparative and FAIMoS used detected probes common to HumanWG- analyses were undertaken with only high-dose treated 6v3 and HumanWG-6v2 (n ¼ 15051), followed by an patients to avoid the potential for false negativity by inclu- unpaired t test of treatment-induced alterations. Detected sion of those receiving low-dose fulvestrant. probes common to HumanHT-12v4, HumanWG-6v3, and HumanWG-6v2 (n ¼ 11122) were assessed in vitro.Biologic Similarities in the transcriptional response to interpretation involved: (i) identification of functional fulvestrant and estrogen deprivation groupings from the Gene Ontology database, by Gene Ontol- The overall transcriptional response to anastrozole and ogy ANOVA in PGS and (ii) network analyses with Ingenuity high-dose fulvestrant in presurgical studies was signifi- Pathway Analysis (IPA; Ingenuity Systems). cantly correlated (Pearson r ¼ 0.61, P < 0.0001; Fig. 2B and C), as were those of estrogen deprivation and fulves- Technical validation of microarray findings for selected trant in vitro (Pearson r ¼ 0.87, P < 0.0001; Fig. 2D). In genes both settings, ERGs (e.g., PDZK1, PGR, GREB1,andTFF1) Expression of two selected genes, CAV1 and SNAI2, was were significantly downregulated by estrogen deprivation assessed by qRT-PCR of the same RNA preparations used for and fulvestrant; full listings are provided in Supplemen- expression profiling of high-dose fulvestrant (n ¼ 20) and 2- tary Tables S2–S4. week anastrozole (n ¼ 31)-treated patients. TaqMan assays Proliferation-associated genes were also downregulated (Applied Biosystems) were used to quantify CAV1 by estrogen deprivation (e.g., TOP2A, CDCA5, CDC20, (Hs0971716_m1) and SNAI2 (Hs0950344_m1), which CCNB2, AURKA, and E2F2) and fulvestrant (e.g., TOP2A, were normalized to FKBP15 (Hs0391480_m1) and PUM1 CCNA2, CCNB2, CCND1, CDCA5, CDCA7, CDCA8, (Hs0982775_m1). CDC2, CDC20, CDC25C, AURKA, and POLE; detailed in Supplementary Tables S2–S4). Proliferation-associated ESR1 knockdown in MCF7 cells Gene Ontology sets, pathways, and networks were prom- MCF7 cells were seeded into DCC at a density of 7 104 inent in transcripts affected by both treatments (Supple- cells/well in 12-well plates. After 24 hours, monolayers were mentary Tables S5–S8). Ki-67 staining was comparably transfected with 50 nmol/L of siRNA targeting ESR1 (ON- reduced in samples from patients receiving anastrozole TARGETplus 003401, Dharmacon, ThermoFisher), or non- (geometric mean of post-/pretreatment expression ¼ targeting siRNA using DharmaFECT 3 reagent. Media (0.1 24.8% after 2 weeks, n ¼ 69) and fulvestrant (19.6%, nmol/L E2 in DCC) were replenished the following day and following high-dose treatment, n ¼ 22). cells were cultured for 24 hours. TaqMan assays were used to quantify ESR1 (Hs01046818_m1) and selected genes: Differences in the transcriptional response to BRI3 (Hs0854645_g1), CAV1, CAV2 (Hs0184597_m1), fulvestrant and estrogen deprivation CCDC34 (Hs0293234_m1), CYP26B1 (Hs01011223_m1), The overall transcriptional response to high-dose fulves- C9orf140 (Hs0746788_s1), FNTA (Hs0357739_m1), trant was of greater magnitude than anastrozole in presur- GTSE1 (Hs0212681_m1), HMGN4 (Hs01549435_m1), gical studies (slope ¼ 0.62, Fig. 2B). This difference was LRP8 (Hs0182998_m1), NRP1 (Hs0826128_m1), NSUN2 supported by the bridging study (slope ¼ 0.56, Fig. 2C), (Hs0214829_m1), RBMS1 (Hs0377856_m1), RECQL4 excluding batch separation as a potential explanation, and (Hs01548660_g1), SEPP1 (Hs01032845_m1), SLC7A5 consistent with the greater impact of fulvestrant compared (Hs0185826_m1), SNAI2, and STRA13 (Hs0414534_m1), with estrogen deprivation in vitro (slope ¼ 0.63, Fig. 2D). To which were normalized to FKBP15. determine whether any transcripts were affected differently by the agents, rigorous comparisons were undertaken following the scheme illustrated in Fig. 3A. Treatment-related Results transcripts were identified that met the following criteria: (i) Transcriptional response to high- and low-dose expression changed significantly after treatment in clinical fulvestrant samples and corresponding in vitro models (FDR<0.05), (ii) The overall transcriptional response to low-dose fulves- expression was affected differently by the two agents in vitro trant was significantly correlated with that to high-dose (discovery set, FDR<0.05) and in presurgical studies

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A B Pearson r = 0.36 (P < 0.0001) Pearson r = 0.61 (P < 0.0001) Slope = 0.29 Slope = 0.62 (post/pre) (post/pre) 2 (post/pre) (post/pre) 2 log log Mean change with 250 mg fulvestrant Mean change with 1 mg anastrozole

Mean change with Mean change with 500 mg fulvestrant 500 mg fulvestrant log (post/pre) 2 log2 (post/pre )

C D Pearson r = 0.64 (P < 0.0001) Pearson r = 0.87 (P < 0.0001) Slope = 0.56 Slope = 0.63 (post/pre) (post/pre) 2 log E2 deprivation deprivation E2 1 mg anastrozole (DCC/0.1 nmol/L E2) nmol/L E2) (DCC/0.1 Mean change with Mean change with 2 log

Mean change with Mean change with 500 mg fulvestrant 10 nmol/L fulvestrant log2 (post/pre ) log2 (10 nmol/L fulvestrant/ 0.1 nmol/L E2)

Figure 2. Scattergrams illustrating the overall transcriptional response to fulvestrant or estrogen deprivation: A, low-dose compared with high-dose fulvestrant. B, high-dose fulvestrant compared with 2 weeks of anastrozole. C, bridging study of high-dose fulvestrant compared with 2 weeks of anastrozole. D, MCF7 cells modeling fulvestrant treatment and estrogen deprivation.

(validation set, uncorrected P < 0.05), and (iii) changes in 17, McNemar test); 11 genes were downregulated signifi- expression were consistent in clinical samples and corre- cantly more by fulvestrant than by estrogen deprivation sponding in vitro models (direction of change and relative (e.g., CCDC34, RECQL4, SLC7A5, and SAPCD2). Of the treatment effect). Transcript sets from Fig. 3A are detailed in three genes significantly upregulated by estrogen depriva- Supplementary Table S2. tion, only one (DCXR) was not similarly affected by fulves- Fulvestrant-related transcripts (n ¼ 41, 13 upregulated trant, whereas for the 13 upregulated by fulvestrant, 11 were and 28 downregulated) and estrogen deprivation–related not similarly affected by estrogen deprivation (e.g., CAV1, transcripts (n ¼ 18, 3 upregulated and 15 downregulated) CAV2, SNAI2, and NRP1, P ¼ 0.004 for the comparison of were then assessed for whether differences were treatment those not similarly upregulated); two transcripts (e.g., specific or quantitative (Fig. 3A). Of the 15 genes down- ZMAT3) were upregulated significantly more by fulvestrant regulated by estrogen deprivation, four (KCNK6, KCNK15, than estrogen deprivation. Treatment-related and -specific RASGRP1, and TUBA3E) were not similarly affected by alterations are summarized in Fig. 3C, i; detailed in Sup- fulvestrant, whereas for the 28 downregulated by fulves- plementary Table S9. trant, 17 were not similarly affected by estrogen deprivation To assess whether transcript differences might be suffi- (e.g., GTSE1, LRP8, NSUN2, and STRA13, P ¼ 0.005 for the cient to influence distinct biologic processes, differentially comparison of those not similarly downregulated, i.e.. 4 vs. affected Gene Ontology sets were identified in the same

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A ED C ED F F C ED C ED F F C

Significantly affected transcripts 3,046 5,011 2,274 1,714 (FDR < 0.05)

Differentially affected transcripts 1,736 1,171 (FDR < 0.05) (P < 0.05) Significantly and differentially affected transcripts 1,010 1,569 232 538

Transcripts common to clinical studies and in vitro models 41 66

Transcripts responding consistently in clinical studies and in vitro models 18 41

Treatment-related or treatment- specific transcript alterations 1 2 11 4 11 17 Figure 3. Summary of analytical strategy with (A) resultant transcripts and (B) Gene Ontology Upregulated Downregulated functional gene sets. Key: In vitro models (yellow), presurgical studies (grey), control conditions in B ED C ED F F C ED C ED F F C vitro/pretreatment clinical samples (C, black), estrogen deprivation Significantly affected GO sets 3,051 4,176 2,583 1,969 (ED, blue), fulvestrant (F, red), ED (FDR < 0.05) and F (green). Transcripts and Differentially affected GO sets Gene Ontology sets resulting from (FDR < 0.05) 2,560 490 comparative analyses are detailed Significantly and differentially affected in Web Appendix 1. C, summary of GO sets 1,655 2,333 170 217 (i) transcripts and (ii) Gene Ontology functional gene sets affected by GO sets common to clinical studies and estrogen deprivation (ED, blue), in vitro models 76 96 fulvestrant (F, red), or both (green), GO sets responding consistently in with treatment-speciﬁc and clinical studies and in vitro models 50 84 quantitative differences indicated by horizontal and vertical stripes, respectively. Treatment-related or treatment-specific 2 12 37 4 32 3 GO set alterations

Upregulated Downregulated C (i) ED F (ii) ED F

Upregulated Upregulated

Downregulated Downregulated 4

manner (Fig. 3B; detailed in Supplementary Table S2). pathway, and negative regulation of: ERK1/2 cascades, Fulvestrant downregulated 32 Gene Ontology sets signifi- TGFb receptor signaling, canonical Wnt receptor signaling, cantly more than estrogen deprivation. Common down- and epithelial cell proliferation, P < 0.001 for the compar- regulated sets were predominantly proliferation related ison of those not similarly upregulated). Some Gene Ontol- (e.g., DNA helicase activity, microtubule motor activity, ogy sets specifically upregulated by fulvestrant pertained and spindle assembly; Supplementary Table S10). Of the to fulvestrant-specific upregulated genes (e.g., caveolae 14 Gene Ontology sets upregulated by estrogen deprivation, assembly and CAV1/CAV2). Fulvestrant upregulated 12 two were not similarly affected by fulvestrant, whereas for Gene Ontology sets significantly more than estrogen dep- the 49 upregulated by fulvestrant, 37 were not similarly rivation (e.g., negative regulation of MAPK cascade). Treat- affected by estrogen deprivation (e.g., apoptotic signaling ment-related and -specific alterations in Gene Ontology

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sets are summarized in Fig. 3C, ii; detailed in Supplemen- directly, whereas those upregulated correlated inversely, tary Table S10. with baseline AURKA and ESR1 (summarized in Supplementary Fig. S2 and detailed in Supplementary Pathway and network analysis of genes differentially Table S11). affected by fulvestrant Canonical pathways associated with fulvestrant-specific Treatment-related gene alterations and genes (n ¼ 28, 11 upregulated and 17 downregulated) antiproliferative response included signaling through heterotrimeric G-proteins To determine whether baseline expression of treatment- (b-/g-subunits) and estrogen-mediated S phase entry (Table related genes (n ¼ 46) was associated with antiproliferative 1). One principal network (IPA score ¼ 40) included 17 of response, their pretreatment levels were correlated with 28 fulvestrant-specific genes with functions related to cell change in AURKA expression. Twenty-six genes correlated cycle, death, survival, and tumor morphology (Fig. 4). Six with response to anastrozole or high-dose fulvestrant fulvestrant-specific upregulated genes were present, with (uncorrected P < 0.05, summarized in Fig. 5A and detailed CAV1/2, SNAI2, and NRP1 forming a core group associated in Supplementary Table S12). Twenty-three fulvestrant- directly and/or indirectly with the focal points of ERa, related genes correlated with response to high-dose fulves- androgen receptor (AR), and TP53. Members of the trant, with five of 23 (C9orf140, POLD2, SAC3D1, MAPK/ERK family and the serine/threonine protein kinase ZMYND19, and STRA13) also doing so in the low-dose (AKT) were prominent. Eleven fulvestrant-specific down- treated cohort (Supplementary Table S12c). Notably, 18 of regulated genes occupied the network periphery. 23 were not associated with response to anastrozole. Ful- To determine whether this functional alliance of vestrant response was specifically associated with low pre- fulvestrant-specific genes was specifically induced by, treatment expression of two genes (SNAI2 and NRP1) and attributable to, congruent response to fulvestrant, found to be upregulated by fulvestrant, and high expression their pretreatment expression was assessed. Fulvestrant- of 16 genes found to be downregulated by fulvestrant; of related genes correlated significantly with one another these C9orf140, LRP8, and CCDC34 had predictive signif- at baseline; upregulated genes were coexpressed, as were icance independent of pretreatment AURKA expression (P < downregulated genes, and these two groups were already 0.05, Fig. 5B, i–iii. inversely correlated, implicating a preexisting regula- Pretreatment expression of five genes had predictive tory system. Fulvestrant downregulated genes correlated significance for both agents and their correlation was

Table 1. Ingenuity-based biologic interpretation of genes differentially affected by fulvestrant in presurgical studies and in vitro models: Network functions and canonical pathways

Canonical pathways P Genes G bg signaling 9.120E03 CAV1, CAV2 Estrogen-mediated S-phase entry 3.890E02 SKP2 Antiproliferative role of TOB in T-cell signaling 4.169E02 SKP2

Cell cycle: G2–M DNA damage checkpoint regulation 6.761E02 SKP2 Semaphorin signaling in neurons 8.128E02 NRP1

Molecules in network Score Focus Top functions molecules Akt, Alphatubulin, AR, CAV1, CAV2, CD55, CHTF18 40 17 Cell cycle, cell death and survival, CXCL12, E2f, EMC9, EP300, ERK, ER, EWSR1, tumor morphology FANCA, FNTA, GTSE1, HBEGF, HIF1A, LRP8, NRP1 (includes EG:18186), P38 MAPK, POLH, PPME1, RBM14, RFC3, SEPP1, SKA2, SKP2 (includes EG:27401), SMARCA4, SNAI2, STRA13, TFF3, TJP2, TP53 (includes RBMS1, SUZ12 2 1 Embryonic development, cancer, skeletal and muscular disorders CD63, SYTL4 2 1 Cell morphology, endocrine system development and function, nervous system development and function FBL, NOP56 2 1 RNA posttranscriptional modiﬁcation, hereditary disorder, neurologic disease AGTR1, NSUN2, PMS2 2 1 Cancer, gastrointestinal disease, hereditary disorder

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Figure 4. Network analysis of genes differentially affected by fulvestrant. Up- and downregulated transcripts are indicated in red and green, respectively. Direct and indirect relationships are indicated by solid and interrupted lines, respectively.

invariably stronger with response to fulvestrant. Anastro- duration of treatment may have biased the identification of zole response was specifically correlated with three genes, alterations in gene expression in favor of fulvestrant. of which KCNK15 was estrogen deprivation–related and SKP2 had predictive significance independent of pretreat- ESR1 knockdown in MCF7 cells ment AURKA expression (Fig. 5B, iv). Further evaluation Knockdown of ESR1 invariably downregulated the of the independence of the predictive performance of expression of genes which were found to be differentially agent-specific genes was not conducted because of limited downregulated by fulvestrant (e.g., LRP8 and GTSE1) and numbers. upregulated the expression of some genes which were differentially upregulated by fulvestrant (e.g., SNAI2 and Validation of differentially affected transcripts SEPP1), but not others (e.g. CAV1 and RBMS1). The major- qRT-PCR of clinical samples confirmed significant upre- ity of genes responded concordantly to fulvestrant and ESR1 gulation of CAV1 (1.87-fold increase, P ¼ 0.0095) and SNAI2 knockdown (Supplementary Fig. S3). (1.88-fold increase, P ¼ 0.0005) by fulvestrant, whereas changes induced by anastrozole were not significant. The differential impact of the two agents on treatment-related Discussion transcripts (n ¼ 46) was consistent between the parent and This study compared, for the first time, transcriptional bridging studies (Pearson r ¼ 0.93, P < 0.0001, slope ¼ 0.97). profiles from breast cancer in situ following fulvestrant or Fulvestrant-related transcripts (n ¼ 41) were concordantly anastrozole and corresponding in vitro models. The robust affected, albeit to a lesser extent, following low-dose treat- integrative strategy avoids the identification of spurious ment (Pearson r ¼ 0.77, P < 0.0001, slope ¼ 0.23). Genes, genes in clinical samples which may reflect increasing which were found to respond differently to 4 weeks of proportions of stroma with treatment response (33). The fulvestrant and 2 weeks of anastrozole, were evaluated in a approach taken may discard alterations inadequately mod- subgroup of patients receiving 16 weeks of anastrozole eled in vitro, including those dependent upon three-dimen- therapy. There was no significant change in any of the 11 sional structure, stromal interactions, and hypoxia, but transcripts differentially upregulated by fulvestrant, or 11 of focuses on those genes which may be subjected to func- the 17 transcripts differentially downregulated (Supplemen- tional interrogation in model systems. The hallmark molec- tary Table S13). This argues against the possibility that ular responses to interrupted estrogenic signaling, including

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High pretreatment expression correlates with good response

Low pretreatment expression correlates with good response

Figure 5. A, summary of treatment- related genes for which pretreatment expression B correlates significantly with treatment-induced changes in (i) (ii) expression of the proliferation- associated gene AURKA. Anastrozole (A, blue), fulvestrant (F, red), or both (green), with agent- specific and quantitative differences indicated by horizontal and vertical stripes, respectively. B, scattergrams illustrating the correlation between change in AURKA expression and three fulvestrant-specific genes (i)–(iii), and one estrogen deprivation– Spearman r = –0.82 (P = 0.000003) Spearman r = –0.60 (P = 0.003) specific gene (iv), with predictive Spearman r = –0.09 (P = 0.45) Spearman r = –0.03 (P = 0.78) significance (P < 0.05) independent of pretreatment AURKA expression in NEWEST (red) and (iii) (iv) FAIMoS (blue).

Spearman r = –0.47 (P = 0.026) Spearman r = 0.36 (P = 0.001) Spearman r = –0.22 (P = 0.053) Spearman r = –0.26 (P = 0.24) suppression of ERGs and proliferative markers, followed arrest of estrogen-independent ERa activity that is unaffect- both fulvestrant and estrogen deprivation. However, dis- ed by estrogen deprivation (27). This greater antiestrogenic tinguishing features were apparent. First, the overall tran- effect is consistent with the greater efficacy of fulvestrant in scriptional response to fulvestrant was of greater magnitude. studies comparing the agents as first-line therapy in Second, differences were not distributed uniformly across advanced disease (16) and in its sequential utility after the transcriptome, but were most marked in a relatively aromatase inhibitor relapse (9, 10). Incompletely overlap- limited cohort of genes. ping transcriptional responses are also consistent with the A small number of differentially affected genes were reported efficacy of combination therapy with fulvestrant specific to estrogen deprivation, potentially attributable to and anastrozole (35). ERa-independent estrogen activity (34). Most were fulves- Genes differentially affected by fulvestrant were associ- trant specific and remained unaffected by extended anastro- ated in networks with ERa, AR, and TP53. ERa activity can zole treatment, raising the possibility of regulation by be influenced by cross-talk with AR signaling (36), which unliganded ERa. Both fulvestrant and estrogen deprivation may exert antiestrogenic/antiproliferative effects in ERa- abrogate estrogen-dependent ERa activity, but only fulves- positive breast cancer, while having contrasting roles in trant, by virtue of ERa depletion, antagonizes estrogen- ERa-negative tumors (37). The discovery of a subset of independent ERa activity, including cross-talk with growth DNA-binding elements and pioneer factors common to factor pathways (26). The greater and differential transcrip- both receptors (38) raises the possibility that activity of tional response to fulvestrant may be attributable to the AR-dependent networks may be influenced by fulvestrant-

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induced loss of the ERa transcriptional program. Tran- baseline patient and tumor characteristics between presur- scripts differentially upregulated by ERa depletion, includ- gical studies with incompletely overlapping entry criteria, ing CAV1/2 and SNAI2, are associated with AR signaling and differences such as details in sample taking, storage, and and the biology of ERa-negative and basal-like breast cancer ethnicity of the populations. Sample sizes may have also (39–42). CAV1 encodes caveolin-1, the principal constitu- restricted the statistical power to identify treatment-induced ent of specialized membrane invaginations called caveolae. changes. Caveolin-1 and -2 are widely expressed and may colocalize. In conclusion, the molecular response to fulvestrant has Caveolae have diverse functions, including: vesicular traf- much in common with estrogen deprivation, but is stronger ficking, lipid homeostasis, subcellular partitioning, and with distinctions potentially attributable to arrest of estro- integrating the activity of signaling molecules. Caveolin-1 gen-independent ERa activity and involvement of AR sig- may facilitate nongenomic/extranuclear and ligand-inde- naling. Genes responding differently to fulvestrant may pendent ERa activity (42, 43). SNAI2 encodes SLUG, a have agent-specific predictive utility. These data are consis- transcription factor implicated in breast cancer progression, tent with the efficacy of first-line fulvestrant versus anastro- nodal involvement, and metastasis (44, 45). Expression is zole in advanced disease, combination therapy in the met- associated with epithelial-to-mesenchymal transition, E- astatic setting, sequential utility after aromatase inhibitor cadherin downregulation, and stem cell-associated gene relapse, and higher dosing regimens. expression (46, 47). NRP1, encoding neuropilin-1, a cor- eceptor for semaphorins and VEGF, also has links with stem Disclosure of Potential Conflicts of Interest cell phenotype (48) and poor prognosis (49). H. Anderson has ownership interests in AstraZeneca. M. Dowsett is a Pretreatment expression of fulvestrant-related genes may consultant/advisory board member for and reports receiving a commercial research grant and speakers’ bureau honoraria from AstraZeneca. No poten- be influenced by, and reflect, inherent tumor proliferation tial conflicts of interest were disclosed by the other authors. and/or estrogenicity. The possible agent-specific predictive utility of the differentially affected genes identified warrants Authors' Contributions further study. This may indicate whether such genes con- Conception and design: N. Patani, A.K. Dunbier, Z. Ghazoui, E. Anderson, tribute mechanistically to the action of the two agents, are J. Lindemann, J. Walker, I. Kuter, L.-A. Martin, M. Dowsett inconsequentially associated with treatment, and/or have Development of methodology: N. Patani, A.K. Dunbier, H. Anderson, Z. Ghazoui, E. Anderson, I. Kuter, L.-A. Martin external validity as predictive biomarkers. The impact of Acquisition of data (provided animals, acquired and managed patients, ESR1 knockdown on the expression of genes differentially provided facilities, etc.): N. Patani, A.K. Dunbier, H. Anderson, R. Ribas, affected by fulvestrant supports ERa destabilization as the E. Anderson, H. Anderson, J. Lindemann, I. Kuter, L.-A. Martin, M. Dowsett Analysis and interpretation of data (e.g., statistical analysis, biosta- mechanism underpinning particular fulvestrant-induced tistics, computational analysis): N. Patani, A.K. Dunbier, H. Anderson, Z. alterations. The identification of such genes may facilitate Ghazoui, R. Ribas, E. Anderson, R. A’hern, A. Mackay, J. Walker, L.-A. Martin Writing, review, and or revision of the manuscript: N. Patani, A.K. the comparative pharmacology of SERDs in development. Dunbier, H. Anderson, E. Anderson, R. A’hern, J. Lindemann, J. Walker, This study also provides the first comparison of transcrip- I. Kuter, L.-A. Martin, M. Dowsett tional responses with fulvestrant dosing regimens, and Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): N. Patani, H. Anderson, Z. Ghazoui, supports the efficacy of low-dose treatment (8–10). The R. Wellings greater transcriptional impact of high-dose therapy is con- Study supervision: A.K. Dunbier, I. Kuter, L.-A. Martin, M. Dowsett sistent with pharmacokinetic models predicting 5-fold Other (assisted with bioinformatic analyses): Q. Gao greater plasma concentrations on day 28 (50), and the increased efficacy observed in the NEWEST (17) and CON- Acknowledgments FIRM trials (13, 14). The authors thank Dr. Scott Brouilette and Jorg€ Mages for providing technical support and assistance with Partek Genomics Suite. Limitations of this study include in vitro modeling using a single cell line, which is also PIK3CA mutated, and expression profiling across different BeadChip versions which Grant Support reduced the number of comparable probes. Potential con- This work was funded by the Mary-Jean Mitchell Green Foundation and AstraZeneca. Prof. Mitch Dowsett and Dr. Lesley-Ann Martin are supported founding factors also include better patient compliance by Breakthrough Breast Cancer and NHS funding to the Royal Marsden NIHR with treatment regimens in favor of fulvestrant given its Biomedical Research Centre. Neill Patani is funded by a Medical Research mode of administration (although good estrogen-suppres- Council Clinical Research Training Fellowship (G1100450). The costs of publication of this article were defrayed in part by the sion was found in the aromatase inhibitor-treated payment of page charges. This article must therefore be hereby marked patients—data not shown) and duration of treatment; advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate extended anastrozole treatment may have induced further this fact. changes in gene expression. Nonrandomized comparisons Received May 26, 2013; revised April 8, 2014; accepted April 28, 2014; may also be influenced by selection bias, with differences in published OnlineFirst June 10, 2014.

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Differences in the Transcriptional Response to Fulvestrant and Estrogen Deprivation in ER-Positive Breast Cancer

Neill Patani, Anita K. Dunbier, Helen Anderson, et al.

Clin Cancer Res Published OnlineFirst June 10, 2014.

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