Era-Dependent E2F Transcription Can Mediate Resistance to Estrogen Deprivation in Human Breast Cancer

Published OnlineFirst July 20, 2011; DOI: 10.1158/2159-8290.CD-11-0101

RESEARCH ARTICLE ERa-Dependent E2F Transcription Can Mediate Resistance to Estrogen Deprivation in Human Breast Cancer

Todd W. Miller1,6, Justin M. Balko2, Emily M. Fox2, Zara Ghazoui8, Anita Dunbier8, Helen Anderson8, Mitch Dowsett8,9, Aixiang Jiang3, R. Adam Smith4,7, Sauveur-Michel Maira10, H. Charles Manning6,4,7, Ana M. González-Angulo11,12, Gordon B. Mills12, Catherine Higham2, Siprachanh Chanthaphaychith2, Maria G. Kuba5, William R. Miller13, Yu Shyr3,6, and Carlos L. Arteaga1,2,6

INTRODUCTION (fulvestrant), or 3) blocking estrogen biosynthesis [aromatase inhibitors (AI)]. Although endocrine therapies Approximately 70% of breast cancers express estrogen have changed the natural history of hormone-dependent receptor α (ER) and/or progesterone receptor (PR), bio- breast cancer, many tumors develop drug resistance (1). markers indicative of hormone dependence. Therapies for The only mechanism of resistance to endocrine therapy such patients inhibit ER signaling by 1) antagonizing li- for which clinical data exist is overexpression of the ErbB2/ gand binding to ER (tamoxifen), 2) downregulating ER HER2 protooncogene (2, 3). However, < 10% of hormone receptor-positive breast cancers express high HER2 levels, + suggesting that for the majority of ER breast cancers, Authors' Affiliations: Departments of 1Cancer Biology, 2Medicine, mechanisms of escape from endocrine therapy remain un- 3Biostatistics, 4Radiology and Radiological Sciences, and 5Pathology; 6Breast defined. We and others have shown that activation of the 7 Cancer Research Program, Vanderbilt-Ingram Cancer Center; Institute of phosphatidylinositol 3-kinase (PI3K) signaling pathway Imaging Sciences, Vanderbilt University, Nashville, Tennessee; 8Breakthrough Breast Cancer Centre, Institute of Cancer Research, and 9Academic also promotes resistance to endocrine therapy (4, 5), al- Department of Biochemistry, Royal Marsden Hospital, London, UK; though demonstration of such a mechanism in the clinic 10Novartis Institute for Biomedical Research, Oncology Disease Area, Basel, awaits confirmation. 11 12 Switzerland; Departments of Breast Medical Oncology and Systems Tamoxifen has been the standard treatment for patients Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 13Breast Research Group, University of Edinburgh, Edinburgh, UK with hormone receptor-positive breast cancer. This drug exhibits dual agonistic/antagonistic effects on ER transcrip- Note: Supplementary data for this article are available at Cancer Discovery Online (http://www.cancerdiscovery.aacrjournals.org). tional activity and cancer cell growth (6). In contrast, AIs Corresponding Author: Carlos L. Arteaga, The Division of Hematology suppress estrogen biosynthesis and thus ligand-induced ER and Oncology, Vanderbilt University School of Medicine, 2220 Pierce activity with no known agonistic effects. In postmenopausal Avenue, 777 PRB, Nashville, TN 37232-6307. Phone: 615-936-3524; patients, AIs are modestly superior to tamoxifen in prevent- Fax: 615-936-1790; E-mail: [email protected] ing disease recurrence. However, a significant fraction of + doi: 10.1158/2159-8290.CD-11-0101 patients with early ER breast cancer receiving adjuvant AIs ©2011 American Association for Cancer Research. recur within the first decade of follow-up (1). Interestingly,

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ABSTRACT Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER+ xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.

SIGNIFICANCE: ERα retains genomic activity and drives a CDK4/E2F- dependent transcriptional program despite estrogen deprivation therapy. Combined inhibition of ER and PI3K induced regression of ER+ xenografts, supporting further development of strong ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for the treatment of antiestrogen-resistant breast cancers. Cancer Discovery; 1(4); 338–51. ©2011 AACR.

a recent comparison of high-dose fulvestrant to the AI anas- hormone-independent growth of both fulvestrant-sen- + trozole as first-line treatment for advanced breast cancer sitive and -insensitive ER cell lines. Furthermore, post- + revealed that fulvestrant provided a longer time-to-progres- menopausal ER tumors with a gene expression signature sion (7 ). In other studies, approximately 30% of patients of E2F activation following neoadjuvant therapy with an who progressed on an AI responded to second-line fulves- AI exhibited a lesser response, suggesting that estrogen- trant (8 , 9 ). These data suggest that in some clinical situ- independent E2F activation promotes resistance to estro- ations, downregulation of ER may be superior or add to gen deprivation. Finally, fulvestrant in combination with + estrogen deprivation. a PI3K inhibitor induced marked regression of ER breast Fulvestrant has been shown to inhibit the growth of cancer xenografts in mice devoid of estrogen supplementa- + ER MCF-7 human breast cancer cells and xenografts un- tion. These results provide a basis for 1) further develop- der estrogen-depleted conditions ( 10 , 11 ). However, the ment of potent ER downregulators and CDK4 inhibitors, + role of ER in estrogen-independent growth remains un- particularly in AI-resistant ER tumors with a gene expres- clear. In order to model the low estrogen levels seen in sion signature of E2F activation, and 2) their combination + patients treated with an AI [≤ 3 pM plasma 17β-estradiol, with PI3K inhibitors for the treatment of ER breast can- E2 (12 )], we generated long-term estrogen-deprived cers that escape estrogen deprivation. + (LTED) derivatives of 4 ER human breast cancer cell lines. These LTED lines exhibit hyperactivation of the PI3K pathway, and variable changes in ER levels and E2 sensi- RESULTS tivity ( 4 ). Herein, we report that the unliganded ER fre- quently plays a role in the hormone-independent growth Unliganded ER Is Required for the Hormone- + of LTED cells by modulating a transcriptional program Independent Growth of ER Breast Cancer Cells directed by E2F proteins. Concurrently, a small interfer- We previously reported that LTED cells show variable ing RNA (siRNA) screen identified that CDK4, a kinase changes in ER levels after selection in hormone-depleted that activates E2F transcription, is required for hormone- medium (10% dextran-charcoal-treated FBS, DCC-FBS). independent cell growth. CDK4 inhibition suppressed the MCF-7/LTED and HCC-1428/LTED cells exhibit higher ER

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Figure 1. ER is required for acquired hormone-independent breast cancer cell growth. A, lysates from cells treated with 10% DCC-FBS ± fulvestrant or 4-OH-T 3 24 hours were analyzed by immunoblotting using ER and actin antibodies. B, cells were treated as in (A) ± E2, 4-OH-T, or fulvestrant. Media and drugs were replenished every 2 to 3 days. Adherent cells were counted after 5 to 10 days. Data are presented as percent parental control, mean of triplicates ± SD. C, cells transfected with siRNA targeting ER or nonsilencing control (siCtl) were treated as in (B), and adherent cells were counted after 6 to 9 days. Data are presented as percent parental siCtl/no hormone, mean of triplicates ± SD. D, immunoblots of lysates from the same batches of cells used in (C). E, parental cells were treated as in (B). When control wells reached 30% to 50% confluence (18–60 days), cells were stained with crystal violet. Representative images are shown. Quantification of staining intensity is noted at bottom as mean of triplicates ± SD (% control). *P < 0.05 by Bonferroni post-hoc test compared to control [or siCtl/no hormone in (C)] of each cell line.

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ERα Drives Resistance to Estrogen Deprivation Therapy RESEARCH ARTICLE levels compared to parental controls, while ZR75-1/LTED 285 genes upregulated by fulvestrant (Fig. 2B). Of these 1,003 and MDA-361/LTED cells show the opposite (Fig. 1A; ref. 4). genes, 671 were available to query the Connectivity Map, a We sought to determine whether ER plays a role in hormone- collection of gene signatures induced by the treatment of independent growth. Treatment with the ER downregulator cells with a panel of 1,309 small molecules (15). Treatment fulvestrant reduced growth of MCF-7/LTED and HCC-1428/ of MCF-7 cells with fulvestrant in 10% FBS induced gene LTED cells in estrogen-depleted medium (P < 0.05 vs. con- signatures (data from Connectivity Map) that were directly trols), but not MDA-361 or ZR75-1 parental or LTED cells correlated with the 671-gene signature derived from fulves- (Fig. 1B). These results suggest that the (upregulated) ER trant-treated LTED cells in the absence of hormones (P < in MCF-7/LTED and HCC-1428/LTED cells drives estrogen- 0.00001). Therefore, fulvestrant induced similar gene ex- independent growth. Conversely, cells which did not require pression profiles in the presence and absence of hormones, ER for hormone-independent growth (MDA-361, ZR75-1) implying that ER regulates overlapping sets of genes under downregulated ER during prolonged estrogen deprivation E2-stimulated as well as hormone-depleted conditions. (Fig. 1A). Treatment of both parental and LTED cells with In order to further evaluate the estrogen-independent 4-hydroxy-tamoxifen (4-OH-T) or fulvestrant suppressed E2- genomic function of ER, we did chromatin immunoprecipita- induced growth (Fig. 1B) and E2-induced ER transcriptional tion followed by next-generation DNA sequencing (ChIP-seq). reporter activity (Supplementary Fig. S1). In agreement with a We identified 3,366 and 4,331 regions (peaks) that were signifi- prior report (11), prolonged hormone deprivation of MCF-7/ cantly enriched following ER-ChIP-seq in MCF-7/LTED and LTED cells (. 6 months) generated an estrogen-insensitive HCC-1428/LTED cells, respectively (Fig. 2C). A comparison of line (MCF-7/LTED-I). MCF-7/LTED-I cells maintained high these data with published hormone-independent, E2-induced, ER levels. Their growth was inhibited by fulvestrant (data not and EGF-induced ER binding regions in MCF-7 cells (14, shown), suggesting that dependence upon ER for hormone- 16–18) showed 18.1% (hormone-independent), 23% to 52.4% independent growth is not a transient phenotype. To confirm (E2-induced), and 46.1% to 53.3% (EGF-induced) overlap with that the effects of fulvestrant were through action on ER, MCF-7/LTED cells, and 4.9% (hormone-independent), 9.3% to we knocked-down ESR1 (ER) expression using siRNA. siER- 26.6% (E2), and 11.8% to 18.1% (EGF) overlap with HCC-1428/ transfected MCF-7/LTED, HCC-1428/LTED, and BT-474 LTED cells (Supplementary Fig. S2). Since a significant frac- cells exhibited reduced growth in hormone-depleted medium tion of E2-induced ER binding sites are distal to transcrip- compared to siControl. In contrast, ER knockdown did not tion start sites of E2-regulated genes, some of which may be alter the hormone-independent growth of MDA-361, ZR75- nonfunctional (14, 16), we focused on ER binding sites within 1, or T47D cells (Fig. 1C). ER knockdown was confirmed by 60 kb of transcription start sites of the 1,003 genes commonly immunoblot (Fig. 1D); results were confirmed using a second deregulated by fulvestrant (Fig. 2C; Supplementary Fig. S3). siRNA targeting ER (data not shown). This analysis revealed 402 and 658 proximal ER binding re- We next assessed whether the lines in which growth was gions in MCF-7/LTED and HCC-1428/LTED cells, respectively. inhibited by ER downregulation were the same in which ER Using ChIP-qPCR, we tested 48 of these sites and found that was required for acquired resistance to estrogen depriva- most were enriched in ER-ChIP compared to IgG-ChIP control tion. Parental cells were reselected for hormone-independent (Fig. 2D). Interestingly, only approximately half of these ER growth in the presence of fulvestrant. Fulvestrant suppressed binding regions contained a proximal estrogen-response-ele- the emergence of hormone-independent MCF-7, HCC-1428, ment (ERE), and many had not been previously identified in and BT-474 cells (P < 0.05 vs. respective controls), but not genome-wide ER-ChIP studies (14, 16–18). Transcription fac- ZR75-1, MDA-361, or T47D cells (Fig. 1E), implying that the tor motif analysis revealed that ER-bound regions were en- requirement for ER in estrogen-independent growth is intrin- riched for motifs which bind ER, FoxA1, RARα, and C/EBP, sic to the parental cells. among others (Supplementary Figs. S4, S5; Supplementary Tables S1 to S4); such proteins are known to functionally cooperate with ER (18–21). We next subcloned 3 ER-bound ER Promotes an Estrogen-Independent, regions (Fig. S6) into a transcriptional reporter vector. In E2F-Mediated Transcriptional Program MCF-7/LTED and HCC-1428/LTED cells, reporter activity in- To determine the role of ER in hormone-independent creased with E2 and decreased with fulvestrant (Fig. 2E). The growth, we did gene expression analysis of “ER-dependent” latter results suggest that these ER binding sites regulate tran- MCF-7/LTED and HCC-1428/LTED cells treated ± fulves- scription in the absence of ER ligands. trant. To assess whether ER regulates similar gene sets in We next examined whether estrogen-independent ER + the presence and absence of estrogen, we derived a signature genomic signaling occurs in primary ER cancers in of 141 genes altered by exogenous E2 in MCF-7 cells from patients subjected to estrogen deprivation. Breast tumors two prior datasets (13, 14). Hierarchical clustering of these in patients treated with AIs can be considered estrogen- 141 genes in LTED cells ± fulvestrant (in the absence of es- deprived since these drugs maximally suppresses plasma trogen) showed that fulvestrant induced a gene expression estrogen levels within 2 weeks (22), providing a setting in pattern opposite to E2 (Fig. 2A). We next determined which which to test estrogen-independent ER activity. Hence, we + genes were commonly altered by fulvestrant in both MCF-7/ did ChIP-qPCR for ER binding events using 3 ER breast LTED and HCC-1428/LTED cells (≥1.5-fold, FDR-adjusted tumors acquired from patients following 10 to 21 days of P ≤ 0.05). This analysis yielded 718 genes downregulated and neoadjuvant therapy with the AI letrozole (clinical trial

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A B C MCF-7/ MCF-7/ LTED LTED ER binding sites 1390 2964

718 402

1713 601 HCC-1428/ genes deregulated by Fulv

>1.5-fold by fulvestrant LTED # genes downregulated 1003 genes MCF-7/ 345 LTED genes deregulated 1487 by Fulv

285 658

916 HCC-1428/LTED HCC-1428/ ER binding sites # genes upregulated

>1.5-fold by fulvestrant LTED 3673

E F

Figure 2. Gene expression and genomic profiling reveal estrogen-independent ER transcriptional activity. A, RNA from MCF-7/LTED and HCC- 1428/LTED cells treated ± fulvestrant 3 48 hours was analyzed using gene expression microarrays. We derived a signature of 141 genes commonly up- or downregulated by E2 stimulation in MCF-7 cells. Using LTED cell gene expression data, we hierarchically clustered these 141 genes (x-axis). Fulvestrant induced expression changes diametrically opposed to those induced by E2. B, we determined probe sets up- or downregulated by fulvestrant in each LTED line (≥1.5-fold, P ≤ 0.05), and used Venn diagrams to identify commonly deregulated probe sets (yielded 1,434 probe sets for 718 down- and 285 upregulated genes). C, ChIP-seq analysis to identify ER genomic binding regions. To focus on functional ER binding sites, we determined which sites were within 60 kb of the transcription start site of a fulvestrant-deregulated gene. D, Forty-eight ER binding regions identified in (C) were tested by ChIP-qPCR. Data presented as heatmap of fold-enrichment in ER-ChIP over IgG-ChIP control, or fold-enrichment score from ChIP-seq. E2-regulated genes deregulated by fulvestrant in (A), presence of a proximal estrogen-response-element (ERE) within 500 bp of a peak, and overlap of peaks with published ER-ChIP datasets are noted below heatmap. ND, not detected. E, ER-bound regions verified in (D) were subcloned into a luciferase reporter vector. Cells transfected with reporter plasmids were treated with 10% DCC-FBS ± fulvestrant or E2.

Luciferase activities were measured after 24 or 48 hours. ERE—contains 2 consensus EREs. Data are presented as log10 (ratio vs. each untreated control), mean of triplicates ± SD. *P < 0.05 by Bonferroni post-hoc test compared to each control. RLU, relative light units (firefly/Renilla). F, ChIP-qPCR as in (D) using human ER+ breast tumor samples acquired from patients following 10 to 21 days of neoadjuvant letrozole. Data presented as fold-enrichment in ER-ChIP over IgG-ChIP control.

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ERα Drives Resistance to Estrogen Deprivation Therapy RESEARCH ARTICLE

NCT00651976). In 2 of 3 tumors, ER-ChIP samples showed GO analysis showed that 37 of 61 genes containing an E2F significant enrichment for ER-bound genomic regions motif that were deregulated by fulvestrant in LTED cells were identified in cell lines compared to IgG controls (Fig. 2F), associated with cell cycle regulation. Tumor Ki67 score has supporting the presence of estrogen-independent ER-DNA been shown to correlate with the expression of genes encoding binding activity in primary human tumors deprived of proteins involved in the cell cycle (30). This analysis suggested estrogens. that the remaining 24 genes, which did not have a cell cycle- To identify the functional output of estrogen-indepen- related GO annotation, could be used to assess E2F activity in- dent ER activity, we classified the proteins encoded by the dependent of the cell cycle (Supplementary Fig. S7A and S7B; 1,003 fulvestrant-deregulated genes (Fig. 2B) using Gene Supplementary Table S5). In order to distinguish between cell Ontology (GO) analysis (23). The top enriched biological cycle-associated and -independent effects of E2F activation, we processes were Cell Cycle, Cell Proliferation, M Phase, Mitotic evaluated both the 61-gene E2F signature and the 24-gene E2F Cell Cycle, and DNA Replication and Chromosome Cycle (all P signature devoid of cell cycle-associated genes. < 0.0001). These data suggest that in the absence of hor- Hierarchical clustering revealed that tumors did not group mones, ER modulates the expression of genes encoding pro- into distinct clusters (Supplementary Fig. S7C and S7D), teins that regulate the cell cycle. We then asked whether suggesting that E2F activation may be better represented as there was an underlying transcriptional program directed a continuous variable. Hence, we generated E2F activation by ligand-independent ER within this large set of modu- scores for each tumor using both pre- and post-anastrozole lated genes. We computed the overlap between 1) the 1,003 gene expression data and compared them to the post-anas- genes altered by fulvestrant in LTED cells, and 2) sets of trozole (2 weeks) Ki67 score. As expected, the pre- and post- genes that share a transcription factor binding site within anastrozole 61-gene E2F scores were significantly associated 2 kb of transcription start sites. Twelve of the top 20 gene with the Ki67 score (ANOVA P = 0.0004, r = 0.412, and P < sets that showed significant overlap with the fulvestrant- 10−4, r = 0.641, respectively). Importantly, both the pre- and induced signature contained E2F binding sites (Fig. 3A). post-anastrozole non-cell cycle 24-gene E2F scores also cor- E2F1 binds predominantly within 2 kb of transcription related significantly with the Ki67 score (ANOVA P = 0.0144, start sites (24). The E2F family contains 8 proteins (E2F1-8) r = 0.293, and P < 10−4, r = 0.493, respectively; Fig. 3B and which modulate the expression of genes that coordinate cell C). These data suggest that an expression signature of E2F + cycle progression, DNA replication, mitosis, and apopto- activation in ER tumors may be linked with resistance to AI- sis (25). Included in the set of fulvestrant-suppressed genes induced estrogen deprivation. were E2F1, -2, -7, and -8. Prior work has shown that E2 We confirmed this correlation in a second cohort of + stimulates E2F1 expression (26) and phosphorylation/inac- 48 patients with ER breast cancer treated with neoadju- tivation of the E2F repressor Rb (27). These findings sug- vant letrozole (31). Again, the pre- and post-2-week letrozole gest that ER also promotes E2F expression and activation 61-gene E2F scores correlated significantly with the Ki67 in an estrogen-independent manner in MCF-7/LTED and score (ANOVA P = 0.043, r = 0.293, and P < 10−4, r = 0.558, HCC-1428/LTED cells. In contrast, fulvestrant did not alter respectively). In this second set, however, the posttreatment estrogen-independent E2F1, -2, -7, or -8 expression in MDA- but not the pretreatment 24-gene E2F score was significantly 361 or ZR75-1 cells (data not shown), suggesting that these associated with Ki67 score (ANOVA P = 0.0026, r = 0.43; + ER cell lines harbor an ER-independent mechanism(s) to Fig. 3D and E). Variable estrogen levels in the pretreatment promote E2F transcription. tumor samples may have increased noise and confounded the analysis of estrogen-independent E2F activation. Considering the E2F signature was derived from LTED cells, and the con- E2F Activation Signature Correlates with + sistent statistical correlations between the posttreatment Resistance to Estrogen Deprivation in ER E2F score and Ki67 score, we speculate the contribution of Primary Tumors E2F signaling to endocrine resistance may be best evaluated Since fulvestrant suppressed hormone-independent under conditions of estrogen deprivation. growth in cells with ER-dependent E2F activity, we pos- We finally determined whether the Ki67 score follow- tulated that an estrogen-independent, E2F-induced gene ing neoadjuvant therapy with an AI correlated with E2F- expression profile would predict for resistance to estrogen regulated expression at the protein level. The promoters of deprivation in human tumors. Thus, we generated a sig- FANCD2 and CDC2 contain E2F binding sites (32, 33) and nature of E2F activation from LTED cells and assessed its were part of the 24-gene and 61-gene E2F signatures, respec- ability to predict response to neoadjuvant therapy with an tively. Antibodies were validated for analysis of these pro- + AI in patients with ER breast cancer. Primary tumor biop- teins using reverse-phase protein arrays (RPPA). In a third + sies were obtained from 68 patients with newly diagnosed cohort of 10 patients with ER breast cancer, we quantified + ER breast cancer before and after 14 days of therapy with levels of FANCD2 and CDK1 (CDC2) by RPPA analysis of anastrozole (28). High tumor cell proliferation measured tumor samples obtained following 10 to 21 days of neoad- by immunohistochemistry (IHC) for the proliferation juvant letrozole (Fig. 3F). FANCD2 levels were significantly marker Ki67 after short-term endocrine therapy has been correlated with post-letrozole Ki67 score, while CDK1 shown to predict a worse long-term disease outcome (29). levels showed a correlative trend (Fig. 3G). In addition to Hence, we used the posttreatment Ki67 score as a surro- FANCD2 and CDC2, mRNA levels of another 8 of 21 and gate of clinical response. 8 of 20 available genes in the 24-gene E2F signature showed

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B C

D E

F G

Figure 3. A gene expression signature of E2F activation correlates with poor tumor response to AIs in patients. A, the set of 1,003 genes commonly deregulated by fulvestrant was used to query TRANSFAC database. Top 20 Genesets are shown, 12 of which contain E2F motifs (highlighted). The number of fulvestrant-deregulated genes as a percentage of the total genes in each set is shown. We derived a set of 24 genes containing E2F motifs but lacking a Gene Ontology cell cycle annotation. B–E, expression values of these 24 genes were used to generate E2F activation scores for ER+ primary breast tumors from patients treated with an AI using pre- and posttreatment gene expression data. E2F scores were standardized to generate Z-scores. Patients were treated with neoadjuvant anastrozole for 14 days (B-C), or with letrozole for 10 to 14 days (D–E). The associations of the pre-anastrozole (B), post-anastrozole (C),

pre-letrozole (D), and post-letrozole (E) E2F Z-scores with log2-transformed post-AI Ki67 score were analyzed by linear regression. Pearson correlation coefficients (r) and ANOVA P values were calculated. F, in a third cohort, protein levels of FANCD2 and CDK1 were measured by RPPA in ER+ primary breast

tumors following 10 to 21 days of neoadjuvant letrozole. Log2-transformed signal values were standardized into Z-scores, which were compared to post- letrozole Ki67 score. G, the protein (Z-score) and mRNA levels (from posttreatment microarrays) of FANCD2 and CDK1 (CDC2) were compared to Ki67 score in all 3 datasets using linear regression. adj, adjusted P-value from multiple regression analysis.

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ERα Drives Resistance to Estrogen Deprivation Therapy RESEARCH ARTICLE

significant univariate correlations with Ki67 score in the phosphorylation at Ser167 and estrogen-independent tran- post-anastrozole and post-letrozole microarray datasets, scriptional activity (39, 40); thus, we examined whether PI3K respectively (Fig. 3C and E, P < 0.05). activates ER in LTED cells. Although we detected higher

levels of P-ERSer167 in MCF-7/LTED and HCC-1428/LTED CDK4 Is Required for Hormone-Independent ER+ cells compared to parental controls in hormone-depleted Breast Cancer Cell Growth conditions, treatment with the PI3K inhibitor BKM120 In a complementary experiment, we screened a siRNA (41) or the TORC1 inhibitor RAD001 (42) only modestly library targeting 779 kinases to identify pathways re- increased and decreased P-ERSer167 levels in MCF-7/LTED quired for hormone-independent growth. siCDK4 ranked and HCC-1428/LTED cells, respectively (Supplementary as the overall first and third strongest hits that inhibited Fig. S10A). Treatment with BKM120 did not alter ER-DNA growth of MCF-7/LTED and MCF-7 cells, respectively binding (Supplementary Fig. S10B) or ER transcriptional (Fig. 4A). CDK4 and CDK6 phosphorylate Rb to derepress reporter activities (data not shown). Conversely, fulvestrant E2F signaling which, in turn, promotes cell cycle progres- did not alter P-AKT levels (Supplementary Fig. S10C), sug- sion (25). Hence, CDK4 knockdown may phenocopy the gesting that ER does not activate PI3K under hormone- depleted conditions. In addition, estrogen deprivation has effects of ER downregulation since both pathways con- + verge on E2F. Verification showed that CDK4 knockdown been shown to induce synthetic lethality in ER breast can- inhibited hormone-independent growth and reduced cer cells treated with a PI3K inhibitor or a siRNA target- + P-Rb levels in 6 of 6 ER breast cancer cell lines (Fig. 4B, ing PI3K (5). These data collectively suggest that PI3K and Supplementary Fig. S8; confirmed using an independent ER modulate non-overlapping pathways in the absence of CDK4 siRNA, data not shown). This is consistent with re- estrogens. We thus reasoned that combined inhibition of + cent reports showing that ER breast cancer cell lines were both pathways may be synergistic. more sensitive to the CDK4/6 inhibitor PD-0332991 (34) Analysis of the effects of BKM120 and fulvestrant on – hormone-independent cell growth showed synergy in 6 of than ER lines (35), and that PD-0332991 inhibits the + growth of antiestrogen-resistant MCF-7 cells (36). In sup- 8 ER lines (Fig. 5A; Supplementary Fig. S11). To assess port of a role of CDK4 in hormone-independent growth, whether these drugs inhibit estrogen-independent growth, PD-0332991 treatment reduced growth and P-Rb lev- mice bearing MCF-7 xenografts (as in Fig. 4E) were ran- + els in 6 of 6 parental and 2 of 2 LTED ER cell lines in domized to vehicle, BKM120, fulvestrant, or the combi- estrogen-depleted conditions (Fig. 4C and D). These find- nation. Although the single-agent therapies significantly ings suggest that CDK4/6-mediated phosphorylation of inhibited tumor growth compared to vehicle, the drug Rb (and E2F activation) is required for both hormone- combination induced near-complete tumor regression and + independent and ER-independent growth of ER cells. was significantly more effective than each single agent (Fig. 18 To validate these findings in vivo, we established MCF-7 5B). We used [ F]FDG-PET as a biomarker of PI3K/AKT xenografts in athymic ovariectomized female mice supple- inhibition. Mice were imaged before and after 9 days of mented with a 14-day-release E2 pellet. Fifteen to twenty-six treatment. BKM120 but not fulvestrant significantly de- 18 days after implantation, tumor-bearing mice were random- creased tumor [ F]FDG uptake compared to baseline (P = ized to treatment with vehicle, PD-0332991, or fulvestrant. 0.013; Fig. 5C; Supplementary Fig. S12). We also observed Both therapies significantly inhibited estrogen-independent a significant correlation between the percent change in tu- 18 tumor growth compared to vehicle (Fig. 4E). Biomarkers of mor [ F]FDG uptake and the percent change in tumor vol- response were assessed by immunoblot and IHC after 5 weeks ume after 9 to 10 days of treatment (r = 0.805, P = 0.0028), of therapy. Fulvestrant decreased ER levels, and both agents suggesting that a reduction in FDG uptake correlates with decreased P-Rb, FANCD2, p107 (RBL1), LAP2 (TMPO), E2F1, early tumor response. and E2F2 (Fig. 4F). FANCD2, RBL1, and TMPO contain proxi- Molecular analyses of tumor lysates showed that fulves- mal E2F motifs and are part of the 24-gene E2F signature trant decreased ER levels, whereas BKM120 decreased P-AKT (Supplementary Fig. S7). These data imply that fulvestrant and P-S6 (Fig. 5D). Fulvestrant also decreased the levels of reduces estrogen-independent, ER-regulated E2F activity in PR, E2F1, E2F2, p107 (RBL1), and c-myc (MYC), whereas vivo. Although both drugs decreased tumor cell prolifera- BKM120 did not. Of note, RBL1 and MYC contain proximal E2F binding motifs and are downregulated by fulvestrant in tion (assessed by P-Histone H3Ser10 IHC), PD-0332991 also induced apoptosis [assessed by IHC using an antibody for LTED cells. These data imply that ER and PI3K signal inde- cleaved caspase-3 which may cross-react with cleaved cas- pendently in estrogen-depleted conditions, and that ER but pase-7 (37), Fig. 4G; Supplementary Fig. S9]. not PI3K modulates E2F activity. Analysis of tumors treated with BKM120 plus fulvestrant for more than 6 weeks revealed decreased tumor cell den- Combined Targeting of ER and PI3K Induces sity and increased fibrosis (Fig. 5E, H&E). BKM120-treated + + Regression of ER Xenografts tumors showed an increase in cleaved caspase-3/7 tumor + We previously reported that in LTED cells, hyperacti- cells compared to controls but no change in Ki67 tumor vation of the PI3K pathway is required for estrogen-inde- cells, suggesting that PI3K inhibition predominantly in- pendent growth (4). Crosstalk between the PI3K and ER duced apoptosis. In contrast, tumors treated with fulves- + pathways has been suggested as a mechanism of endocrine trant and the combination showed a reduction in Ki67 + resistance (38). PI3K activation was shown to induce ER cells but no change in cleaved-caspase-3/7 cells. Whether

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RESEARCH ARTICLE Miller et al.

A MCF-7 MCF-7/LTED

CDK4 CDK4

E F G

P-RBs780

Figure 4. CDK4 is required for the hormone-independent growth of ER+ breast cancer cells. A, MCF-7 and MCF-7/LTED cells transiently transfected with a siRNA library targeting 779 kinases were reseeded in 10% DCC-FBS. Cell viability was measured 4 to 5 days later. Cell growth for each kinase siRNA relative to nonsilencing controls (siCtl) was transformed to a Z-score, and the median Z-score across 3 to 4 independent experiments was calculated. B, cells transfected with an independent siRNA targeting CDK4 or siCtl were treated with 10% DCC-FBS ± 1 μM fulvestrant. Media and drugs were replenished every 2 to 3 days. Adherent cells were counted after 6 to 9 days. Data are presented as percent parental siCtl/no hormone, mean of triplicates ± SD. We were unable to achieve CDK4 knockdown in HCC-1428 lines. C, cells were treated with 10% DCC-FBS ± fulvestrant or 0.2 μM PD-0332991. Adherent cells were counted after 6 to 16 days. Data are presented as percent parental control, mean of triplicates ± SD. *P < 0.05 by Bonferroni post-hoc test compared to control [or siCtl/no hormone in (B)] for each cell line. D, immunoblots of lysates from cell treated as in (C) 3 24 hours. E, mice bearing established MCF-7 xenografts were randomized to the indicated treatments. Mean tumor volumes ± SEM are shown. *P < 0.05 by general linear model compared to vehicle control at the indicated time point. F, immunoblot analysis of lysates from tumors from (E) using the indicated antibodies. G, IHC scoring for phospho-Histone < H3Ser10 and cleaved caspase-3/7 was done on tumors from (E). *P 0.05 by Bonferroni post-hoc test. ns, not significant.

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ERα Drives Resistance to Estrogen Deprivation Therapy RESEARCH ARTICLE

A BC

D E

Figure 5. Combined downregulation of ER and inhibition of PI3K induces regression of ER+ xenografts. A, cells were treated with 10% DCC-FBS ± fulvestrant, BKM120, or both. Combination index (CI) values were calculated using the Median Effect method at the IC50, IC75, and IC90 for both drugs. CI < 1 is indicative of synergy. B, mice bearing MCF-7 tumors were randomized to the indicated treatments. Mean tumor volumes ± SEM are shown. *P < 0.05 by general linear model compared to vehicle control at the indicated time point. #P < 0.05 compared to both single-agent treatment arms. C, tumor-bearing mice were imaged before and after 9 days of treatment by [18F]FDG-PET. Representative images show [18F]FDG uptake pre- and post-BKM120 (T, tumor). Quantified in Supplementary Fig. S12. D, immunoblot analysis of lysates of tumors from (B) using the indicated antibodies. E, H&E staining and IHC for Ki67 and cleaved caspase-3/7 of tumors from (B). Quantification is shown below. *P < 0.001 by Bonferroni post-hoc test. ns, not significant. F, model depicting the synergistic effects of fulvestrant and BKM120.

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RESEARCH ARTICLE Miller et al. fulvestrant inhibited the pro-apoptotic effect of BKM120 by predict that the combination arm will show the best clini- blocking tumor cell proliferation, and/or there was an early cal outcome. Although high-dose fulvestrant (500 mg) in- wave of cell death not captured by the time of tumor collec- duces only partial ER downregulation (48), a 500-mg dose tion is unclear. However, the marked drug synergy observed was shown to increase time-to-progression more effectively + and the late timing of this assessment in small residual than a 250-mg dose in patients with advanced ER breast tumors does not support the former speculation. Thus, the cancer (49). Therefore, the optimal therapeutic dose of ful- combination of fulvestrant and BKM120 likely induced tu- vestrant remains undefined, and there is a need for discovery mor regression by both inhibition of tumor cell prolifera- of strong ER downregulators to completely neutralize ER tion and induction of apoptosis (Fig. 5F). activity in breast cancers that adapt to estrogen deprivation but continue to utilize ER for growth. + Data shown herein suggest that some ER cells uti- DISCUSSION lize the unliganded ER to drive an E2F transcriptional + We identified a role for ER in the acquired hormone- program and cell cycle progression, while other ER cells independent growth of human breast cancer cells. utilize ER-independent means. In effect, the latter cells Fulvestrant treatment suppressed the emergence of hor- behave as “ER-negative” under estrogen-depleted condi- + mone-independent cells from 3 of 6 ER breast cancer lines. tions. We therefore predict that, in patients treated with Gene expression profiling revealed that ER was required AIs, only a fraction of estrogen-independent cancers will for the estrogen-independent modulation of an E2F tran- continue to rely on the ER for growth and, thus, will re- scriptional program in “ER-dependent” but not in “ER- spond clinically to therapeutic downregulation of the ER. independent” cells. A kinome-wide siRNA screen showed At this time, short of testing an ER downregulator and/or that CDK4, which derepresses E2F signaling, is required analyzing biopsies of drug-resistant recurrent metastases for hormone-independent growth. Expression of an LTED- to assess ER levels and activity, there is no clinical assay derived E2F activation signature in primary breast cancers that would infer continued reliance on ER in tumors that following short-term therapy with an AI correlated with high progress in patients in whom estrogen production is sup- levels of Ki67, a predictive biomarker of worse treatment pressed. In this study, a gene expression signature of E2F + outcome, suggesting that estrogen-independent E2F signal- activation in ER tumors after short-term estrogen depri- ing may confer resistance to estrogen deprivation. Finally, vation with an AI correlated with high post-AI tumor cell fulvestrant synergized with a PI3K inhibitor to suppress hor- proliferation (Fig. 3). This suggests that an E2F activation + mone-independent growth in vitro and induce marked tumor signature may identify those ER tumors that are resistant regressions in vivo. to estrogen deprivation but may respond to an ER down- In parallel to the role of ER in estrogen-independent regulator or E2F/Rb-directed therapy. breast cancer cell growth, androgen receptor (AR) has Estrogen-independent, ER-dependent E2F transcription been implicated in androgen-independent prostate cancer may promote endocrine resistance but this can be abro- growth. AR is analogous to ER and conventionally acts as a gated by an ER downregulator (Figs. 2 and 3). In contrast, ligand-inducible transcription factor. The majority of pros- both hormone-independent and ER-independent growth + tate cancers are androgen-dependent at the time of diagno- of all ER cells was suppressed by CDK4/6 inhibition sis as most patients initially respond to androgen-ablation (Fig. 4). Therefore, we speculate that a CDK4/6 inhibitor therapy. However, recurrent cancers are typically androgen may be more broadly applicable than an ER downregula- (ligand)-independent and AR+ (43). AR gene amplification tor for the treatment of breast cancers progressing after an has been observed in some recurrent prostate cancers follow- AI. The benefit of CDK4/6-directed therapy is being tested + ing androgen deprivation therapy (44). Wang and colleagues in a clinical trial where patients with advanced ER breast (45) reported that ligand-independent AR upregulates cancer are randomized to treatment with letrozole ± PD- M-phase cell cycle genes in androgen-independent prostate 0332991 (50). cancer cells. Similarly, we observed that gene sets downregu- We previously reported that LTED cells exhibit hyperac- lated following fulvestrant-induced ER downregulation were tivation of the PI3K pathway, which is required for estro- significantly enriched for cell cycle genes, suggesting that gen-independent growth (4). Despite reports suggestive of some breast cancers utilize ER in a ligand-independent fash- crosstalk between PI3K and ER signaling (39, 40), we were ion to drive cell proliferation. We should note that ESR1 unable to detect such interactions in LTED cells, implying (ER) amplification has also been detected in primary breast that both ER and PI3K signaling are required for hormone- cancers (46); however, its association with disease outcome independent growth. Therefore, we examined the effects + remains unclear. of combined inhibition of ER and PI3K in ER xenografts. The involvement of ER in estrogen-independent breast Although single-agent therapies targeting these path- cancer progression is being assessed in a randomized clini- ways slowed tumor growth, the combination of fulvestrant cal trial [SoFEA (47)] in postmenopausal patients with and BKM120 induced regression of established tumors in + advanced ER breast cancer who progressed on an AI. This mice devoid of estrogen supplementation. These data col- trial compares treatment with fulvestrant (250 mg), an al- lectively suggest that ER and PI3K modulate independent ternative AI, or the combination. If this study were to be pathways critical for tumor maintenance, growth, and resis- + enriched with patients harboring tumors that remain ER- tance to estrogen deprivation in ER tumors. Hence, in pa- + dependent under conditions of estrogen suppression, we tients with ER breast cancer that progresses on AI therapy,

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ERα Drives Resistance to Estrogen Deprivation Therapy RESEARCH ARTICLE particularly in those harboring an E2F activation signature, experiment, tumor-bearing mice received vehicle, BKM120 (30 mg/ combined treatment with an ER downregulator (or a CDK4 kg/d), fulvestrant, or BKM120/fulvestrant. Tumor diameters were inhibitor) and a PI3K inhibitor is a strategy worthy of clinical measured twice per week. General linear modeling was used to evalu- investigation. ate differences between treatment groups at each time point. Tumors

were harvested and snap-frozen in liquid N2 or fixed in 10% formalin prior to paraffin embedding and IHC. METHODS

Cell Lines [18F]FDG-PET Imaging Parental lines (ATCC) were maintained in IMEM/10% FBS [18F]FDG-PET imaging was done before and 9 days after treatment (Gibco), and were authenticated on March 15, 2011 by short tandem with vehicle, BKM120 (60 mg/kg/d), or fulvestrant (5 mg on days 0 repeat profiling using Sanger sequencing. LTED cells were generated and 7). upon long-term culture in phenol red-free IMEM/10% DCC-FBS Further details are provided in Supplemental Experimental (Hyclone) as described (4). Procedures.

Cell Proliferation Assays Disclosure of Possible Conflicts of Interest Cells were treated with 10% DCC-FBS ± E2, 4-OH-T, fulvestrant, M. Dowsett receives research grant funds and has received hono- or PD-0332991 (gift from Pfizer) for 5 to 10 days before being tryp- raria for consulting and advisory board work from AstraZeneca and sinized and counted using a Coulter counter, or fixed and stained Novartis. S-M. Maira is an employee of Novartis. No potential con- with crystal violet. In siRNA transfection experiments, cells were re- flicts of interest were disclosed by the other authors. verse-transfected, then reseeded and treated as above. Grant Support Statistical Analyses This work was supported by NIH F32CA121900 (T.W. Miller), In the above assays, significant differences (P < 0.05) were de- K99CA142899 (T.W. Miller), Breast Cancer Specialized Program termined by ANOVA and Bonferroni post-hoc tests (multiple of Research Excellence (SPORE) grant P50CA98131, Vanderbilt- testing-corrected). Ingram Cancer Center Support Grant P30CA68485, U24CA126588 (South-Eastern Center for Small-Animal Imaging), R01CA140628 Gene Expression Analyses (H.C. Manning), RC1CA145138 (H.C. Manning), K25CA127349 (H.C. Manning); a Vanderbilt Institute for Clinical and Translational MCF-7/LTED and HCC-1428/LTED cells were treated ± 1 μM Research grant (T.W. Miller); a grant from the Breast Cancer Research fulvestrant for 48 hours. RNA was analyzed using gene expression Foundation (C.L. Arteaga); ACS Clinical Research Professorship arrays (GEO #GSE22533). Breast tumor gene expression data were Grant CRP-07-234 (C.L. Arteaga); the Lee Jeans Translational Breast obtained from GEO (GSE5462) and Array Express (E-MTAB-520). Cancer Research Program (C.L. Arteaga); Stand Up to Cancer/ AACR Dream Team Translational Cancer Research Grant SU2C- Chromatin Immunoprecipitation AACR-DT0209 (C.L. Arteaga, G.B. Mills). ChIP was done using MCF-7/LTED and HCC-1428/LTED cells, Received May 4, 2011; revised July 3, 2011; accepted July 5, 2011; and primary human breast tumors. ChIP DNA was analyzed by high- published OnlineFirst July 20, 2011. throughput sequencing (GEO #GSE27300) and real-time PCR.

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ERα-Dependent E2F Transcription Can Mediate Resistance to Estrogen Deprivation in Human Breast Cancer

Todd W. Miller, Justin M. Balko, Emily M. Fox, et al.

Cancer Discovery 2011;1:338-351. Published OnlineFirst July 20, 2011.

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