The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a diverse, clinically relevant panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.
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The authors thank C. Hoffmann, N. Dean and the teams supporting them for consenting and recruiting tissue donors, as well as tissue donors themselves. We thank A. Welm for PDX model provision; J. Robinson, M. Pickering, Z. Kikhtyak and N. Ryan for experimental assistance; and our institutional animal care facilities and genomics cores. This work was supported by grants from the National Health and Medical Research Council of Australia (W.D.T., J.S.C., T.E.H., E.L., L.A.S., E.E., S.N.B., C.P. and A.S.; ID 1084416, ID 1130077; K.M.C.; ID 1093801), the National Breast Cancer Foundation (NBCF; W.D.T.; ID PS-15-041; C.E.C.; ID ECF-17-002) and a Movember & National Breast Cancer Foundation Collaboration Initiative grant (MNBCF-17-012 to W.D.T., J.S.C., E.L., T.E.H. and L.A.S.), Cancer Australia (W.D.T., E.L., T.E.H., J.S.C.; ID 1107170), Cancer Institute of New South Wales (H.H.M.; ID ECF-17-1156) and The Hospital Research Foundation (T.E.H., L.A.S., W.D.T.). T.E.H. held fellowship awards from the US Department of Defense Breast Cancer Research Program (ID BC103923) and the Royal Adelaide Hospital Research Foundation (Adelaide, Australia). W.Z. and S.S. were supported by the Movember Foundation (ID NKI01), Dutch Cancer Society/Alpe D’HuZes (ID 10084) and the Netherlands Organisation for Scientific Research Vidi grant (ID 91716401). C.A.S. and J.F.S. were supported by NIHR01CA140985 and the Breast Cancer Research Foundation. J.S.C. and C.P. are supported by CRUK Funding (grant no. A17197 to J.S.C.). E.L. is supported by an NBCF Endowed Chair. K.M.C. is an NHMRC PhD Scholar. T.E.H. is currently supported by an NBCF Fellowship (IIRS-19-009) and the University of Adelaide.
S.N.B. is a major shareholder in Havah Therapeutics Pty Ltd. Research support was received from AstraZeneca (W.Z.), Astellas Pharma (W.Z.) and GTx Inc. (W.D.T. and C.P.). All other authors declare no competing interests.
Peer review information Javier Carmona was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Immunogenic AR protein levels predict breast cancer-specific survival in the Nottingham Tenovus Primary Breast Cancer Series14. Kaplan–Meier plots depicting the association between immunogenic AR positivity (dichotomized by high ≥ 78% and low < 78% nuclear staining based on an optimal cut-point criterion8) and breast cancer-specific survival. a, All cases; b, ER-positive cases (defined by immunogenic ER positivity of >1% as per current ASCO guidelines); c, High-risk (defined by Nottingham Prognostic Index ≥ 3.4) ER-positive cases treated with adjuvant endocrine therapy (Tamoxifen; Tam); d, ER-positive cases not treated with adjuvant Tamoxifen. Survival probabilities were estimated using a univariate Cox proportional hazards model. Differences between survival rates were tested for significance using the log-rank test. Hazard ratios (HR), 95% confidence intervals (95% CI), and p values are provided within each graph. All tests were two-sided.
Extended Data Fig. 2 Anti-proliferative effects of androgen in patient-derived explants (PDEs) of breast tissue exposed to estrogen.
a, Quantification of BrdU immunohistochemistry in ER + malignant patient-derived explants (PDEs; n = 9) treated with estrogen (E2, 10 nM) or E2 plus androgen (DHT, 10 nM). Data represented as mean ± SEM. Data was analyzed using a two-tailed Wilcoxon matched-pairs signed rank test (W = -45.0, r = 0.1149, **p = 0.0039). b, Correlation between immunohistochemical quantification of both BrdU- and Ki67-positive cells shown in (a) and (Fig. 1b). At least 1,000 cells, or all visible epithelial cells, whichever threshold was reached first, were examined in n = 9 independent cases of ER + malignant PDEs, treated with either E2 (left panel) or E2 + DHT (right panel). Data was analyzed by linear regression (trend line) and a two-tailed Pearson’s correlation test (E2 treatment; r = 0.697 [0.061, 0.931]; E2 + DHT treatment; r = 0.912 [0.627, 0.982]. P values are provided within each graph. c,d, RNA-seq data derived from PDEs of (c) normal breast tissue (n = 17 cases) and (d) ER + invasive breast cancer (n = 8 cases) treated with E2 (10 nM) or E2 + DHT (10 nM each). Heat maps depict the top 200 differentially expressed genes. e, Gene set enrichment plots for the Hallmark G2M Checkpoint gene set derived from RNA-seq data representing normal breast tissues (left panel) or ER + breast cancers (right panel).
a, Motif analysis of E2-stimulated ER binding sites associated with Fig. 2a,b, which are altered in the presence of androgen (DHT) in ZR-75-1 cells. GRHL (grainy-head-like motif); ERE (estrogen response element); ARE (androgen response element). b, Cropped immunoblot showing effective siRNA-mediated knock-down of AR (left panel) and ER (right panel) in ZR-75-1 cells treated with estrogen (E; 10 nM) alone or in combination with androgen (E + D; 10 nM each) for 4 hours. β-Actin was used as a loading control. Blots are representative of three experiments corresponding to independent passages of cells. c, Left panels: Genome browser images showing ER and AR ChIP-seq signals (associated with Fig. 2b) at E2-stimulated ER binding sites (BCL2, PGR) that are reduced in the presence of androgen (DHT, 10 nM). Right panels: Corresponding ER ChIP-PCR for BCL2 (upper panel) and PGR (lower panel) following AR knockdown and subsequent treatment with estrogen (E2, 10 nM) alone or in combination with androgen (E2 + DHT, 10 nM each) for 4 hours in ZR-75-1 cells. Data was analyzed using a one-sided unpaired t-test. Upper panel test details: t = 5.320, 0.2325, and 0.2274, for siCon (p = 0.0030), siAR (1) (p = 0.4138), and siAR (2) (p = 0.4915), respectively. Lower panel test details: t = 2.074, 0.2437, and 0.3767 for siCon (p = 0.0534), siAR (1) (p = 0.4097), and siAR (2) (p = 0.3628), respectively; df = 4 for each test. d, Left panels: Genome browser images showing AR and ER ChIP-seq signals (associated with Fig. 2b) at DHT-stimulated AR binding sites (ZBTB16, SEC14L2). Right panels: Corresponding AR ChIP-qPCR for ZBTB16 (upper panel) and SEC14L2 (lower panel) following ER knockdown and subsequent treatment with estrogen (E2, 10 nM) alone or in combination with androgen (E2 + DHT, 10 nM each) for 4 hours in ZR-75-1 cells. Each genome track in (c) and (d) depicts the average read density of all ChIP-seq replicates for the designated receptor and treatment condition. Data was analyzed using a one-sided unpaired t-test. Test details: Upper-left panel; t = 4.587, 1.102, and 0.6163, for siCon (p = 0.0051), siER (1) (p = 0.1662), and siER (2) (p = 0.2885), respectively. Upper right panel; t = 2.596, 4.443, and 6.906, for siCon (p = 0.0301), siAR (1) (p = 0.0057), and siAR (2) (p = 0.0012), respectively. Lower-left panel; t = 4.301, 1.221, and 1.198, for siCon (p = 0.0063), siER (1) (p = 0.1446), and siER (2) (p = 0.1485), respectively. Lower-right panel; t = 2.321, 2.504, and 1.678, for siCon (p = 0.0405), siAR (1) (p = 0.0332), and siAR (2) (p = 0.0843), respectively; df = 4 for each test. ChIP-PCR data is represented as mean ± SEM of three independent passages of cells. Asterisks denote statistical significance; NS = not significant. e, Average read density plots for ER, AR, and H3K27ac at ER binding sites within 100 kb of genes down-regulated in E2 + DHT versus E2 treatment arms in ZR-75-1 cells. f, Heatmap of RT-qPCR analysis of ER target gene expression in ZR-75-1 and T-47D cells treated for 6 hours with Vehicle (Veh), DHT (10 nM), E2 (10 nM) or E2 + DHT (ED, 10 nM each). g, Average read density plots for AR, ER, and H3K27ac at AR binding sites within 100 kb of genes up-regulated by E2 + DHT versus E2 in ZR-75-1 cells. h, Heatmap of RT-qPCR analysis of AR target gene expression in ZR-75-1 and T-47D cells treated for 6 hours with Vehicle (Veh), DHT (10 nM), E2 (10 nM) or E2 + DHT (ED, 10 nM each). Heatmap data shown in (f) and (h) represents the average normalized gene expression of four independent passages of cells.
Extended Data Fig. 4 AR-mediated modification of the ER cistrome requires an agonist ligand and AR DNA binding capacity.
a, Genome browser image of ER and AR chromatin binding events after treatment for 4 hours with either estrogen (E2, 10 nM) or estrogen plus androgen (E2 + DHT, 10 nM each) in ZR-75-1 cells at the MYB gene locus based on ChIP-seq data from Fig. 2. Each genome track depicts the average read density of all ChIP-seq replicates for the designated receptor and treatment condition. Enh., Enhancer. Tracks are scaled to facilitate visualization of the Intron 1 locus. b, ZR-75-1 in vitro ChIP-qPCR validation data for ER, H3K27ac and AR at three loci associated with the MYB gene locus as depicted in (a). Bar graphs depict mean ± SEM of 3 independent passages of cells. Data was analyzed using a one-sided unpaired t-test. ER ChIP; t = 1.345, 1.17, and 0.5662 for Enh. 1 (p = 0.1249), Intron 1 (p < 0.0001), and Enh. 2 (p = 0.3101), respectively. H3K27ac ChIP; t = 0.3320, 2.310, and 0.5664 for Enh. 1 (p = 0.3783), Intron 1 (p = 0.0410), and Enh. 2 (p = 0.3007), respectively. AR ChIP; t = 3.555, 1.480, and 3.993 for for Enh. 1 (p = 0.0118), Intron 1 (p = 0.1064), and Enh. 2 (p = 0.0085), respectively; df = 4 for each test. Grey asterisks denote statistical significance. c, Average read density plot of ER ChIP-seq enrichment at ER binding sites proximal (<100 kb) to genes down-regulated in E2 + DHT versus E2 treatment arms in ZR-75-1 cells, as per Extended Data Fig. 3e. Data shows ER enrichment in ZR-75-1 cells following in vitro treatment with Vehicle (Veh), estrogen (E2, 1 nM), an AR agonist (DHT, 1 nM), an AR antagonist (Bicalutamide; Bic 1 µM), alone or with indicated combinations. The data independently recapitulates the DHT-induced reduction in the E2-stimulated ER signal observed in Extended Data Fig. 3c,e at 10 nM doses of hormones, and using an independent ER antibody; Millipore 06-935. Treatment with an AR antagonist does not induce loss of ER at these loci. d, Genome browser images of ER binding sites in ZR-75-1 cells at ER target genes (BCL2, PGR), showing reduced binding following treatment with an AR agonist (DHT) but not an AR antagonist (Bicalutamide). Each genome track depicts the average read density of all ChIP-seq replicates for the designated receptor and treatment condition. e, Growth curve showing that estrogen (E2, 1 nM)-stimulated in vitro growth of ZR-75-1 cells is inhibited by an AR agonist (DHT, 1 nM) but not an AR antagonist (Bicalutamide, 1 µM). Data represents mean ± SD of three replicate cell culture wells per condition, and is a representative of two independent experiments. f-i, Experiments in MCF7 breast cancer cells transformed to overexpress a constitutively active, truncated AR (1-707 aa; AR) or a mutant AR that is unable to bind DNA (ARmut). AR expression is induced upon treatment with doxycycline (Dox). f Cropped immunoblot showing Dox-induced expression of truncated AR in cells treated with Vehicle or estrogen (E2, 1 nM), and resultant ER expression. β-Actin was used as a loading control. Blots are representative of three experiments corresponding to independent passages of cells. g Heatmap of RT-qPCR data for two ER target genes (MYB, CCND1) and two AR target genes (SEC14L2; ZBTB16) in cells treated with Vehicle (Veh) or estrogen (E2, 1 nM). Data represents the average normalized gene expression from three independent passages of cells replicates. h ER ChIP-qPCR at loci associated with ER target genes (MYB, CCND1) (top panels) and an AR target gene (ZBTB16). AR ChIP-qPCR is also shown for the AR target gene. Data was analyzed by a two-way ANOVA followed by Tukey’s multiple comparisons test. Upper-left panel; F = 125.9, 11.75, and 3.143 for presence of E2 (p < 0.0001), AR status (p = 0.0015), and their interaction (p = 0.0799), respectively. Asterisks denote statistical significance, where **p = 0.0060, NSp = 0.9998. Upper-right panel; F = 81.79, 13.85, and 1.565 for presence of E2 (p < 0.0001), AR status (p = 0.0008), and their interaction (p = 0.2489), respectively; where **p = 0.0094, NSp > 0.9999. Lower-left panel; F = 140.8, 284.6, and 94.59 for presence of E2 (p < 0.0001), AR status (p < 0.0001), and their interaction (p < 0.0001), respectively; where ****p < 0.0001. Lower-right panel; F = 0.1975, 147.1, and 0.09898 for presence of E2 (p = 0.6647), AR status (p < 0.0001), and their interaction (p = 0.9065), respectively; where ****p < 0.0001. df = 4 for each test. Bar graphs depict mean ± SEM of 3 independent passages of cells. NS = Not significant; **p < 0.01; ****p < 0.0001. i, Growth curve showing that induction of constitutively-active AR inhibits E2-stimulated growth but induction of the constitutively-active ARmut does not. Data represents mean ± SD of three replicate cell culture wells per condition, and is a representative of two independent experiments.
a, Replicate data for p300 ChIP-seq in vitro experiments in ZR-75-1 cells associated with Fig. 2e. Venn diagrams show the overlap of three independent experiments representing consecutive passages of cells treated with vehicle (Veh), estrogen (E2; 10 nM) or estrogen plus androgen (E2 + DHT; 10 nM each). Peaks present in at least 2 of 3 replicates were used to generate a consensus cistrome, indicated below the Venn diagrams, for further comparative analyses. b, Overlap of consensus p300 cistromes under E2 or E2 + DHT hormone treatments, after subtracting peaks present under basal (Veh) conditions to generate as set of hormone-regulated peaks. c, Consensus p300 ChIP-seq data from (b), associated with Fig. 2e, showing average read density plots (top panels) and heatmaps (bottom panels), illustrating changes in hormone-regulated p300 chromatin binding sites following treatment with E2 + DHT. d, Average read density plots for p300 binding at ER binding sites (ERBS) proximal (<100 kb) to genes down-regulated by androgen under estrogenic conditions (left panel), and AR binding sites (ARBS) proximal (<100 kb) to genes up-regulated under the same conditions (right panel). e, Example genome browser images showing averaged p300 ChIP-seq signals at binding sites associated with an ER target gene (PGR; left panel) and AR target gene (SEC14L2; right panel) in ZR-75-1 cells. Data represents the average signal of three replicates. f, p300 ChIP-qPCR at enhancers of ER-regulated cell cycle genes (as per Fig. 2g) in T-47D cells treated in vitro under designated hormone conditions. Data was analyzed by a two-way ANOVA (F = 93.45, 80.19, and 14.78 for hormone treatment (p < 0.0001), test site (p < 0.0001), and their interaction (p < 0.0001), respectively; df = 30). Data represented as mean ± SEM of 3 independent passages of cells. Post-hoc analyzes were performed using Tukey’s multiple comparisons test, where asterisks denote statistical significance; *p = 0.0275; *** p = 0.0040; ****p < 0.0001. g, Heatmap of RT-qPCR data for genes associated with Fig. 2g assessed in ZR-75-1 and T-47D cells treated in vitro with estrogen (E2, 10 nM) alone or in the presence of androgen (E2 + DHT, 10 nM each). Data represents the average normalized gene expression of four experiments conducted on independent passages of cells.
Extended Data Fig. 6 Activation of AR relocates SRC-3 from ER to AR target genes; correlating with p300 chromatin localization.
a, Replicate data for SRC-3 ChIP-seq experiments. Venn diagrams show the overlap of three independent experiments representing consecutive passages of ZR-75-1 cells treated with vehicle (Veh), estrogen (E2; 10 nM) or estrogen plus androgen (E2 + DHT; 10 nM each). Peaks present in at least 2 of 3 replicates were used to generate a consensus cistrome, indicated below the Venn diagrams, for further comparative analyses. b, Overlap of consensus SRC-3 cistromes under E2 or E2 + DHT hormone treatments. c, Two-factor log ratio (M) plot displaying DHT-induced changes in E2-stimulated p300 and SRC-3 enrichment at consensus p300 binding sites in ZR-75-1 cells (as per Fig. 2e). Point color denotes p300 consensus peak occupancy; hormone-responsive E2-unique (pink), hormone-responsive E2 + DHT-unique (purple) and basal (black; plotted at rear). Point co-ordinates denote enrichment scores at consensus binding sites, derived from an average of three replicates. Example p300 binding sites associated with ER (yellow-orange) and AR (orange-red) target genes are highlighted. d, SRC-3 ChIP-seq data showing read density plots (left panel) and genome browser images (right panels), illustrating E2 + DHT-induced changes in SRC-3 enrichment at ER binding sites (ERBS) proximal (<100 kb) to genes down-regulated by androgen under estrogenic conditions. Data represents an average of three replicates. e, Average read density plots for SRC-3 binding at AR binding sites (ARBS) proximal (<100 kb) to genes up-regulated by androgen under estrogenic conditions (left panel). Right panels: Genome browser images displaying averaged SRC-3 ChIP-seq signals at example ARBS; SEC14L2 and ZBTB16. Data represents an average of three replicates. f, SRC-3 ChIP-PCR at enhancers of ER-regulated cell cycle genes (as per Fig. 2g; Extended Data Fig. 5f) in ZR-75-1 cells (upper panel) and T-47D cells (lower panel), treated in vitro under designated hormone conditions. Data was analyzed by a two-way ANOVA. Upper panel F = 235.4, 61.41, and 7.338 for hormone treatment (p < 0.0001), test site (p < 0.0001), and their interaction (p < 0.0001), respectively; df =30. Lower panel F = 33.84, 29.62, and 5.343 for hormone treatment (p < 0.0001), test site (p < 0.0001), and their interaction (p = 0.0003), respectively; df = 30. ChIP-PCR data is represented as mean ± SEM of 3 independent passages of cells. Post-hoc analyzes were performed using Tukey’s multiple comparisons test, where asterisks denote statistical significance; *p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001. Exact p-values for upper panel: MYC p = 0.0051 for E2 vs. E2 + DHT; BCL2 p = 0.0066 for E2 vs. E2 + DHT; FOXM1 p = 0.296 for E2 vs. E2 + DHT; and p < 0.0001 for all other comparisons indicated by ****. Lower panel: MYB p = 0.0002 for E2 vs. E2 + DHT; CCND1 p = 0.0371 for E2 vs. E2 + DHT; FOXM1 p = 0.0198 for E2 vs. E2 + DHT; p < 0.0001 for all other comparisons that are indicated by ****.
a, Growth curves for ZR-75-1 xenograft tumors treated with E2 alone (n = 6) or in combination with AR agonists (DHT or Enobosarm (SARM); n = 6 and 7 tumors, respectively) or an AR antagonist (Enzalutamide; n = 6). Data represented as mean ± SEM. b, Representative IHC for ER, PR (encoded by ER target gene PGR), AR, and SEC14L2 (encoded by AR target gene SEC14L2) for ZR-75-1 xenograft tumors harvested 5 days post-treatment. Scale bars represent 100 µm. c, Quantification of Ki67-positivity from n = 4 (E2 + Veh, E2 + SARM and E2 + Enz) or n = 5 (E2 + DHT) ZR-75-1 xenograft tumors harvested 5 days post-treatment. Boxes in the graph show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes). Data was analyzed using a two-tailed, unpaired t-test (t = 17.36, 11.37, and 0.7941; df = 7, 6, and 7; for E2 + Veh vs. E2 + DHT (p < 0.0001), E2 + SARM (p < 0.0001), and E2 + Enza (p = 0.4574), respectively). Asterisks denote statistical significance; ****p < 0.0001, d, Ki67 IHC images in each treatment arm associated with (c). Scale bars represent 100 µm. Images are representative of n = 4 (E2 + Veh, E2 + SARM and E2 + Enz) or n = 5 (E2 + DHT) independent tumors. e, RNA-seq heatmap of all differentially expressed genes (FDR < 0.05) in 5 day-treated ZR-75-1 tumors demonstrates clustering of AR agonists (DHT and SARM) away from Vehicle (Veh) and the AR antagonist Enzalutamide (Enz). f, Independent RT-qPCR validation of select ER and AR target genes. Data represents an average normalized gene expression of 4 (E2 + Veh, E2 + Enz, E2 + SARM) or 5 (E2 + DHT) replicate tumors. NS, not significant.
Extended Data Fig. 8 ZR-75-1 xenograft sequencing data representing tumors harvested 5 days post-AR agonist treatment in vivo.
a, Read density plots (upper panel) and heatmaps (lower panel) of ER ChIP-seq data from ZR-75-1 xenograft tumors, illustrating the loss of ER binding upon activation of AR by agonists (SARM, DHT). Data is presented as an average of 4 (E2 + Veh) or 5 (E2 + SARM, E2 + DHT) replicate tumors. b, Two-factor log-ratio (M) plot showing SARM-induced changes in ER and AR enrichment at consensus ER (yellow-orange), AR (blue), or Shared (that is AR and ER co-occupied; grey-brown; plotted at rear) binding sites seen across the Vehicle, SARM, and DHT treatment arms. Example binding sites near ER target genes and AR target genes are highlighted in pink and purple, respectively. Enrichment scores were calculated from an average of 4 (ER, E2) or 5 (ER, E2 + SARM, AR E2, E2 + SARM) tumors. c, Read density plots (upper panel) and heatmap (lower panels) depictions of ER ChIP-seq presented in (a,b). d, Genome browser images of ER and AR binding in ZR-75-1 xenograft tumors treated with E2 or E2 + SARM at loci associated with ER target genes (MYB, CCND1; left panels) and AR target genes (SEC14L2, ZBTB16; right panels). Data represents an average of 4-5 replicates as described in (b). e, Normalized enrichment scores for signature gene sets correlated with E2 or E2 + SARM treatments, derived using RNA-seq data associated with Extended Data Fig. 7e.
Extended Data Fig. 9 AR modulation in patient-derived xenograft (PDX) models of ER+ endocrine-resistant breast cancer.
a, IHC showing baseline expression of ER, AR, progesterone receptor (PR), Ki67, and human cytokeratin 8/18 (CK8/18) in HCI-005 and GAR15-13 PDX models. Scale bars represent 50 µm. b, Growth curves showing inhibition of E2-stimulated PDX tumor growth (UCD4; UCD65) by co-treatment with DHT at time of tumor inoculation. Tumor volumes at ethical end-point were analyzed using a two-tailed, unpaired t-test, for UCD4; vehicle (n = 6) vs. DHT (n = 6 tumors each; t = 4.213, df = 10, p = 0.0018) and UCD65; vehicle (n = 12) vs. DHT (n = 5; t = 5.572, df = 15, p < 0.0001). c, AR agonist Enobosarm (SARM) inhibits in vivo growth of the UCD65 PDX. Tumors were grown in the presence of E2 to ~100 mm3 and subsequently treated with E2 + SARM (n = 10) or E2 n = 7), indicated by the red arrow. Graph on right shows quantification of tumor mass at end-point for E2 vs. E2 + SARM (n = 9 tumors each), analyzed by a two-tailed, unpaired t-test (t = 2.635, df = 16, p = 0.018). HER2, human epidermal growth factor receptor 2 (where ‘HER2-’ indicates lack of overexpression or gene amplification). d, Quantification of Ki67 positivity in PDX model HCI-005, from Fig. 3b, harvested 5 days post-treatment (t = 32.43 and 14.30 for E2 vs. E2 + DHT and E2 + SARM, respectively; df = 6 and p < 0.0001 for each test). At least 1,000 cells, or all visible epithelial cells, whichever threshold was reached first, were examined over n = 4 independent tumors per condition. e, HCI-005 PDX ER ChIP-seq data depicting loss of E2-stimulated ER chromatin binding at the MYC gene locus following 5 days treatment with AR agonists; Enobosarm (SARM) and DHT. f, HCI-005 PDX ER ChIP-seq data from Fig. 3b, showing average read density plots (top panels) and heatmaps (bottom panels) to illustrate genome-wide changes in E2-stimulated ER chromatin binding sites following 5 days treatment with AR agonists; Enobosarm (SARM) or DHT. g, Growth curve representing the UCD4 PDX cell line42 treated with estrogen (E2, 1 nM) alone or in combination with an AR agonist (DHT, 1 nM), AR antagonist (Enzalutamide; Enz, 1 µM) or the AR agonist and antagonist combined (Combo). Graph depicts mean± SEM of twelve replicate cell culture wells. h, Quantification of Ki67 positivity and i, corresponding representative images of Ki67 IHC in PDX model GAR15-13 from Fig. 3f, harvested 5 days post-treatment, with 5 tumors analyzed per condition by a two-tailed, unpaired t-test. Test details: Veh vs. DHT (t = 9.231, df = 8, p < 0.0001); Veh vs. SARM (t = 4.361, df = 8, p = 0.0024); Veh vs. Enz (t = 0.7958, df = 8, p = 0.4491). Scale bars in (i) represent 50 µm. j, Growth curve representing GAR15-13 PDX tumors treated with either Vehicle (n = 6), AR agonist (DHT, n = 4), or AR antagonist (Enzalutamide; Enza, n = 5) alone or in combination (DHT + Enza, n = 5). k, Heatmap of RT-qPCR data representing the average normalized gene expression from GAR15-13 and HCI-005 PDX tumors harvested 5 days post-treatment (GAR15-13: Veh, n = 6; DHT, n = 8; Enza, n = 8; DHT + Enza, n = 8) and (HCI-005: Veh, n = 5; DHT, n = 6; Enza, n = 7; DHT + Enza, n = 5). All graphed data depicts mean ± SEM. Asterisks denote statistical significance; *p < 0.05; **p < 0.01; ****p < 0.0001.
a, Hallmark gene sets enriched in the AR signature. The signature was separated into up-regulated (n = 59) and down-regulated (n = 83) genes and enriched gene sets (FDR < 0.05) identified by GSEA. b, Average read density plots for H3K27ac binding in HCI-005 PDX tumors, found at ER chromatin binding sites (left panel) and AR chromatin binding sites (right panel) proximal (<100 kb) to AR signature genes, associated with Fig. 5c. Data illustrates AR agonist (SARM, DHT)-dependent loss of H3K27ac binding at ER binding sites (ERBS) associated with down-regulated AR signature genes, and AR agonist-dependent gain of H3K27ac binding at AR binding sites (ARBS) associated with up-regulated AR signature genes. c, Genome browser image of H3K27ac binding in HCI-005 PDX tumors at a locus associated with an ER target gene down-regulated by AR agonists (PGR; left panel), and an AR target gene up-regulated by AR agonists (ZBTB16; right panel) as found in the AR signature. Data represents an average of three (E2 + DHT) or four (E2, E2 + SARM) replicate tumors.
Supplementary Figs. 1–4, unprocessed western blots related to Extended Data and Supplementary Figs. and raw data exemplifying FACS analysis.
Data associated with Fig. 1 and Extended Data Figs. 1 and 2, collated over seven tabs, including the regression model analysis for 20 years of breast cancer–specific survival for both all and ER-positive breast cancer cases, clinical information associated with patient-derived explants used for Ki67 quantification and RNA-seq, and clinical cohort data associated with breast tissue microarray.
Data associated with Fig. 2, providing a list of genes differentially expressed by dihydrotestosterone (DHT) in ZR-75-1 cells, as determined by RNA-seq, and MACS2 peak calling statistics from AR and ER ChIP-seq with ZR-75-1 tumors.
Data associated with development of the AR signature, providing a list of genes differentially expressed by DHT in an E2-dependent ZR-75-1 xenograft model and two PDX models (E2-dependent HCI-005 and E2-independent GAR15-13). A list of genes found in the AR gene signature is provided alongside results of the SigCheck analysis.
Primer sequences used in ChIP–PCR and RT–PCR experiments.
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Hickey, T.E., Selth, L.A., Chia, K.M. et al. The androgen receptor is a tumor suppressor in estrogen receptor–positive breast cancer. Nat Med 27, 310–320 (2021). https://doi.org/10.1038/s41591-020-01168-7
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