Triple-negative breast cancer (TNBC) is an aggressive subtype, with a peak recurrence rate within the first few years after diagnosis. Few targeted therapies are available to treat this breast cancer subtype, defined by the lack of estrogen receptor (ER) and progesterone receptor and without amplification of human epidermal growth factor receptor 2 (HER2). Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are approved for treatment of ER-positive (ER+) breast cancer, they have not proven effective as monotherapy in patients with TNBC. The androgen receptor (AR) has emerged as a therapeutic target in a subset of TNBCs and with significant clinical benefit observed in multiple trials. The purpose of this study was to investigate the preclinical activity of the CDK4/6 inhibitor, abemaciclib, in combination with an agent that targets both androgen biosynthesis and AR activity, seviteronel, using TNBC cell lines expressing high AR, cell line xenografts, and an AR-positive (AR+), androgen-responsive TNBC patient-derived xenograft (PDX). Single-cell RNA sequencing demonstrated heterogeneity in AR levels, even in a highly AR+ cell line, and identified cell cycle pathway activation in ARHigh- versus ARLow-expressing cells. Combination treatment with the cell cycle CDK4/6 inhibitor, abemaciclib, and seviteronel showed synergy in an AR+ TNBC model compared with each drug alone. Although cell cycle inhibitors are FDA approved for use in ER+ breast cancer, our studies suggest that they may also be effective in AR+ TNBC, perhaps combined with AR-targeted agents.

Breast cancer is the most common cancer among women in the United States, and approximately one in eight women will be diagnosed with breast cancer within their lifetime (1). Breast cancers can be divided into three main subtypes: estrogen receptor alpha (ERα)-positive, human epidermal growth factor receptor 2 (HER2)-positive, or triple-negative breast cancer (TNBC); and treatment regimens are determined on the basis of the subtype at diagnosis (2). TNBCs, which account for approximately 15% to 20% of diagnosed breast cancer cases (2), lack expression of ERα and progesterone receptor (PR) and do not have amplified HER2. TNBC is notoriously difficult to treat, particularly once chemoresistant metastatic disease develops, and few targeted therapies are available for the majority of patients. In addition, TNBC is the most aggressive breast cancer subtype with a high rate of metastasis in the first 3 years and a low 5-year survival rate (3). Thus, development of additional therapeutic strategies for TNBC is an important avenue of study.

The androgen receptor (AR) has emerged as a potential therapeutic target in breast cancer. More than 80% of all breast cancers and up to 50% of TNBCs overexpress AR (4–9). In TNBC, AR supports cancer stem cell–like properties, including anchorage-independent survival, mammosphere formation, and tumor initiation (10). AR activation also promotes cell survival through regulation of the cell cycle (11). Androgen withdrawal results in G1-phase arrest (12), and AR-dependent gene expression is highest during G1-phase and decreases throughout the cell cycle (13). In fact, AR-positive (AR+) TNBC cells are highly sensitive to cyclin-dependent kinase (CDK) 4/6 inhibition due to their dependence on these proteins to progress through the cell cycle (14). Combining standard-of-care chemotherapy with AR antagonists and/or CDK4/6 inhibitors may prove effective in this patient population and this strategy is being employed in clinical trials with older-generation drugs. Given the recent advancements in both AR antagonist and CDK4/6 inhibitor drug design, newer generation inhibitors may prove even more effective.

Anti-androgens have been under investigation in breast cancer for more than a decade (15, 16). First-generation anti-androgens, such as bicalutamide, function as competitive inhibitors that block AR transcriptional activity. The second-generation anti-androgen, enzalutamide, is a competitive inhibitor that blocks nuclear localization of AR, and consequently has less partial agonist activity than first-generation anti-androgens (17, 18). Both of these AR antagonists are well-tolerated in patients and have shown beneficial clinical activity in TNBC, with subsets of patients showing prolonged stable disease (19–21).

To maximize response rates of AR+ TNBC to anti-androgens, studies are ongoing to determine the best diagnostic cut-off levels of AR expression, identify gene expression signatures predictive of AR dependency (22), and evaluate newer generation AR-targeted therapies that block AR activity through the inhibition of androgen biosynthesis. One such therapy is abiraterone acetate, a cytochrome P450 family 17 (CYP17) hydroxylase inhibitor that blocks androgen synthesis and has shown promise in the clinical setting against AR+ TNBC (23). However, a consequence of inhibiting CYP17 hydroxylase is the interruption of cortisol production, which necessitates supplementation with the corticosteroid, prednisone (23, 24). Seviteronel (INO-464) is a dual CYP17 lyase inhibitor and AR antagonist (Supplementary Fig. S1), with preclinical activity in multiple tumor types, including castration-resistant prostate cancer (CRPC), ER-positive (ER+) breast cancer, TNBC, and glioblastoma (GBM; refs. 25–30). It has undergone clinical investigation in CRPC, breast cancer (31, 32), and GBM (NCT03600467). Because seviteronel preferentially inhibits CYP17 lyase over hydroxylase, there is less impact on cortisol production compared with hydroxylase inhibitors, such as abiraterone (33). Thus, seviteronel's dual mechanism of action of CYP17 lyase inhibition and AR antagonism combines the actions of enzalutamide and abiraterone, potentially providing increased efficacy in treating AR+ TNBCs. The purpose of this study was to investigate the preclinical activity of seviteronel in AR+ TNBC and further explore the efficacy of targeting both androgen biosynthesis and AR activity in combination with CDK4/6 inhibition (34).

Cell culture and reagents

Human TNBC cell lines were cultured in 5% CO2. Luminal AR MDA-MB-453 cells were purchased from the ATCC and maintained in DMEM with 10% FBS. SUM159PT cells, expressing moderate levels of AR (35), were obtained in 2013 from the University of Colorado Cancer Center (UCCC) Tissue Culture Core (Aurora, CO) and maintained in Ham/F-12 with 5% FBS, 1% HEPES, 1 μg/mL hydrocortisone, and 5 μg/mL insulin. Only cells of under 10 passages were used in this study. All cell lines were routinely tested for Mycoplasma contamination, and human cell lines were authenticated in 2017 by short tandem repeat analysis in the UCCC Tissue Culture Core (Aurora, CO). The androgen dihydrotestosterone (DHT) (Sigma-Aldrich Corporation) used for in vitro experiments was diluted in ethanol. The AR and CYP17 lyase inhibitor, seviteronel, was provided by Innocrin Pharmaceuticals, Inc. The CDK4/6 inhibitor, abemaciclib, was purchased from Selleck Chemical LLC. All drugs were diluted in DMSO.

Drug sensitivity assays

Cells were plated in 96-well plates in quadruplicate or quintuplicate, and treated with either DMSO or increasing concentrations of seviteronel or abemaciclib. After 5 days of drug treatment, cells were fixed with 10% formalin, stained with 0.1% crystal violet dye, and dye was then solubilized with 10% acetic acid. Absorbance was measured at 570 nm. Data are presented as percentage cell growth and normalized to the mean absorbance of DMSO-treated cells. Synergy was calculated with CalcuSyn Software (BioSoft Inc.) using mean percentage growth inhibition values from crystal violet cell viability assays. CalcuSyn software uses the median effect method to determine synergy, where a combination index (CI) <0.9 indicates synergy, CI = 0.9–1.1 indicates additivity, and CI > 1.1 indicates antagonism (36).

In vivo preclinical models

All in vivo experiments were performed in accordance with NIH Guidelines of Care and Use of Laboratory Animals. Mice were euthanized by CO2 inhalation, followed by cervical dislocation. Tissue was immediately frozen whole in liquid nitrogen for RNA analysis or fixed in 10% buffered formalin for histologic analysis. Seviteronel experiments were limited to 4 weeks because rodents experience induction metabolism in which seviteronel is degraded by activated liver enzymes following 4 weeks of treatment (27).

Xenograft studies

A total of 1 × 106 MDA-MB-453 cells were bilaterally, orthotopically injected into the mammary fat pads of cycling female nu/nu mice. There was no hormone supplementation in this experiment. When tumors reached a size of approximately 100 mm3, mice were randomized and matched into treatment groups (n = 5–12 mice/group). Varying doses of seviteronel and/or abemaciclib were administered by oral gavage daily for 4 weeks. Tumors were measured weekly by caliper.

Patient-derived xenograft studies

HCI-009 is a TNBC patient-derived xenograft (PDX) originally developed in the laboratory of Dr. Alana Welm (from the University of Utah, Huntsman Cancer Institute) (37) and propagated in immunodeficient NOD/SCID/gamma (NSG) mice. In the DHT experiment (n = 8 mice/group), cycling female NSG mice were implanted with cellulose control or slow release 8-mg DHT pellets, prepared in Dow Corning Silastic Tubing (Thermo Fisher Scientific). PDX tumor portions were then bilaterally, orthotopically injected into the mammary fat pads, and tumors were measured weekly by caliper for 9 weeks. In the seviteronel experiment, PDX tumor portions were bilaterally, orthotopically injected into the mammary fat pads of cycling female NSG mice. When tumors reached a size of approximately 100 mm3, mice were randomized and matched into treatment groups (n = 4–5 mice/group). Seviteronel (150 mg/kg/day) was administered daily by oral gavage for 2 weeks. Tumors were measured weekly by caliper.

Histology

For the analysis of MDA-MB-453–cultured cells, cells were fixed in 10% buffered formalin and pelleted in HistoGel from Thermo Fisher Scientific Inc. The UC Denver Tissue Biobanking and Processing Core (Denver, CO) performed all tissue and cell processing and paraffin embedding. Five-micrometer-thick sections of formalin-fixed, paraffin-embedded samples were used for analyses.

IHC

Sections were deparaffinized in a series of xylenes and ethanol, and antigens were heat retrieved in either 10 mmol/L citrate buffer, pH 6, or 10 mmol/L Tris and 1 mmol/L EDTA, pH 9. Antibodies used included the following: rabbit mAb specific for AR (No. 200R clone SP107, Cell Marque), rabbit polyclonal antibody specific for FKBP5 (No. 8245, Cell Signaling Technology), rabbit polyclonal antibody specific for PSA (No. A0562, Agilent Technologies Inc.), goat polyclonal antibody specific for GDF15 (No. AF957, R&D Systems), and rabbit polyclonal antibody specific for CHI3L1 (No. ab77528, Abcam). TBS with 0.05% Tween 20 was used for all washes. AR, FKBP5, PSA, and CHI3L1 antibodies were detected with EnVision-HRP Anti-rabbit Polymer (No. K4003, Agilent Technologies), while the GDF15 antibody was detected with a biotinylated donkey anti-goat secondary antibody (No. 705-065-147, Jackson ImmunoResearch Laboratories, Inc.), followed by streptavidin-horseradish peroxidase (HRP; No. P0397, Agilent Technologies). Representative images were taken using a BX40 Microscope (Olympus) with a SPOT Insight Mosaic 4.2 Camera and Software (Diagnostic Instruments, Inc.). Expression levels were scored visually by an experienced histotechnician for the entire sample and presented as an IHC score, calculated by multiplying the average staining intensity by the percentage of positive cells.

Tumor necrosis

Mammary tumor sections were hematoxylin and eosin (H&E) stained and analyzed for necrosis by a board-certified veterinary pathologist. Data were presented as the percentage of each tumor that was composed of necrotic dead cells.

Bioinformatics

Bulk RNA sequencing

RNA isolated from frozen HCI-009 PDX mammary tumors was used to prepare Illumina HiSeq libraries according to the manufacturer's instructions for the TruSeq Stranded RNA Kit (Illumina Inc). The mRNA template libraries were then sequenced as single-pass 50-bp reads on the Illumina HiSeq4000 platform at the UC Genomics and Sequencing Core Facility (Denver, CO). Derived sequences were analyzed by applying a custom computational pipeline consisting of the open-source gSNAP, Cufflinks, and R for sequence alignment and ascertainment of differential gene expression (38). In short, reads generated were mapped to the human genome (GRCh38) by gSNAP (39), expression was (FPKM) derived by Cufflinks (40), and differential expression was analyzed with ANOVA in R. Differentially expressed genes (FDR < 0.05 for DHT and P < 0.05 for seviteronel, due to a lack of statistical power) with a minimum expression ratio of 1.15 were used for downstream analyses. These data are available in the Gene Expression Omnibus (GEO) database as GSE152246 for the DHT experiment (Supplementary Fig. S2A) and GSE152318 for the seviteronel experiment (Supplementary Fig. S2B).

Single-cell RNA sequencing

A total of 5 × 105 MDA-MB-453 cells were plated in two T25 flasks. After 48 hours, cells were trypsinized, washed, and prepared for single-cell RNA sequencing (scRNA-seq) analysis via the 10X Genomics platform and Illumina NovSeq 6000 platforms (41) at the UC Genomics and Sequencing Core Facility (Denver, CO) using the 10X Genomics Sample Preparation Protocol. A total of 3,000 cells were sequenced with a minimum read-depth of 75,000 reads per cell. Read mapping and expression quantification were performed using a combination of the 10X Cellranger pipeline and custom analytic scripts. Briefly, single-cell reads were mapped to the human genome (GRCh38) and assigned to genes using the standard CellRanger pipeline. The R packages, Monocle (42–44) and Seurat (45), were used for differential expression. The cell cycle heatmap, using 10 cell aggregates, was generated using the Morpheus software package. These data are available in the GEO database as GSE152315. Downstream RNA sequencing (RNA-seq) analyses were performed using Ingenuity Pathway Analysis (IPA, Qiagen) and gene set enrichment analyses (GSEA; refs. 46, 47). Overlap between significantly differentially expressed genes from the MDA-MB-453 scRNA-seq and HCI-009 seviteronel experiments was compared using the web application BioVenn (48).

Statistical analysis

Statistically significant differences (P < 0.05) were calculated using the GraphPad Prism 7.0 statistical program. Single-variable comparisons were made with two-tailed unpaired t tests. A repeated measure two-way ANOVA was used for in vivo tumor data analysis.

Seviteronel effectively limits AR activity in TNBC

Seviteronel has been shown to target AR transcriptional activity in prostate and breast cancer, with preclinical activity similar to previously approved AR-targeted therapies (25–27, 49). Chromatin immunoprecipitation qPCR indicated that seviteronel significantly blocks DHT-induced binding of AR to the promoters of aquaporin 3, transmembrane serine protease 2, and fatty acid synthase (49). Similarly, seviteronel blocked the DHT-induced transcription of multiple AR-target genes (49). In both cases, seviteronel inhibited AR activity with an efficiency similar to that observed with the second-generation AR antagonist, enzalutamide (49). Moreover, a recent publication found that seviteronel sensitizes TNBC cells to standard radiation (50). To determine the effect of seviteronel on TNBC proliferation, AR+ MDA-MB-453 cells were treated with increasing concentrations of seviteronel, resulting in the inhibition of cell proliferation at concentrations greater than 10 μmol/L (Fig. 1A). This was further confirmed in the TNBC cell line SUM159PT (Fig. 1A), which express moderate levels of AR (10). Given the ability of seviteronel to inhibit androgen synthesis in addition to inhibiting AR directly (49), as compared with other AR antagonists, we examined its effectiveness against TNBC tumor growth in vivo using a xenograft model. Normal cycling female mice were used as a more clinically relevant model of female patients with low levels of androgen synthesis. Seviteronel significantly inhibited tumor volume and growth rate in a dose-dependent manner (Fig. 1B and C). Together, these data indicate that seviteronel effectively targets the pro-proliferative activities of AR in TNBC cell lines in culture and in vivo.

Figure 1.

TNBC sensitivity to seviteronel (sevi) in a preclinical model. A, TNBC cell lines were cultured in full-serum media. Cell viability was determined by crystal violet assay after a 5-day exposure to DMSO (0) or increasing concentrations of seviteronel. Data were normalized to the mean absorbance of DMSO-treated cells. Mean ± SD. B, MDA-MB-453 cells were injected into the mammary fat pads of cycling female nu/nu mice. Supplemental DHT was not given in this experiment. Seviteronel was administered daily. C, Tumor volume was measured by calipers and the rate of tumor growth per day over 4 weeks was calculated. Mean ± SEM (*, P < 0.05; ****, P < 0.0001).

Figure 1.

TNBC sensitivity to seviteronel (sevi) in a preclinical model. A, TNBC cell lines were cultured in full-serum media. Cell viability was determined by crystal violet assay after a 5-day exposure to DMSO (0) or increasing concentrations of seviteronel. Data were normalized to the mean absorbance of DMSO-treated cells. Mean ± SD. B, MDA-MB-453 cells were injected into the mammary fat pads of cycling female nu/nu mice. Supplemental DHT was not given in this experiment. Seviteronel was administered daily. C, Tumor volume was measured by calipers and the rate of tumor growth per day over 4 weeks was calculated. Mean ± SEM (*, P < 0.05; ****, P < 0.0001).

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PDXs are preclinical models that more closely mimic patient tumor heterogeneity and clinical responses to therapeutic agents than traditional cultured cell lines (51). HCI-009 is an AR+ TNBC PDX with a distinctly heterogeneous AR staining pattern (Fig. 2A). In mice supplemented with AR agonist DHT, HCI-009 PDX tumor growth was significantly increased (Fig. 2B) and AR protein levels were upregulated (Fig. 2C), indicating that this PDX model is androgen responsive. To evaluate the transcriptional activity of AR in these tumors, known AR-regulated genes were examined for upregulation at the protein level by IHC. Expression of the AR-regulated proteins, FKBP prolyl isomerase 5 (FKBP5), prostate-specific antigen (PSA), growth differentiation factor 15 (GDF15), and chitinase 3-like 1 (CHI3L1), was significantly upregulated in HCI-009 PDX tumors from mice supplemented with DHT (Fig. 2D). Similarly, RNA-seq of HCI-009 tumors from mice supplemented with DHT (Supplementary Fig. S2A) showed upregulation of known AR-regulated genes, such as KLK3, the gene that encodes PSA (Fig. 2E; Supplementary Table S1), and many others, such as KLK1 (kallikrein 1), KLK2, AZGP1 (zinc-alpha-2-glycoprotein 1), PARM1 (prostate androgen-regulated mucin-like protein 1), SCGB1A1 (secretoglobin family 1A member 1), PLA2G2A (phospholipase A2 group IIA), CLDN8 (claudin 8), and HMGCS2 (3-hydroxy-3-methylglutaryl-coA synthase 2).

Figure 2.

Androgen-responsive TNBC PDX model. A, TNBC PDX HCI-009 was propagated in NSG mice. Tumor tissue was stained for AR by IHC. AR staining in MDA-MB-453 cells is shown for comparison. B–E, HCI-009 PDX tumor portions were injected into the mammary fat pads of female NSG mice supplemented with cellulose, as a control, or DHT (8 mg/pellet). B, Tumor volume was measured with calipers. C, AR was stained by IHC. D, IHC staining of known AR-regulated genes. Quantification of staining is presented as an IHC score, combining cell positivity and staining intensity. E, Shown are the top 30 up- and downregulated genes identified by RNA-seq in HCI-009 PDX tumors from mice treated with DHT. Mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bars, 50 μm).

Figure 2.

Androgen-responsive TNBC PDX model. A, TNBC PDX HCI-009 was propagated in NSG mice. Tumor tissue was stained for AR by IHC. AR staining in MDA-MB-453 cells is shown for comparison. B–E, HCI-009 PDX tumor portions were injected into the mammary fat pads of female NSG mice supplemented with cellulose, as a control, or DHT (8 mg/pellet). B, Tumor volume was measured with calipers. C, AR was stained by IHC. D, IHC staining of known AR-regulated genes. Quantification of staining is presented as an IHC score, combining cell positivity and staining intensity. E, Shown are the top 30 up- and downregulated genes identified by RNA-seq in HCI-009 PDX tumors from mice treated with DHT. Mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bars, 50 μm).

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After confirming that HCI-009 PDX tumors have active AR and are growth stimulated by exogenous androgen, we tested responsiveness to seviteronel in mice that did not have exogenous androgen supplementation. Mice with HCI-009 PDX mammary tumors were treated with 150 mg/kg/day seviteronel, and tumor volume and rate of growth were significantly decreased by seviteronel treatment (Fig. 3A and B). Seviteronel increased tumor necrosis (Fig. 3C) and significantly decreased AR protein levels (Fig. 3D). RNA-seq showed significant changes in gene expression following seviteronel treatment, inducing a distinctive seviteronel gene signature that included known AR-regulated genes (Fig. 3E; Supplementary Fig. S2B; Supplementary Table S2). Pathway analysis performed on RNA-seq data further demonstrated that while seviteronel affects several biological pathways, the androgen response pathway (normalized enrichment score = 1.69) was one of the top altered pathways (Fig. 3F). Overall, these data support the function of seviteronel as a potent inhibitor of AR and tumor growth in this AR+ TNBC PDX.

Figure 3.

AR-positive TNBC PDX sensitivity to AR-targeted therapy. HCI-009 PDX tumor portions were injected into the mammary fat pads of female NSG mice. Mice were treated daily with vehicle or 150 mg/kg/day seviteronel (sevi). Tumor volume was measured with calipers (A) and the rate of tumor growth per day over 2 weeks was calculated (B). The percentage of tumor necrosis (C) and AR+ cells per tumor (D) were quantified; representative images of H&E staining. Scale bars, 200 and 50 μm, respectively. Shown are the top 30 up- and downregulated genes (E) and most highly correlated biological pathways (F) identified by GSEA in RNA-seq data from HCI-009 PDX tumors from mice treated with seviteronel. Mean ± SEM (*, P < 0.05; **, P < 0.01). NES, normalized enrichment score.

Figure 3.

AR-positive TNBC PDX sensitivity to AR-targeted therapy. HCI-009 PDX tumor portions were injected into the mammary fat pads of female NSG mice. Mice were treated daily with vehicle or 150 mg/kg/day seviteronel (sevi). Tumor volume was measured with calipers (A) and the rate of tumor growth per day over 2 weeks was calculated (B). The percentage of tumor necrosis (C) and AR+ cells per tumor (D) were quantified; representative images of H&E staining. Scale bars, 200 and 50 μm, respectively. Shown are the top 30 up- and downregulated genes (E) and most highly correlated biological pathways (F) identified by GSEA in RNA-seq data from HCI-009 PDX tumors from mice treated with seviteronel. Mean ± SEM (*, P < 0.05; **, P < 0.01). NES, normalized enrichment score.

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AR is a positive regulator of the cell cycle

While MDA-MB-453 cells are highly AR+, IHC demonstrated heterogeneity in cell-specific AR levels within this cell line (Fig. 2A). To examine gene expression associated with varying levels of AR, scRNA-seq was performed on MDA-MB-453 cells cultured in 10% serum containing endogenous levels of androgens (Supplementary Fig. S3A). MDA-MD-453 cells were divided into ARHigh (1,205 cells) and ARLow (948 cells) populations based on the degree of AR mRNA expression (Fig. 4A; Supplementary Tables S3 and S4 for gene lists). Pathway analysis on genes associated with ARHigh versus ARLow levels indicated that the cell cycle (z = 4.12; P = 1.13E-45) was one of the top pathways associated with high AR expression (Fig. 4B; Supplementary Fig. S4B). A large percentage (54%) of cell cycle–related genes were upregulated in high AR–expressing MDA-MB-453 cells (Fig. 4C; Supplementary Table S5). Interestingly, when the genes associated with ARHigh MDA-MB-453 cells were compared with the genes altered in HCI-009 PDX tumors treated with seviteronel (Fig. 3), approximately 65% of genes altered by seviteronel overlapped with the MDA-MB-453 ARHigh–related gene signature (Supplementary Fig. S4; Supplementary Table S6), providing further evidence to support the AR-directed effects of seviteronel in TNBC, although non-AR–directed seviteronel effects cannot be ruled out.

Figure 4.

The association between high AR expression and cell cycle pathway activation. scRNA-seq was performed on untreated MDA-MB-453 cells cultured in full-serum media. Cells were divided into groups based on their expression of AR, ARLow and ARHigh. A, Histogram of the distribution of AR expression in single MDA-MB-453 cells, including AR level group determination. B, IPA was performed on genes differentially expressed between ARLow and ARHigh groups. Shown are the top 15 most significantly altered disease and function pathways. C, Differential expression of genes involved in cell cycle indicating cell cycle activation associated with high AR expression, shown as 10 cell aggregates.

Figure 4.

The association between high AR expression and cell cycle pathway activation. scRNA-seq was performed on untreated MDA-MB-453 cells cultured in full-serum media. Cells were divided into groups based on their expression of AR, ARLow and ARHigh. A, Histogram of the distribution of AR expression in single MDA-MB-453 cells, including AR level group determination. B, IPA was performed on genes differentially expressed between ARLow and ARHigh groups. Shown are the top 15 most significantly altered disease and function pathways. C, Differential expression of genes involved in cell cycle indicating cell cycle activation associated with high AR expression, shown as 10 cell aggregates.

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Combination targeting of AR and CDK4/6 is effective in AR+ TNBC

On the basis of the link between AR levels and cell cycle activation in TNBC cells (Fig. 4), we aimed to evaluate dual targeting of AR and the cell cycle as a potential treatment strategy for AR+ TNBC. A previous investigation of combination therapy, using different early-generation inhibitors, showed some promise clinically (NCT02605486; palbociclib with bicalutamide in patients with AR+ metastatic breast cancer; refs. 52–54). We tested combination treatment with seviteronel and the CDK4/6 inhibitor, abemaciclib (55), in a preclinical TNBC model. In MDA-MB-453 cells, dual treatment showed synergy at multiple concentration combinations, analyzed by the Chou–Talalay method (36), indicated by combination index (CIs) less than 0.9 (Fig. 5A). While abemaciclib is currently FDA approved for use in patients with ER+ breast cancer (56), our preclinical analysis showed that it is effective in inhibiting MDA-MB-453 tumor growth as well (Fig. 5B). These data led us to examine how dual treatment with seviteronel and abemaciclib affected MDA-MB-453 tumor growth in vivo. Combination treatment significantly inhibited tumor growth when compared with vehicle alone and was more effective than abemaciclib treatment alone (Fig. 5C). Interestingly, the antitumor effects of combined treatment predominantly occurred during the final week of treatment (weeks 3–4) where the combination treatment led to tumor shrinkage indicated by an average negative rate of growth. Eight of 12 (67%) tumors shrank during the last week of treatment in the combination of seviteronel plus abemaciclib group as compared with just three out of 12 (25%) that shrank with abemaciclib alone (Fig. 5D and E). Moreover, the combination treatment was well-tolerated with no negative effects on mouse weight gain (Supplementary Fig. S5). These data suggest that the combination of AR-targeted therapies with cell cycle inhibitors may prove to be an effective treatment for patients with AR+ TNBC.

Figure 5.

Combination (combo) treatment of TNBC cells with an anti-androgen and cell cycle inhibitor. A, MDA-MB-453 cells were cultured in full-serum media. Cell viability was determined by crystal violet assay after a 5-day exposure to DMSO (0) or increasing concentrations of seviteronel (sevi) and (abemaciclib (abem; CDK4/6 inhibitor). Data were normalized to the mean absorbance of DMSO-treated cells and presented as mean percentage growth inhibition. CalcuSyn software was used to calculate the degree of synergy between combination treatments, represented as the CI. CIs < 0.9 indicate synergy. B, MDA-MB-453 cells were bilaterally injected into the mammary fat pads of cycling female nu/nu mice. Abemaciclib was administered daily. Tumor volume was measured with calipers. C–E, MDA-MB-453 cells were injected into the mammary fat pads of cycling female nu/nu mice. Abemaciclib and/or seviteronel were administered daily. Tumor volume was measured with calipers (C) and the rate of tumor growth per day over 4 weeks was calculated (D). Rate of tumor growth was broken down to illustrate the differences in tumor growth over the first 3 weeks of treatment compared with the final week alone. E, Change in tumor volume, either growth or reduction, during the final week of treatment is depicted for each individual mouse. Mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 5.

Combination (combo) treatment of TNBC cells with an anti-androgen and cell cycle inhibitor. A, MDA-MB-453 cells were cultured in full-serum media. Cell viability was determined by crystal violet assay after a 5-day exposure to DMSO (0) or increasing concentrations of seviteronel (sevi) and (abemaciclib (abem; CDK4/6 inhibitor). Data were normalized to the mean absorbance of DMSO-treated cells and presented as mean percentage growth inhibition. CalcuSyn software was used to calculate the degree of synergy between combination treatments, represented as the CI. CIs < 0.9 indicate synergy. B, MDA-MB-453 cells were bilaterally injected into the mammary fat pads of cycling female nu/nu mice. Abemaciclib was administered daily. Tumor volume was measured with calipers. C–E, MDA-MB-453 cells were injected into the mammary fat pads of cycling female nu/nu mice. Abemaciclib and/or seviteronel were administered daily. Tumor volume was measured with calipers (C) and the rate of tumor growth per day over 4 weeks was calculated (D). Rate of tumor growth was broken down to illustrate the differences in tumor growth over the first 3 weeks of treatment compared with the final week alone. E, Change in tumor volume, either growth or reduction, during the final week of treatment is depicted for each individual mouse. Mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

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AR signaling has emerged as an effective targetable pathway in a subset of TNBCs, and first- and second-generation anti-androgens, such as bicalutamide and enzalutamide, have shown promising clinical activity in patients with AR+ TNBC. Seviteronel is unique in its ability to directly antagonize AR, as well as inhibit the androgen-synthesizing enzyme, CYP17 lyase, with less interference on corticosteroid production than earlier generations of anti-androgens (27). Similar to what has been reported previously in prostate cancer (25), seviteronel is as effective as enzalutamide in inhibiting AR activity in TNBC cells (49). Because of its mechanistic differences from earlier generation AR anti-androgens, seviteronel's effectiveness was predicted to be particularly pronounced in vivo because of its ability to decrease ligand availability (25, 49). Our data demonstrate that seviteronel significantly inhibited an AR+ TNBC cell line xenograft in a dose-dependent manner (Fig. 1). Our data are the first evidence that seviteronel is able to inhibit proliferation of an AR+ TNBC PDX tumor model as well (Fig. 3). Thus, seviteronel shows promising antitumor activity in both cell line and PDX preclinical models of TNBC.

Although the activity of seviteronel as an inhibitor of both AR activity and ligand synthesis has been demonstrated (25, 49), there are likely other mechanisms of action contributing to its effect on TNBC growth. Our data indicate that a proportion of the antitumor effects of seviteronel is AR directed, as shown by the “androgen response pathway” being one of the top biological pathways associated with seviteronel treatment and tumor shrinkage in vivo (Fig. 3F) and the large overlap between genes altered by seviteronel treatment and upregulated in ARHigh MDA-MB-453 cells (Supplementary Fig. S4). Pathway analysis (Fig. 3F) also indicates that cholesterol homeostasis, myogenesis, and mTOR complex 1 signaling are also highly altered by seviteronel treatment. The influence of seviteronel on these pathways has yet to be investigated, but may be linked to its role as a dual CYP17A1 and AR inhibitor because the CYP17A1 enzyme catalyzes multiple reactions involved in the synthesis of steroids and other lipids, including cholesterol (57). AR inhibition has been shown previously to activate mTOR signaling in hepatocellular carcinoma (58), and our laboratory identified a feedback loop between AR and mTOR signaling in breast cancer (28), providing a potential link between seviteronel and mTOR pathway activation. Finally, androgens are known to regulate myogenic differentiation (59, 60); thus, seviteronel likely has dramatic effects on myogenesis as well. Future studies are needed to confirm the antitumor contribution of these and other pathways affected by seviteronel.

In addition to targeting AR directly and inhibiting ligand synthesis, targeting additional points within the AR signaling cascade may be therapeutically beneficial because clinical data from prostate cancer show that acquired resistance to AR-targeted therapies can develop over time (61). While this has yet to be shown for breast cancer, being mindful of acquired resistance from the outset could lead to more effective combination therapies for patients prior to their clinical need. Our data indicate that blocking the cell cycle may be an effective cooperative therapeutic approach to add to anti-androgen therapy in AR+ TNBC (Fig. 4).

During the cell cycle, CDK4/6 binds to cyclin D to mediate phosphorylation and deactivation of Rb protein, which allows for G0–G1- to S-phase progression through the cell cycle. ER+ breast cancers often overexpress cyclin D, promoting cell cycle activation, making CDK4/6-targeted therapies an attractive therapeutic option (62), and the FDA has approved multiple CDK4/6 inhibitors for use in the treatment of ER+ breast cancer, including palbociclib, ribociclib, and abemaciclib, between 2015 and 2017 (63). While a portion of TNBCs have RB1 mutations and/or loss, making CDK4/6 inhibition impractical, AR and Rb expression are positively associated and AR promotes cyclin D-CDK4/6 activation (63–65), suggesting the AR+ TNBCs may benefit from CDK4/6 blockade. In fact, our data indicate that not only do AR+ TNBC xenografts respond well to the CDK4/6 inhibitor, abemaciclib, but that the combination of abemaciclib and seviteronel was more effective than abemaciclib alone (Fig. 5). The synergistic effect is likely due to the interconnectedness and known overlap between the AR signaling and cell cycle pathways (34, 64). While CDK4/6 inhibitors are not currently approved for use in TNBC, our preclinical data suggest that this may be an effective treatment strategy in AR+ TNBC, especially when combined with AR-targeting therapies.

M.M. Williams reports grants from the NIH/NCI during the conduct of the study. G.D. Trahan reports other support from 10x Genomics outside the submitted work. J.R. Eisner reports a patent for seviteronel + dexamethasone pending. J.K. Richer reports grants from Innocrin during the conduct of the study. No disclosures were reported by the other authors.

J.L. Christenson: Conceptualization, data curation, formal analysis, investigation, visualization, writing–original draft, project administration, writing–review and editing. K.I. O'Neill: Software, formal analysis, visualization, writing–review and editing. M.M. Williams: Formal analysis, investigation, writing–review and editing. N.S. Spoelstra: Validation, investigation, visualization, methodology, project administration, writing–review and editing. K.L. Jones: Resources, software, formal analysis, writing–review and editing. G.D. Trahan: Software, formal analysis, writing–review and editing. J. Reese: Investigation, methodology, project administration. E.T. Van Patten: Writing–original draft. A. Elias: Writing–review and editing. J.R. Eisner: Conceptualization, funding acquisition, writing–original draft, project administration, writing–review and editing. J.K. Richer: Conceptualization, resources, supervision, funding acquisition, writing–original draft, writing–review and editing.

The authors acknowledge the shared resources of the University of Colorado Cancer Center NCI Support Grant (P30CA046934), particularly the Genomics Shared Resource and the Office of Laboratory Animal Resources. The authors would also like to thank Dr. Jim Lambert for his assistance with cell line preparations for in vivo xenograft experiments. Financial support included NIH NRSA T32 CA190216-01A1 (to J.L. Christenson), DOD CTRA W81SWH-13-1-0091/90 (to A. Elias and J.K. Richer), and NIH R01CA187733 (to J.K. Richer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data